WO2014147873A1 - Antibody that binds specifically with hmgb1 decomposition product, and method and reagent for assaying hmgb1 decomposition product - Google Patents

Antibody that binds specifically with hmgb1 decomposition product, and method and reagent for assaying hmgb1 decomposition product Download PDF

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WO2014147873A1
WO2014147873A1 PCT/JP2013/076177 JP2013076177W WO2014147873A1 WO 2014147873 A1 WO2014147873 A1 WO 2014147873A1 JP 2013076177 W JP2013076177 W JP 2013076177W WO 2014147873 A1 WO2014147873 A1 WO 2014147873A1
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antibody
hmgb1
degradation product
thrombin
sample
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PCT/JP2013/076177
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French (fr)
Japanese (ja)
Inventor
東　義則
山田　晋吾
幸恵 小野
丸山　征郎
伊藤　隆史
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株式会社シノテスト
国立大学法人鹿児島大学
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Priority to JP2015506540A priority Critical patent/JPWO2014147873A1/en
Publication of WO2014147873A1 publication Critical patent/WO2014147873A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6875—Nucleoproteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons

Definitions

  • the present invention relates to an antibody that specifically binds to a degradation product of HMGB1 with reduced cytotoxicity compared to HMGB1, and a method and reagent for measuring the degradation product of HMGB1.
  • the present invention is useful in the field of life science such as clinical examination, clinical pathology, immunology and medicine, and in the field of chemistry such as analytical chemistry.
  • HMGB High Mobility Group Box Protein
  • HMG High Mobility Group Protein
  • HMGB1 high mobility group box protein 1
  • HMGB2 high mobility group box protein 2
  • HMGB3 high mobility group box protein 3
  • HMGB8 high mobility group box protein 8
  • HMGB17 high mobility group box protein 17
  • HMGB I High Mobility Group Box Protein I
  • HMGB Y High Mobility Group Box Protein Y
  • HMGB I Y
  • HMGB I-C High Mobility Group Box Protein I-C
  • the present inventors analyzed the homology of bovine HMGB1 was 98 with respect to human HMGB1.
  • HMGB1 The homology of porcine HMGB1 was 99.1%.
  • human HMGB2 has a homology of 81.2% with respect to human HMGB1, a homology with bovine HMGB2 of 72.3%, and a homology with porcine HMGB2 of 79. 4%.
  • Wang et al. Quantitatively measured HMGB1 in serum (in blood) for the first time by Western blotting using a polyclonal antibody prepared using HMGB1 itself as an immunogen. As a result, Wang et al. Showed that HMGB1 can be a marker for sepsis.
  • Non-Patent Document 1 It has been previously shown that antibodies used for HMGB1 measurement, that is, antibodies that bind to HMGB1, can be prepared by Parkinen et al., Lepp et al. (See Patent Document 3). Using this antibody, Rep et al. Stated that a solid-phase enzyme immunoassay is possible for HMGB1. In addition, a method for preparing human HMGB1 and human HMGB2 has been shown by Cabart et al. (See Non-Patent Document 4).
  • HMGB1 is also induced by inflammation, and literatures that this is considered to be a cause of mass secretion of various cytokines have been published (see Non-Patent Document 5, Non-Patent Document 6 and Non-Patent Document 7). .
  • the present inventors previously obtained an antibody that binds to human HMGB1 but does not bind to human HMGB2, and human HMGB1 that can obtain an accurate measurement value without error without measuring HMGB2.
  • An immunological measurement reagent and an immunological measurement method were developed (see Patent Document 1).
  • the present inventors previously described an anti-human HMGB1 antibody that is a high-titer antibody that has a high binding ability to human HMGB1 and that can obtain the antibody with a high probability, Has developed an immunological measurement method and an immunological measurement reagent for human HMGB1 in a highly sensitive sample capable of accurately measuring even human HMGB1 contained in the above (see Patent Document 2).
  • the present inventors cleave the 10th arginine (R10) -11th glycine (G11) of human HMGB1 by thrombin or thrombin thrombomodulin complex, It was found that a peptide consisting of the amino acid residues of HMGB1 was separated to produce a degradation product of HMGB1 having a newly exposed N-terminal “GKMSS...”. (The inventors named this degradation product of human HMGB1 as “des-HMGB1”.) As a result of the study by the present inventors, it was found that the degradation product of HMGB1 has a lower cytotoxicity than HMGB1.
  • HMGB1 is decomposed (cleaved) as described above using thrombin or thrombin / thrombomodulin complex, and converted to a degradation product of HMGB1 having a lower cytotoxicity.
  • thrombin or thrombin-thrombomodulin complex quantitative measurement of the degradation product of HMGB1 was required and a measurement method was examined.
  • Conventional methods are based on degradation of HMGB1.
  • HMGB1 and the like are also measured together with the product, and only the degradation product of HMGB1 cannot be specifically measured (see Non-Patent Document 8).
  • the conventional antibody is not highly specific for the degradation product of HMGB1, and the affinity for the degradation product of HMGB1 is similar to that for HMGB1.
  • the conventional measurement method and reagent are not highly specific for the HMGB1 degradation product, and not only measure the HMGB1 degradation product but also measure HMGB1 and the like, that is, the measured value Included a positive error derived from HMGB1 and the like.
  • the gist of the present invention is as follows. (1) The degradation product of HMGB1 by the thrombin or thrombin / thrombomodulin complex is at least 1.5 times the affinity for the degradation product by the thrombin or thrombin / thrombomodulin complex by comparison with the affinity for HMGB1. An antibody that binds. (2) The affinity of HMGB1 for the degradation product by thrombin or thrombin-thrombomodulin complex is at least 10 times greater than the affinity for HMGB2 and the affinity of HMGB2 for the degradation product by thrombin or thrombin-thrombomodulin complex, respectively.
  • the antibody that binds to the degradation product of HMGB1 by thrombin or thrombin / thrombomodulin complex is a monoclonal antibody, and binds to the degradation product of HMGB1 by thrombin or thrombin / thrombomodulin complex according to (1) or (2) above antibody.
  • a solid-phase antibody and a labeled antibody wherein either one of the antibody (a) and the antibody (b) is used as a solid-phase antibody, and the other antibody
  • a reagent for immunological measurement of degradation products of thrombin or thrombin / thrombomodulin complex of HMGB1 contained in a sample comprising the following antibodies (a) and (b): (A) The antibody according to any one of (1) to (3).
  • the antibody of the present invention is an antibody whose affinity for degradation products of thrombin or thrombin-thrombomodulin complex of HMGB1 is at least 1.5 times higher than the affinity for HMGB1, ie, specific for the HMGB1 degradation product It is a highly potent antibody.
  • the measurement method and reagent of the present invention have high specificity for the degradation product of HMGB1 by thrombin or thrombin / thrombomodulin complex, and the measurement of HMGB1 and the like is suppressed, that is, positively derived from HMGB1 and the like. Is suppressed, and only the HMGB1 degradation product can be accurately quantitatively measured.
  • FIG. 1 is a photograph of a gel when HMGB1, the HMGB1 degradation product, HMGB2, and the HMGB2 degradation product were respectively migrated.
  • Lane 1 is a molecular weight marker
  • lane 2 is HMGB1
  • lane 3 is the HMGB1 degradation product
  • lane 4 is HMGB2
  • lane 5 is the HMGB2 degradation product
  • lane 6 is thrombin.
  • FIG. 2 is a photograph of Western blotting confirming the reactivity (affinity) of the anti-HMGB1 degradation product antibody (5D1) with HMGB1, the HMGB1 degradation product, HMGB2, and the HMGB2 degradation product.
  • 5D1 anti-HMGB1 degradation product antibody
  • Lane 1 shows a molecular weight marker
  • lane 2 shows HMGB1, lane 3 shows the HMGB1 degradation product
  • lane 4 shows HMGB2, and lane 5 shows the HMGB2 degradation product.
  • FIG. 3 is a photograph of Western blotting confirming the reactivity (affinity) of the anti-HMGB1 degradation product antibody (2H6) with HMGB1, the HMGB1 degradation product, HMGB2, and the HMGB2 degradation product.
  • Lane 1 shows a molecular weight marker
  • lane 2 shows HMGB1
  • lane 3 shows the HMGB1 degradation product
  • lane 4 shows HMGB2 degradation product.
  • FIG. 1 shows a molecular weight marker
  • lane 3 shows the HMGB1 degradation product
  • lane 4 shows HMGB2 degradation product.
  • FIG. 4A shows the reactivity of the antibody (2D4) to the HMGB1 degradation product and the like immobilized on a microtiter plate.
  • the horizontal axis represents the concentration of antibody in each antibody solution used as a sample, and the vertical axis represents the absorbance value when measured by ELISA.
  • FIG. 4B is a diagram showing the reactivity of the antibody (4F12) to the HMGB1 degradation product and the like immobilized on a microtiter plate.
  • the horizontal axis represents the concentration of antibody in each antibody solution used as a sample, and the vertical axis represents the absorbance value when measured by ELISA.
  • FIG. 4A shows the reactivity of the antibody (2D4) to the HMGB1 degradation product and the like immobilized on a microtiter plate.
  • the horizontal axis represents the concentration of antibody in each antibody solution used as a sample, and the vertical axis represents the absorbance value when measured by ELISA.
  • FIG. 4C is a view showing the reactivity of the antibody (8H4) to the HMGB1 degradation product and the like immobilized on a microtiter plate.
  • the horizontal axis represents the concentration of antibody in each antibody solution used as a sample, and the vertical axis represents the absorbance value when measured by ELISA.
  • FIG. 4D is a view showing the reactivity of the antibody (2H6) to the HMGB1 degradation product and the like immobilized on a microtiter plate.
  • the horizontal axis represents the concentration of antibody in each antibody solution used as a sample, and the vertical axis represents the absorbance value when measured by ELISA.
  • FIG. 4E shows the reactivity of the antibody (5D1) to the HMGB1 degradation product and the like immobilized on a microtiter plate.
  • the horizontal axis represents the concentration of antibody in each antibody solution used as a sample, and the vertical axis represents the absorbance value when measured by ELISA.
  • FIG. 4F is a view showing the reactivity of the antibody (2A10) to the HMGB1 degradation product and the like immobilized on a microtiter plate.
  • the horizontal axis represents the concentration of antibody in each antibody solution used as a sample, and the vertical axis represents the absorbance value when measured by ELISA.
  • FIG. 4G is a view showing the reactivity of the antibody (6H3) to the HMGB1 degradation product and the like immobilized on a microtiter plate.
  • the horizontal axis represents the concentration of antibody in each antibody solution used as a sample, and the vertical axis represents the absorbance value when measured by ELISA.
  • FIG. 4H is a view showing the reactivity of the antibody (MD78) to the HMGB1 degradation product and the like immobilized on a microtiter plate.
  • the horizontal axis represents the concentration of antibody in each antibody solution used as a sample, and the vertical axis represents the absorbance value when measured by ELISA.
  • FIG. 4I shows the reactivity of the antibody (MD77) to the HMGB1 degradation product and the like immobilized on a microtiter plate.
  • the horizontal axis represents the concentration of antibody in each antibody solution used as a sample, and the vertical axis represents the absorbance value when measured by ELISA.
  • FIG. 4J is a view in which the reactivity of the antibody (4C3) to the HMGB1 degradation product and the like immobilized on a microtiter plate is confirmed.
  • the horizontal axis represents the concentration of antibody in each antibody solution used as a sample, and the vertical axis represents the absorbance value when measured by ELISA.
  • FIG. 4K shows the reactivity of the antibody (J2E1) to the HMGB1 degradation product and the like immobilized on a microtiter plate.
  • the horizontal axis represents the concentration of antibody in each antibody solution used as a sample, and the vertical axis represents the absorbance value when measured by ELISA.
  • FIG. 4L shows the reactivity of the antibody (HAP46.5) to the HMGB1 degradation product and the like immobilized on a microtiter plate.
  • the horizontal axis represents the concentration of antibody in each antibody solution used as a sample, and the vertical axis represents the absorbance value when measured by ELISA.
  • FIG. 5 is a diagram showing the results of measuring HMGB1 and the HMGB1 degradation product by ELISA (sandwich method) using an antibody that binds to the HMGB1 degradation product and an antibody that binds to other HMGB1.
  • the upper part of the abscissa indicates the letters indicating the antibody producing cell line of the POD-labeled antibody solution used in the measurement, and the lower part of the abscissa indicates the antibody producing cell line of the antibody-immobilized microplate used for the measurement. Letters are shown, and the vertical axis represents absorbance values measured by ELISA.
  • FIG. 6 is a diagram showing calibration curves when HMGB1 and the HMGB1 degradation product are measured by ELISA method (sandwich method) as a conventional reagent / method.
  • the horizontal axis represents the concentration of HMGB1 or the degradation product of HMGB1 in the sample (0 to 80 ng / mL), and the vertical axis represents the absorbance value when measured by the ELISA method.
  • FIG. 7 is a diagram showing a calibration curve when HMGB1 and the HMGB1 degradation product are measured by ELISA method (sandwich method) according to the present invention.
  • FIG. 8 shows the anti-HMGB1 degradation product antibody (2H6) as the antibody (a) according to the present invention and the anti-HMGB1 degradation product antibody (5D1) as the antibody (b) by ELISA (sandwich method). It is a figure which shows a calibration curve when each of HMGB1 degradation product, HMGB2, and the said HMGB2 degradation product is measured.
  • FIG. 9 shows the anti-HMGB1 degradation product antibody (2H6) as the antibody (a) according to the present invention and the anti-HMGB1 degradation product antibody (2A10) as the antibody (b) by ELISA (sandwich method). It is a figure which shows a calibration curve when each of HMGB1 degradation product, HMGB2, and the said HMGB2 degradation product is measured.
  • FIG. 10 shows the anti-HMGB1 degradation product antibody (2H6) as the antibody (a) according to the present invention and the anti-HMGB1 degradation product antibody (6H3) as the antibody (b) by ELISA (sandwich method). It is a figure which shows a calibration curve when each of HMGB1 degradation product, HMGB2, and the said HMGB2 degradation product is measured.
  • the horizontal axis represents the concentration (0 to 80 ng / mL) of HMGB1, HMGB1 degradation product, HMGB2, or HMGB2 degradation product in the sample, and the vertical axis represents the value of the absorbance difference when measured by the ELISA method.
  • Anti-HMGB1 degradation product antibody of the present invention is a thrombin or thrombin-thrombomodulin complex of HMGB1, wherein the affinity for the degradation product of thrombin or thrombin-thrombomodulin complex of HMGB1 is at least 1.5 times compared to the affinity for HMGB1.
  • an antibody that binds to a degradation product by (Hereinafter, this antibody of the present invention may also be referred to as “the present anti-HMGB1 degradation product antibody”.)
  • the affinity of HMGB1 for the degradation product by thrombin or thrombin / thrombomodulin complex is “at least 1.5 times” compared to the affinity for HMGB1, and the concentration of the antibody is 0.625 to 5 ng / mL. At least one concentration in the range (preferably all concentrations in the range) means that the affinity is at least 1.5 times as measured by the “method for measuring affinity” described later.
  • the affinity of HMGB1 for the degradation product of thrombin or thrombin / thrombomodulin complex is compared with the affinity for HMGB2 and the affinity of HMGB2 for the degradation product of thrombin or thrombin / thrombomodulin complex, respectively.
  • those that are at least 10 times each are preferred.
  • Antibody concentration at least one concentration in the range of 0.625 to 5 ng / mL (preferably all concentrations in the range)
  • the anti-HMGB1 degradation product antibody is preferably a monoclonal antibody.
  • the anti-HMGB1 degradation product antibody is a polyclonal antibody, an antiserum containing a polyclonal antibody, a monoclonal antibody, or a fragment of these antibodies (Fab, F (ab ′)). 2 Or Fab ′ or the like.
  • HMGB1 degradation products The “degradation product of HMGB1 by thrombin or thrombin-thrombomodulin complex” in the present invention is a protein or peptide produced by hydrolysis of human HMGB1 by thrombin or thrombin-thrombomodulin complex.
  • the 10th arginine (R10) -11th glycine (G11) of human HMGB1 is cleaved by thrombin or thrombin-thrombomodulin complex, and a peptide comprising 10 amino acid residues of “MGKGDPKKPR” at the N-terminus of HMGB1 Refers to a degradation product of HMGB1 having a newly exposed N-terminal “GKMSS...” Generated by separation.
  • the amino acid sequence of human HMGB1 is shown in the sequence listing as SEQ ID NO: 1, and the amino acid sequence of the degradation product of human HMGB1 by thrombin or thrombin-thrombomodulin complex is shown as SEQ ID NO: 2.
  • deletion, substitution, or insertion of one to several amino acid residues in the amino acid sequence shown as SEQ ID NO: 2 is used.
  • it may be a protein comprising an amino acid sequence obtained by addition or modification, or a saccharide or lipid bound to this protein.
  • the number of amino acid residues in the deletion, substitution, insertion, addition or modification of the amino acid residues is usually 1 to 4, preferably 1 to 3, more preferably 1 to 2, particularly preferably 1. It is a piece.
  • the thrombin or thrombin / thrombomodulin complex is not only a thrombin or thrombin / thrombomodulin complex present in the human body, but also an artificially prepared one such as a gene recombinant or the like It includes those that have been artificially isolated.
  • “degradation product of HMGB1 by thrombin or thrombin-thrombomodulin complex” may be referred to as “the HMGB1 degradation product” hereinafter.)
  • the anti-HMGB1 degradation product antibody has an affinity for the HMGB1 degradation product that is at least 1.5 times the affinity for HMGB1.
  • the method for measuring the affinity of this antibody is not particularly limited, and can be performed, for example, as follows.
  • HMGB1 degradation products Each of HMGB1, HMGB1 degradation product, HMGB2 and HMGB2 degradation product by thrombin or thrombin-thrombomodulin complex (hereinafter sometimes referred to as “the HMGB2 degradation product”) is 1 ⁇ g / mL with phosphate buffered saline.
  • Each of the 96-well microtiter plates [Thermo Fisher Scientific Inc. (Ind., Illinois, USA)] was injected into a well of 100 ⁇ L, allowed to stand at 25 ° C.
  • HMGB1, HMGB1 degradation product, HMGB2 and HMGB2 degradation product were respectively added to the wells of the microtiter plate.
  • TBS Tris buffered saline
  • POD-labeled anti-mouse IgG antibody solution POD-labeled anti-mouse IgG antibody [DakoCytomation (Denmark)] was diluted 1000-fold with 50 mM Tris-HCl buffer (pH 8.0) containing 0.5% sodium caseinate and 100 mM sodium chloride. This was used as a POD-labeled anti-mouse IgG antibody solution.
  • Cleaning solution Phosphate buffered saline containing 0.05% Tween 20 was used as a washing solution.
  • the measurement when the measurement is performed as described above using a solution of an antibody whose affinity is to be measured (for example, a concentration of 2.5 ng / mL) as a sample, it is obtained in a well in which the HMGB1 degradation product is immobilized.
  • this antibody When the absorbance difference value is 1.35 and the absorbance difference value obtained in the well in which HMGB1 is immobilized is 0.9, this antibody is a product of degradation of HMGB1. It can be said that the affinity for is at least 1.5 times that of HMGB1. That is, in this case, this antibody is the present anti-HMGB1 degradation product antibody.
  • (4) Immunogen The immunogen for obtaining the anti-HMGB1 degradation product antibody will be described below.
  • Examples of the immunogen for obtaining this anti-HMGB1 degradation product antibody include, for example, human HMGB1, HMGB1 of animals having high homology with the amino acid sequence of human HMGB1 (for example, bovine or pig), or the HMGB1 degradation product The whole or a part can be mentioned.
  • Examples of the HMGB1 degradation product include a peptide comprising 20 amino acid residues of “GKMSYAFFVQTCREEHKK” at the N-terminus of the HMGB1 degradation product.
  • the immunogen for obtaining the anti-HMGB1 degradation product antibody is a deletion, substitution, insertion, addition or modification of one to several amino acid residues in the protein or peptide as the immunogen described so far.
  • It may be a protein or peptide obtained by applying.
  • the number of amino acid residues in the deletion, substitution, insertion, addition or modification of the amino acid residues is usually 1 to 4, preferably 1 to 3, more preferably 1 to 2, particularly preferably 1. It is a piece.
  • carrier carrier
  • carrier carrier about the protein or peptide as an immunogen described so far may be sufficient.
  • the protein or peptide as the immunogen has a low molecular weight, the antibody production ability is improved by binding to the carrier, and therefore, it is preferable to use the one bound to the carrier as the immunogen.
  • the carrier examples include mussel hemocyanin (KLH), bovine serum albumin (BSA), human serum albumin (HSA), chicken serum albumin, poly-L-lysine, polyalanyl lysine, dipalmityl lysine, Known carriers such as tetanus toxoid or polysaccharide can be used.
  • KLH mussel hemocyanin
  • BSA bovine serum albumin
  • HSA human serum albumin
  • chicken serum albumin poly-L-lysine
  • polyalanyl lysine dipalmityl lysine
  • Known carriers such as tetanus toxoid or polysaccharide can be used.
  • Method for obtaining immunogen etc. of this anti-HMGB1 degradation product antibody A method for obtaining an immunogen or the like for immunizing an animal or the like in order to obtain the present anti-HMGB1 degradation product antibody will be described below.
  • the anti-HMGB1 degradation product antibody is an immunogen for obtaining the human HMGB1 described in (4) above, or an animal HMGB1 having a high homology with the amino acid sequence of human HMGB1. Extraction and purification from body fluids, cells, tissues, organs, etc. of mammals other than humans (pigs, cows, rabbits, goats, sheep, mice, rats, etc.) having high homology with the amino acid sequence of HMGB1 by known methods And so on.
  • the HMGB1 degradation product may be obtained by, for example, bringing human HMGB1 or HMGB1 of an animal having high homology with the amino acid sequence of human HMGB1 (for example, bovine or pig) into contact with the thrombin or thrombin / thrombomodulin complex. It can be obtained by hydrolysis, and extraction and purification by a known method.
  • the immunogen can be synthesized by a peptide synthesis method such as a liquid phase method or a solid phase method, and an automatic peptide synthesizer may be used. For example, “Biochemistry Experiment Course 1 Protein Chemistry IV”, Tokyo Kagaku Dojin, 1975; Izumiya et al.
  • the immunogen may be prepared from DNA or RNA having a corresponding nucleobase sequence by using genetic engineering technology, edited by the Japanese Biochemical Society, “Second Biochemistry Experiment Course 1, Gene Research Method I”, Tokyo Chemical. Doujin, 1986; “Sequential Biochemistry Experiment Course 1 Genetic Research Method II” edited by the Japanese Biochemical Society, Tokyo Chemical Doujin, 1986; or “The Biochemistry Experimental Course 1 Genetic Research Method III” edited by the Japanese Biochemical Society, Tokyo Chemical Doujin 1987 and the like.
  • the amino acid sequence of human HMGB1 represented by SEQ ID NO: 1 or the amino acid sequence of the degradation product of HMGB1 represented by SEQ ID NO: 2, or a gene corresponding to a part of these amino acid sequences is incorporated into an expression vector such as a plasmid.
  • an expression vector such as a plasmid.
  • a host cell such as Escherichia coli and culturing the resulting transformant, a protein or peptide comprising the above amino acid sequence or a part thereof can be expressed.
  • Examples of a method for cloning a gene base sequence include a PCR method, a recombinant PCR method, a ligation method, and a linker ligation method.
  • the immunogen when the immunogen is a low-molecular substance, it is common to immunize an animal or the like with a carrier (carrier) bound to the immunogen, but a peptide having 5 amino acids is used as an immunogen. Since there is also a report (Kiyama et al., “Abstract 3 of the 112th Annual Meeting of the Japanese Pharmaceutical Society”, page 122, published in 1992) that a specific antibody was produced, it is not essential to use a carrier.
  • a carrier when immunizing an animal or the like with the above protein or peptide bound to a carrier mussel hemocyanin (KLH), bovine serum albumin (BSA), human serum albumin (HSA), chicken serum albumin
  • KLH mussel hemocyanin
  • BSA bovine serum albumin
  • HSA human serum albumin
  • Any known carrier such as poly-L-lysine, polyalanyl lysine, dipalmityl lysine, tetanus toxoid or polysaccharide can be used.
  • the protein or peptide and the carrier may be bound by the glutaraldehyde method, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide method, maleimidobenzoyl-N-hydroxysuccinimide ester method, bisdiazotized benzidine method or Known coupling methods such as the N-succimidyl-3- (2-pyridyldithio) propionic acid method can be used.
  • sucked the said protein or peptide to carriers such as a nitrocellulose particle
  • the polyclonal antibody or antiserum can be obtained by the following operation.
  • a mammal a mouse, a rabbit, a rat, a sheep, a goat, a horse, etc.
  • a bird a chicken, etc.
  • the immunity of the immunogen or the conjugate of the immunogen and the carrier is determined by the type of animal to be immunized, the site of immunization, and the like.
  • the immunogen or the combined immunogen and carrier is preferably added and mixed with an adjuvant for immunization injection.
  • an adjuvant known ones such as Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide adjuvant or pertussis adjuvant can be used.
  • Immunization may be performed at a site such as subcutaneous, intravenous, intraperitoneal or back. After the initial immunization, booster injections of the immunogen or a conjugate of the immunogen and the carrier are given at sites such as subcutaneous, intravenous, intraperitoneal or back at intervals of 2 to 3 weeks.
  • the immunogen or the conjugate of the immunogen and the carrier is preferably boosted by adding an adjuvant and mixing.
  • the antibody titer in the sera of the immunized animal is repeatedly measured by ELISA or the like. When the antibody titer reaches a plateau, the whole blood is collected, and the serum is separated to contain the anti-HMGB1 degradation product antibody. Get.
  • the antiserum is subjected to antibody purification by a salting-out method using ammonium sulfate, sodium sulfate or the like, ion exchange chromatography, gel filtration method or affinity chromatography, or a combination of these methods to obtain a polyclonal antibody.
  • the polyclonal antibody obtained here comprises an antibody (this anti-HMGB1 degradation product antibody) having an affinity for the HMGB1 degradation product of at least 1.5 times that of HMGB1, and the HMGB1 degradation product. Since both of the antibodies (conventional antibodies) having an affinity for HMGB1 of less than 1.5 times compared to the affinity for HMGB1 are included, this is further used for affinity chromatography immobilized on a solid phase using HMGB1 as a ligand. Pass through the column. The conventional antibody binds to the solid phase via the ligand (HMGB1) of this column and is collected.
  • this anti-HMGB1 degradation product antibody having an affinity for the HMGB1 degradation product of at least 1.5 times that of HMGB1, and the HMGB1 degradation product. Since both of the antibodies (conventional antibodies) having an affinity for HMGB1 of less than 1.5 times compared to the affinity for HMGB1 are included, this is further used for affinity chromatography immobilized on a solid phase using HMGB1 as a
  • the anti-HMGB1 degradation product antibody is difficult to bind to the ligand (HMGB1) of this column, and many of these columns pass through this column.
  • the anti-HMGB1 degradation product antibody is obtained. Can be obtained. This is passed through an affinity chromatography column immobilized on a solid phase with the HMGB1 degradation product as a ligand, and the anti-HMGB1 degradation product antibody is passed through the solid phase via the ligand (the HMGB1 degradation product) of this column. Other antibodies are separated by passing through this column, and then the anti-HMGB1 degradation product antibody bound to the solid phase is separated from the ligand (the HMGB1 degradation product) and separated from this column.
  • the anti-HMGB1 degradation product antibody with higher purity can be obtained.
  • an animal or the like is immunized using a conjugate of an immunogen and a carrier, an antibody against this carrier exists in the obtained antiserum or polyclonal antibody. It is preferable to perform the removal process.
  • a carrier is added to the obtained polyclonal antibody or antiserum solution to remove aggregates generated, or the carrier is immobilized on an insolubilized solid phase and removed by affinity chromatography, etc. Can be used.
  • a monoclonal antibody can be obtained by the following operation.
  • Monoclonal antibodies are antibody-producing cells such as hybridomas by the cell fusion method of Keller et al. (G. Koehler et al., Nature, 256, 495-497, published in 1975), or tumorigenic cells by viruses such as Epstan-Barr virus. Can be obtained.
  • Preparation of a monoclonal antibody by the cell fusion method can be performed by the following operation.
  • a mammal (mouse, nude mouse, rat, etc., for example, BALB / c of an inbred mouse) or a bird (chicken, etc.) is immunized with the immunogen or a conjugate of the immunogen and a carrier.
  • the immunization amount of the immunogen or the conjugate of the immunogen and the carrier can be appropriately determined depending on the type of immunized animal, the site of immunization, and the like.
  • 0.1 ⁇ g to 5 mg of the immunogen or a combination of the immunogen and a carrier is immunized at a time.
  • the immunogen or the conjugate of the immunogen and the carrier is preferably immunized by adding an adjuvant and mixing.
  • adjuvants such as Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide adjuvant, or pertussis adjuvant can be used as the adjuvant.
  • Immunization may be performed at a site such as subcutaneous, intravenous, intraperitoneal or back.
  • booster injections of the immunogen or a conjugate of the immunogen and the carrier are given at sites of subcutaneous, intravenous, intraperitoneal, or back at 1-2 week intervals.
  • the number of booster injections is generally 2 to 6 times.
  • the immunogen or the combined immunogen and carrier is preferably boosted by adding an adjuvant and mixing.
  • the antibody titer in the sera of the immunized animal is repeatedly measured by ELISA or the like.
  • the immunogen or the combined immunogen and carrier is added to physiological saline.
  • a solution dissolved in (0.9% sodium chloride aqueous solution) is injected intravenously or intraperitoneally to obtain final immunization.
  • cells having antibody-producing ability such as spleen cells, lymph node cells or peripheral lymphocytes of immunized animals are obtained.
  • the cell having antibody-producing ability obtained from this immunized animal is fused with myeloma cells (myeloma cells) of mammals (mouse, nude mouse, rat, etc.).
  • myeloma cells myeloma cells
  • mammals mae, nude mouse, rat, etc.
  • a cell line deficient in an enzyme such as guanine phosphoribosyl transferase (HGPRT) or thymidine kinase (TK) is preferred.
  • HGPRT guanine phosphoribosyl transferase
  • TK thymidine kinase
  • ATCC P3-X63-Ag8 strain which is a HGPRT-deficient cell line derived from BALB / c mice.
  • JCRB 0028 P3-X63-Ag8-U1 strain
  • JCRB 0009 P3-NS1-1-Ag4-1 strain
  • JCRB 0028 P3-X63-Ag8.653 strain
  • SP2 / O-Ag-14 strain JCRB 0029 or the like is used.
  • Cell fusion can be performed using a fusion promoter such as polyethylene glycol (PEG) of various molecular weights, liposomes or Sendai virus (HVJ), or by electrofusion.
  • PEG polyethylene glycol
  • HVJ Sendai virus
  • myeloma cells are of HGPRT-deficient strain or TK-deficient strain
  • fusion of cells capable of producing antibodies and myeloma cells by using a selection medium (HAT medium) containing hypoxanthine / aminopterin / thymidine Only cells (hybridomas) can be selectively cultured and propagated.
  • the hybridoma culture supernatant thus obtained is subjected to immunological measurement such as ELISA or Western blot using the immunogen, the conjugate of the immunogen and the carrier, or the HMGB1 degradation product.
  • a hybridoma that produces this anti-HMGB1 degradation product antibody can be selected.
  • a hybridoma that produces an antibody that does not bind to HMGB1 or the like is selected by measuring the culture supernatant of the hybridoma by immunoassay such as ELISA or Western blot using HMGB1 or the like. Can do.
  • the anti-HMGB1 degradation product antibody (monoclonal antibody), that is, “degradation product of HMGB1 by thrombin or thrombin / thrombomodulin complex” is performed by combining these two kinds of hybridoma selection methods and known cloning methods such as limiting dilution.
  • An antibody that binds to a degradation product of HMGB1 by thrombin or thrombin-thrombomodulin complex (monoclonal antibody) having an affinity for HMGB1 that is at least 1.5 times that of HMGB1 Can be obtained.
  • This monoclonal antibody-producing cell line can be cultured in an appropriate medium, and the anti-HMGB1 degradation product antibody (monoclonal antibody) can be obtained from the culture supernatant.
  • a serum-free medium or low-concentration serum medium can be used. In this case, it is preferable from the viewpoint of easy purification of the antibody, and a medium such as DMEM medium, RPMI 1640 medium, or ASF medium 103 can be used.
  • a monoclonal antibody-producing cell line is injected into the abdominal cavity of a mammal that is compatible with this and prestimulated with pristane or the like, and after a certain period of time, the anti-HMGB1 degradation product antibody (monoclonal antibody) from ascites collected in the abdominal cavity. ) Can also be obtained.
  • the monoclonal antibody thus obtained was purified by a salting-out method using ammonium sulfate, sodium sulfate or the like, a method such as ion exchange chromatography, gel filtration or affinity chromatography, or a combination of these methods.
  • This anti-HMGB1 degradation product antibody (monoclonal antibody) can be obtained.
  • the immunological measurement method of HMGB1 degradation product contained in the sample of the present invention includes the following (a): The measurement method using the antibody of (b) and the antibody of (b).
  • the antibody (b) includes HMGB1 thrombin or thrombin at a concentration of the antibody in the range of 0.625 to 2.5 ng / mL (preferably all concentrations in the range).
  • the absorbance value obtained when measuring the amount of the antibody bound to the degradation product is the absorbance of the antibody produced from the hybridoma MD78 (FERM P-18405), which is the reference antibody-producing cell. Antibodies that are more than 6 times the value Is preferred.
  • the affinity of HMGB1 for the degradation product by thrombin or thrombin / thrombomodulin complex is the same as the affinity for HMGB2 and the affinity for HMGB2 by thrombin or thrombin / thrombomodulin complex. Those that are each at least 10 times greater than the affinity for the degradation products are preferred.
  • Antibody concentration at least one concentration in the range of 0.625 to 5 ng / mL (preferably all concentrations in the range)
  • the antibody (a) (anti-HMGB1 degradation product antibody) and / or the antibody (b) are preferably monoclonal antibodies.
  • the antibody (a) (anti-HMGB1 degradation product antibody) and / or the antibody (b) are polyclonal antibodies, antisera containing polyclonal antibodies, monoclonal antibodies, or fragments of these antibodies ( Fab, F (ab ′) 2 Or Fab ′ or the like.
  • the measurement method of the present invention uses a solid-phased antibody and a labeled antibody, and any one of the antibody (a) and the antibody (b) is solid-phased. It may be used as an antibody and the other antibody may be used as a labeled antibody.
  • the measurement method of the present invention uses the antibody (a) and the antibody (b), and has high specificity for the HMGB1 degradation product and accurately measures only the HMGB1 degradation product. Can do.
  • the antibody (a) can be used without particular limitation as long as it is an antibody as described above.
  • the antibody (b) can be used without any particular limitation as long as it is an antibody as described above.
  • the antibody (a) and the antibody (b) are not limited to one type, and a plurality of types may be used simultaneously.
  • (2) Antibody of (a) above Details of the antibody (a), that is, the present anti-HMGB1 degradation product antibody, are as described in the above section “[I] Anti-HMGB1 degradation product antibody”.
  • the antibody of (b) is “an antibody that binds to the HMGB1 degradation product and has high affinity for the HMGB1 degradation product”.
  • HMGB1 thrombin or at least one of the concentrations of the antibody in the range of 0.625 to 2.5 ng / mL (preferably all concentrations in the range) is used.
  • the ELISA method in which the degradation product by the thrombin / thrombomodulin complex is immobilized (A) an antibody having a value of 0.5 or more when the absorbance value obtained when measuring the amount of the antibody bound to the degradation product is divided by the concentration value of the antibody, or (B)
  • the absorbance value obtained when measuring the amount of the antibody bound to the degradation product is the absorbance of the antibody produced from the hybridoma MD78 (FERM P-18405), which is the reference antibody-producing cell.
  • the antibody (b) is an antibody that binds to the HMGB1 degradation product and has high affinity for the HMGB1 degradation product.
  • HMGB1 thrombin or at least one of the concentrations of the antibody in the range of 0.625 to 2.5 ng / mL (preferably all concentrations in the range) is used.
  • HMGB1 degradation product solid-phased microplate The HMGB1 degradation products prepared with phosphate buffered saline to a concentration of 1 ⁇ g / mL were each prepared in 96-well microtiter plates [Thermo Fisher Scientific Inc. Incorporated (Illinois, USA)] was injected at 100 ⁇ L and allowed to stand at 25 ° C. for 18 hours, and the HMGB1 degradation product was immobilized on the wells of the microtiter plate.
  • TBS Tris buffered saline
  • POD-labeled anti-mouse IgG antibody solution POD-labeled anti-mouse IgG antibody [DakoCytomation (Denmark)] was diluted 1000-fold with 50 mM Tris-HCl buffer (pH 8.0) containing 0.5% sodium caseinate and 100 mM sodium chloride. This was used as a POD-labeled anti-mouse IgG antibody solution.
  • Cleaning solution Phosphate buffered saline containing 0.05% Tween 20 was used as a washing solution.
  • Substrate solution A 60 mM disodium phosphate aqueous solution (pH 4.3) containing 5 mM hydrogen peroxide, 41 mM citric acid, and 0.2 mM EDTA ⁇ disodium was used as a substrate solution.
  • Chromogenic substrate The chromogenic solution and the substrate solution were returned to room temperature before use, and mixed in equal amounts at the time of use to obtain a chromogenic substrate.
  • reaction stop solution 0.7N sulfuric acid was used as a reaction stop solution.
  • the antibody solutions having the above three concentrations were prepared by dilution.
  • each well was washed 3 times with 400 ⁇ L of the washing liquid of [iii] in [A].
  • 100 ⁇ L of the POD-labeled anti-mouse IgG antibody solution of [ii] in [A] above was dispensed into each well and allowed to stand at 25 ° C. for 1 hour to cause antigen-antibody reaction.
  • the absorbance of each well was measured (primary wavelength: 450 nm, subwavelength: 550 nm), and the absorbance when each of the antibody solutions of the antibody (b) at the three concentrations was measured as a sample.
  • the absorbance value was set to a value indicating the affinity of the antibody (b) with the HMGB1 degradation product. For example, when the measurement is performed as described above using a solution of an antibody whose affinity is to be measured (for example, a concentration of 2.5 ng / mL) as a sample, it is obtained in a well in which the HMGB1 degradation product is immobilized.
  • [A] to [C] in “(ii-1) Method of measuring the absorbance of the antibody (b)” The same can be done.
  • the measurement was performed as described above in the range where the concentration of the antibody whose affinity was to be measured was 0.625 to 2.5 ng / mL, it was obtained in a well in which the HMGB1 degradation product was immobilized.
  • the value of the absorbance difference obtained when measuring the amount of the antibody bound to the degradation product is 1.2, and is produced from the hybridoma MD78 (FERM P-18405), which is a standard antibody-producing cell.
  • Immunogen As the immunogen for obtaining the antibody (b), for example, the immunogen described in “(4) Immunogen” of the “[I] anti-HMGB1 degradation product antibody” can be used. .
  • the acquisition method described in “Method for acquiring antibody immunogen and the like” can be used.
  • a polyclonal antibody or antiserum can be obtained by the following operation.
  • A First, a mammal (a mouse, a rabbit, a rat, a sheep, a goat, a horse, etc.) or a bird (a chicken, etc.) is immunized with the immunogen or a conjugate of the immunogen and a carrier.
  • the immunity of the immunogen or the conjugate of the immunogen and the carrier is determined by the type of animal to be immunized, the site of immunization, and the like.
  • the immunogen or the combined immunogen and carrier is preferably added and mixed with an adjuvant for immunization injection.
  • an adjuvant known ones such as Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide adjuvant or pertussis adjuvant can be used.
  • Immunization may be performed at a site such as subcutaneous, intravenous, intraperitoneal or back. After the initial immunization, booster injections of the immunogen or a conjugate of the immunogen and the carrier are given at sites such as subcutaneous, intravenous, intraperitoneal or back at intervals of 2 to 3 weeks.
  • the immunogen or the conjugate of the immunogen and the carrier is preferably boosted by adding an adjuvant and mixing.
  • the antibody titer in the sera of the immunized animal is repeatedly measured by ELISA or the like. When the antibody titer reaches a plateau, the whole blood is collected, and the serum is separated to contain the antibody (b). Obtain serum.
  • the antiserum is subjected to antibody purification by a salting-out method using ammonium sulfate, sodium sulfate or the like, ion exchange chromatography, gel filtration method or affinity chromatography, or a combination of these methods to obtain a polyclonal antibody.
  • the affinity of the antibody is measured as described in (ii) above, and an antibody having a high affinity for the HMGB1 degradation product is selected.
  • Antibodies can be obtained.
  • the concentration of the antibody whose affinity is to be measured is at least in the range of 0.625 to 2.5 ng / mL.
  • An ELISA method in which a degradation product of HMGB1 by thrombin or thrombin-thrombomodulin complex is immobilized at any concentration (preferably all concentrations in the range), (A) an antibody having a value of 0.5 or more when the absorbance value obtained when measuring the amount of the antibody bound to the degradation product is divided by the concentration value of the antibody, or (B) The absorbance value obtained when measuring the amount of the antibody bound to the degradation product is the absorbance of the antibody produced from the hybridoma MD78 (FERM P-18405), which is the reference antibody-producing cell. Antibodies that are more than 6 times the value Can be said to have a high affinity for the HMGB1 degradation product.
  • the HMGB1 degradation product is contacted through an affinity chromatography column immobilized on a solid phase with the HMGB1 degradation product as a ligand, and the antibody (b) is immobilized via the ligand (the HMGB1 degradation product) of this column.
  • the antibody (b) is immobilized via the ligand (the HMGB1 degradation product) of this column.
  • other antibodies are separated by passing through this column, and then the antibody (b) bound to the solid phase is separated from the ligand (the HMGB1 degradation product)
  • the antibody (b) having a higher purity can be obtained.
  • C When an animal or the like is immunized using a conjugate of an immunogen and a carrier, an antibody against this carrier exists in the obtained antiserum or polyclonal antibody.
  • the removal process it is preferable to perform the removal process.
  • a carrier is added to the obtained polyclonal antibody or antiserum solution to remove aggregates generated, or the carrier is immobilized on an insolubilized solid phase and removed by affinity chromatography, etc. Can be used.
  • the monoclonal antibody can be obtained by the following operation.
  • Monoclonal antibodies are antibody-producing cells such as hybridomas by the cell fusion method of Keller et al. (G. Koehler et al., Nature, 256, 495-497, published in 1975), or tumorigenic cells by viruses such as Epstan-Barr virus. Can be obtained.
  • Preparation of a monoclonal antibody by the cell fusion method can be performed by the following operation.
  • a mammal mammal, nude mouse, rat, etc., for example, BALB / c of an inbred mouse
  • a bird chicken, etc.
  • the immunization amount of the immunogen or the conjugate of the immunogen and the carrier can be appropriately determined depending on the type of immunized animal, the site of immunization, and the like.
  • 0.1 ⁇ g to 5 mg of the immunogen or a combination of the immunogen and a carrier is immunized at a time.
  • the immunogen or the conjugate of the immunogen and the carrier is preferably immunized by adding an adjuvant and mixing.
  • Known adjuvants such as Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide adjuvant, or pertussis adjuvant can be used as the adjuvant.
  • Immunization may be performed at a site such as subcutaneous, intravenous, intraperitoneal or back. After the first immunization, booster injections of the immunogen or a conjugate of the immunogen and the carrier are given at sites of subcutaneous, intravenous, intraperitoneal, or back at 1-2 week intervals. The number of booster injections is generally 2 to 6 times.
  • the immunogen or the combined immunogen and carrier is preferably boosted by adding an adjuvant and mixing.
  • the antibody titer in the sera of the immunized animal is repeatedly measured by ELISA or the like.
  • the immunogen or the combined immunogen and carrier is added to physiological saline.
  • a solution dissolved in (0.9% sodium chloride aqueous solution) is injected intravenously or intraperitoneally to obtain final immunization.
  • cells having antibody-producing ability such as spleen cells, lymph node cells or peripheral lymphocytes of immunized animals are obtained.
  • the cell having antibody-producing ability obtained from this immunized animal is fused with myeloma cells (myeloma cells) of mammals (mouse, nude mouse, rat, etc.).
  • myeloma cells myeloma cells
  • mammals mae, nude mouse, rat, etc.
  • a cell line deficient in an enzyme such as guanine phosphoribosyl transferase (HGPRT) or thymidine kinase (TK) is preferred.
  • HGPRT guanine phosphoribosyl transferase
  • TK thymidine kinase
  • ATCC P3-X63-Ag8 strain which is a HGPRT-deficient cell line derived from BALB / c mice.
  • JCRB 0028 P3-X63-Ag8-U1 strain
  • JCRB 0009 P3-NS1-1-Ag4-1 strain
  • JCRB 0028 P3-X63-Ag8.653 strain
  • SP2 / O-Ag-14 strain JCRB 0029 or the like is used.
  • Cell fusion can be performed using a fusion promoter such as polyethylene glycol (PEG) of various molecular weights, liposomes or Sendai virus (HVJ), or by electrofusion.
  • PEG polyethylene glycol
  • HVJ Sendai virus
  • myeloma cells are of HGPRT-deficient strain or TK-deficient strain
  • fusion of cells capable of producing antibodies and myeloma cells by using a selection medium (HAT medium) containing hypoxanthine / aminopterin / thymidine Only cells (hybridomas) can be selectively cultured and propagated.
  • the hybridoma culture supernatant thus obtained is subjected to immunological measurement such as ELISA or Western blot using the immunogen, the conjugate of the immunogen and the carrier, or the HMGB1 degradation product.
  • a hybridoma producing an antibody having a high affinity for the HMGB1 degradation product can be selected.
  • the affinity of the antibody is measured as described in (ii) above, and an antibody having a high affinity for the HMGB1 degradation product is selected.
  • a hybridoma producing the antibody of (b) can be selected.
  • the monoclonal antibody-producing cell line can be cultured in an appropriate medium, and the antibody (b) (monoclonal antibody) can be obtained from the culture supernatant.
  • the medium include serum-free medium and low-concentration serum medium.
  • the antibody can be easily purified, and a medium such as DMEM medium, RPMI 1640 medium, or ASF medium 103 can be used.
  • a monoclonal antibody-producing cell line is injected into the abdominal cavity of a mammal that is compatible with this and previously stimulated with pristane or the like, and after a certain period of time, the antibody (monoclonal) (b) from the ascites collected in the abdominal cavity. Antibody).
  • the monoclonal antibody thus obtained was purified by a salting-out method using ammonium sulfate, sodium sulfate or the like, a method such as ion exchange chromatography, gel filtration or affinity chromatography, or a combination of these methods.
  • the above-mentioned antibody (b) (monoclonal antibody) can be obtained.
  • the immunological measurement method of the present invention is an immunological measurement method of the HMGB1 degradation product contained in a sample, the antibody (a) [anti-HMGB1 degradation product antibody] and the (b) above. If an antibody is used, the measurement principle is not particularly limited, and the desired effect is achieved.
  • immunological measurement method examples include enzyme immunoassay (ELISA, EIA), fluorescence immunoassay (FIA), radioimmunoassay (RIA), luminescence immunoassay (LIA), enzyme antibody method, fluorescence Antibody method, immunochromatography method, immunoturbidimetric method, latex turbidimetric method, latex agglutination measurement method, erythrocyte agglutination method, particle agglutination method, JP-A-9-229936 and JP-A-10-132919 A specific binding substance for the described measurement target substance (test substance) is immobilized, and a carrier having a surface coated with this, and particles on which the specific binding substance for the measurement target substance (test substance) is fixed.
  • ELISA enzyme immunoassay
  • FIA fluorescence immunoassay
  • RIA radioimmunoassay
  • LIA luminescence immunoassay
  • enzyme antibody method fluorescence Antibody method
  • immunochromatography method immunoturbidimetric method
  • the immunological measurement method of the present invention can be applied to any method such as a sandwich method, a competitive method, or a homogeneous method (homogeneous method).
  • the measurement in the immunological measurement method of the present invention may be performed by a method or using an apparatus such as an analyzer.
  • Sample Samples in the immunological measurement method of the present invention include human blood, serum, plasma, urine, semen, spinal fluid, saliva, sweat, tears, ascites or amniotic fluid; stool; organs such as blood vessels or liver; Samples such as tissues; cells; or biological samples such as extracts of stool, organs, tissues, cells, or the like that may contain the HMGB1 degradation products are targeted.
  • the immunological measurement method of the present invention comprises a labeled antibody (or labeled antigen) in which a labeling substance is bound to an antibody (or antigen) and an immobilized antibody (or in which the antibody (or antigen) is immobilized on a solid phase carrier (or
  • immunoassay methods such as enzyme immunoassay, fluorescence immunoassay, radioimmunoassay, or luminescent immunoassay using a solid phase antigen
  • use the sandwich method or competition method use the sandwich method or competition method.
  • the immunological measurement method of the present invention is carried out by the sandwich method, either one of the antibody (a) and the antibody (b) is used as the immobilized antibody.
  • the other antibody may be used as a labeled antibody.
  • the solid phase carrier used for the solid phase antibody (or solid phase antigen) used in the immunological measurement method include polystyrene, polycarbonate, polyvinyl toluene, polypropylene, polyethylene, polyvinyl chloride, nylon, polymethacrylate, poly Microcapsules, beads, microplates (microtiter plates), test tubes, sticks, test pieces, etc. made of materials such as acrylamide, latex, liposomes, gelatin, agarose, cellulose, sepharose, glass, ceramics, metals or magnetic materials A solid support in the shape can be used.
  • the solid-phased antibody is an antibody such as the antibody (a) or the antibody (b) or an antigen and a solid phase carrier, a physical adsorption method, a chemical binding method or the like. It can be prepared by adsorbing and binding by a known method such as combination use.
  • the physical adsorption method the antibody (or antigen) and the solid phase carrier are mixed and brought into contact with a solution such as a buffer solution according to a known method, or the antibody (or antigen) dissolved in the buffer solution or the like is fixed. It can be performed by bringing a phase carrier into contact.
  • the chemical binding method is used, the Japanese Society of Clinical Pathology, “Special Issue on Extraordinary Clinical Pathology No.
  • the antibody (or antigen) and the solid support are divalent, such as glutaraldehyde, carbodiimide, imide ester or maleimide. It is possible to carry out the reaction by mixing and bringing into contact with a functional crosslinking reagent and reacting with the amino group, carboxyl group, thiol group, aldehyde group or hydroxyl group of the antibody (or antigen) and the solid phase carrier.
  • the surface or inner wall surface of the solid phase carrier on which the antibody (or antigen) is immobilized for example, Treated with a known method such as bovine serum albumin (BSA), human serum albumin (HSA), ovalbumin, casein, gelatin or a salt thereof, a surfactant, or skim milk powder, etc.
  • BSA bovine serum albumin
  • HSA human serum albumin
  • ovalbumin ovalbumin
  • casein gelatin or a salt thereof
  • surfactant or skim milk powder
  • peroxidase POD
  • alkaline phosphatase ALP
  • ⁇ -galactosidase urease
  • catalase glucose oxidase
  • lactate dehydrogenase or amylase can be used as the labeling substance. it can.
  • fluorescence immunoassay for example, fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, substituted rhodamine isothiocyanate or dichlorotriazine isothiocyanate can be used.
  • radioimmunoassay for example, tritium, iodine 125, iodine 131, or the like can be used.
  • luminescence immunoassay for example, NADH-FMNH 2
  • a substance related to a reaction system such as a luciferase reaction system, a luminol-hydrogen peroxide-POD reaction system, an acridinium ester reaction system, or a dioxetane compound reaction system can be used.
  • the method for binding an antibody (or antigen) such as the antibody (a) or the antibody (b) and a labeling substance such as an enzyme is described in the “Special Issue on Clinical Pathology Special Issue No.
  • the antibody (or antigen) and the labeling substance are mixed with and contacted with a bivalent cross-linking reagent such as glutaraldehyde, carbodiimide, imide ester or maleimide, and the amino group, carboxyl group or thiol of each of the antibody (or antigen) and the labeling substance is contacted.
  • Bonding can be performed by reacting with a group, an aldehyde group or a hydroxyl group.
  • the measurement operation method in the immunoassay method such as the enzyme immunoassay method, the fluorescence immunoassay method, the radioimmunoassay method or the luminescence immunoassay method described above is a known method (“Special Issue on Clinical Pathology” 53, Immunoassay for Clinical Testing -Technology and Application- ", Clinicopathology Publishing Society, published in 1983; edited by Yuji Ishikawa et al.,” Enzyme immunoassay “, 3rd edition, Medical School, published in 1987; Ed. “Protein Nucleic Acid Enzyme Separate Volume No. 31 Enzyme Immunoassay”, Kyoritsu Shuppan, published in 1987).
  • solid-phase carrier-antibody a solid-phased antibody
  • antibody-labeled substance a labeled antibody
  • the amount (concentration) of the HMGB1 degradation product contained in the sample can be measured by measuring the amount of unbound labeled antibody.
  • an enzyme immunoassay a substrate is reacted with an enzyme labeled with an antibody under the optimum conditions, and the amount of the enzyme reaction product is measured by an optical method or the like.
  • the fluorescence immunoassay the fluorescence intensity by the fluorescent substance label is measured.
  • the radiation dose due to the radioactive substance label is measured. Furthermore, in the case of a luminescence immunoassay, the amount of luminescence by the luminescence reaction system is measured. (7) Immunological measurement method by agglutination method
  • the formation of immune complex aggregates is measured by optically measuring the transmitted light or scattered light, or by visually measuring the HMGB1 degradation contained in the sample. It can also be carried out by immunoassay methods such as immunoturbidimetry, latex turbidimetry, latex agglutination, erythrocyte agglutination, or particle agglutination, which measure the amount (concentration) of the product.
  • the solid phase carrier examples include polystyrene, styrene-styrenesulfonate copolymer Polymer, acrylonitrile-butadiene-styrene copolymer, vinyl chloride-acrylic acid ester copolymer, vinyl acetate-acrylic acid copolymer, polyacrolein, styrene-methacrylic acid copolymer, styrene-glycidyl (meth) acrylic acid copolymer Materials such as polymers, styrene-butadiene copolymers, methacrylic acid polymers, acrylic acid polymers, latex, gelatin, liposomes, microcapsules, erythrocytes, silica, alumina, carbon black, metal compounds, ceramics, metals or magnetic materials The particles can be used.
  • the method of immobilizing the antibody (a) and / or the antibody (b) on a solid support is performed by a known method such as physical adsorption, chemical binding, or a combination thereof. be able to.
  • a known method such as physical adsorption, chemical binding, or a combination thereof.
  • the antibody and the solid phase carrier are mixed and brought into contact in a solution such as a buffer solution, or the antibody dissolved in the buffer solution or the like is brought into contact with the solid phase carrier, etc. It can be carried out.
  • the chemical binding method is used, the Japanese Society of Clinical Pathology, “Special Issue on Extraordinary Clinical Pathology No.
  • the antibody and solid phase carrier are divalent cross-linking reagents such as glutaraldehyde, carbodiimide, imide ester or maleimide. It is possible to carry out the reaction by reacting with the amino group, carboxyl group, thiol group, aldehyde group or hydroxyl group of the antibody and the solid phase carrier.
  • the antibody (a) and / or the antibody (b) may be immobilized.
  • a bovine serum albumin (BSA), human serum albumin (HSA), ovalbumin, casein, gelatin or a salt thereof, a surfactant, skim milk powder or the like is coated on the surface or inner wall surface of the solid support.
  • the solid phase carrier may be subjected to a blocking process (masking process) by a known method.
  • the particle size of the latex particles used as the solid phase carrier is not particularly limited, but the latex particles are the antibody (a) or the antibody (b).
  • the particle size of the latex particles is determined based on the average particle size for reasons such as the degree of formation of an aggregate by binding through the substance to be measured (the HMGB1 degradation product) and the ease of measurement of the generated aggregate. Is preferably 0.04 to 1 ⁇ m.
  • the concentration of the latex particles obtained by solidifying the antibody (a) and / or the antibody (b) is included in the sample.
  • the concentration of the latex particles obtained by immobilizing the antibody (a) and / or the antibody (b) is 0.
  • the concentration of the antibody and / or the above-mentioned (a) is such that the concentration in the reaction mixture is such that the concentration is 005 to 1% (w / v).
  • the latex reagent obtained by immobilizing the antibody (b) is included in the measurement reagent.
  • the particle size of the particles used as the solid phase carrier is not particularly limited, but the average particle The diameter is preferably in the range of 0.01 to 100 ⁇ m, more preferably in the range of 0.5 to 10 ⁇ m.
  • the specific gravity of these particles is preferably in the range of 1 to 10, and more preferably in the range of 1 to 2.
  • a container used for the measurement when an indirect agglutination reaction method such as a latex agglutination reaction method an erythrocyte agglutination reaction method or a particle agglutination reaction method is used as a measurement principle, for example, glass, polystyrene, polyvinyl chloride, polymethacrylate, etc.
  • a test tube, a microplate (microtiter plate), a tray, and the like for example, glass, polystyrene, polyvinyl chloride, polymethacrylate, etc.
  • a test tube, a microplate (microtiter plate), a tray, and the like a test tube, a microplate (microtiter plate), a tray, and the like.
  • the bottom surface of the solution storage portion (such as a well of a microplate) of these containers preferably has a shape having an inclination from the center to the periphery of the bottom, such as U-type, V-type, or
  • an immunoassay method such as an immunoturbidimetric method, latex turbidimetric method, latex agglutination method, hemagglutination method or particle agglutination method
  • an immunoassay method such as an immunoturbidimetric method, latex turbidimetric method, latex agglutination method, hemagglutination method or particle agglutination method
  • a phosphate buffer, a glycine buffer, a Tris buffer, a Good buffer, or the like may be used, and a reaction accelerator such as polyethylene glycol or a nonspecific reaction inhibitor may be further included.
  • the measuring operation method in the immunological measurement method such as the immunoturbidimetric method, latex turbidimetric method, latex agglutination method, erythrocyte agglutination method or particle agglutination method can be performed by a known method or the like.
  • the sample and the above-mentioned “the antibody of (a) and / or the antibody of (b)” or “the sample and the solid phase carrier immobilized on the solid phase” are used.
  • the antibody (a) and / or the antibody (b) is reacted, and transmitted light or scattered light is measured by an endpoint method or a rate method.
  • the above-mentioned antibody (a) and / or (b) above which is solid-phased on a sample and a solid support in a container such as a plate or a microplate.
  • the “antibody” is reacted and the state of aggregation is visually determined.
  • Reagent for immunoassay of HMGB1 degradation product contained in sample (1)
  • the reagent for immunological measurement of the degradation product of HMGB1 contained in the sample of the present invention (hereinafter sometimes referred to as “immunological measurement reagent of the present invention” or “measurement reagent of the present invention”) is the following (a): An assay reagent comprising an antibody and the antibody (b).
  • the antibody (b) includes HMGB1 thrombin or thrombin at a concentration of the antibody in the range of 0.625 to 2.5 ng / mL (preferably all concentrations in the range).
  • A an antibody having a value of 0.5 or more when the absorbance value obtained when measuring the amount of the antibody bound to the degradation product is divided by the concentration value of the antibody, or
  • B The absorbance value obtained when measuring the amount of the antibody bound to the degradation product is the absorbance of the antibody produced from the hybridoma MD78 (FERM P-18405), which is the reference antibody-producing cell.
  • the affinity of HMGB1 for the degradation product by thrombin or thrombin / thrombomodulin complex is the same as the affinity for HMGB2 and the affinity for HMGB2 by thrombin or thrombin / thrombomodulin complex. Those that are each at least 10 times greater than the affinity for the degradation products are preferred.
  • Antibody concentration at least one concentration in the range of 0.625 to 5 ng / mL (preferably all concentrations in the range)
  • the antibody (a) (anti-HMGB1 degradation product antibody) and / or the antibody (b) are preferably monoclonal antibodies.
  • the antibody (a) (anti-HMGB1 degradation product antibody) and / or the antibody (b) are polyclonal antibodies, antisera containing polyclonal antibodies, monoclonal antibodies, or fragments of these antibodies ( Fab, F (ab ′) 2 Or Fab ′ or the like.
  • the measurement reagent of the present invention uses a solid-phased antibody and a labeled antibody, and any one of the antibody (a) and the antibody (b) is solid-phased. It may be used as an antibody and the other antibody may be used as a labeled antibody.
  • the measurement reagent of the present invention uses the antibody (a) and the antibody (b), and has high specificity for the HMGB1 degradation product and accurately measures only the HMGB1 degradation product. Can do.
  • the antibody (a) can be used without particular limitation as long as it is an antibody as described above.
  • the antibody (b) can be used without any particular limitation as long as it is an antibody as described above.
  • the antibody (a) and the antibody (b) are not limited to one type, and a plurality of types may be used simultaneously.
  • the immunological measurement method (sandwich method or the like) using a labeling substance such as enzyme immunoassay, fluorescent immunoassay, radioimmunoassay or luminescence immunoassay is used as the measurement principle. Competing methods, etc.), or immunological measurement methods that measure the formation of immune complex aggregates such as immunoturbidimetry, latex turbidimetry, latex agglutination, erythrocyte agglutination, or particle agglutination It can be applied without particular limitation.
  • a labeling substance such as enzyme immunoassay, fluorescent immunoassay, radioimmunoassay or luminescence immunoassay. Competing methods, etc.
  • immunological measurement methods that measure the formation of immune complex aggregates such as immunoturbidimetry, latex turbidimetry, latex agglutination, erythrocyte agglutination, or particle agglutination It can be applied without particular limitation.
  • the antibody (a) and the (b) Of these antibodies any one of the antibodies may be used as a solid-phase antibody, and the other antibody may be used as a labeled antibody.
  • an antibody to be immobilized on a solid phase carrier such as latex particles May be the antibody (a) and / or the antibody (b), and in the measurement reagent based on the immunoturbidimetric method, the antibody (a) and the antibody The antibody of (b) may be used.
  • the immunoassay reagent of the present invention is characterized by containing the antibody (a) and the antibody (b), and therefore contained in the immunoassay reagent of the present invention.
  • the details of “the antibody of (a)” described above are as described in the above section “[I] Anti-HMGB1 degradation product antibody” and the like, and are contained in the immunoassay reagent of the present invention.
  • the details of “the antibody of (b)” are as described in the section “(3) Antibody of (b)” above in “[II] Immunological measurement method of HMGB1 degradation product contained in the sample” above.
  • the details of the measurement principle and the like of the immunological measurement reagent of the present invention are as described in the above-mentioned section “[II] Immunological measurement method of HMGB1 degradation product contained in sample”.
  • Other reagent components In the immunological measurement reagent of the present invention, various aqueous solvents can be used as the solvent.
  • aqueous solvent examples include purified water, physiological saline, and various buffer solutions such as Tris buffer, phosphate buffer, and phosphate buffered saline.
  • the pH of the buffer solution may be appropriately selected and used as appropriate. Although there is no particular limitation, it is general to select and use a pH within the range of pH 3 to 12.
  • the immunoassay reagent of the present invention includes a “solid phase antibody” obtained by immobilizing an antibody such as the antibody (a) or the antibody (b) on a solid phase carrier, and / or
  • a labeling substance such as an enzyme, bovine serum albumin (BSA), human serum albumin (HSA), proteins such as ovalbumin, casein, gelatin or salts thereof; various salts; various sugars; skim milk powder; various animal sera such as normal rabbit serum; various preservatives such as sodium azide or antibiotics;
  • a reaction promoting substance, a sensitivity increasing substance such as polyethylene glycol, a nonspecific reaction inhibiting substance, or various nonionic surfactants, amphoteric surfactants or anionic surfactants.
  • concentration of these in the measurement reagent is not particularly limited, but is preferably 0.001 to 10% (w / v), and particularly preferably 0.01 to 5% (w / v). .
  • surfactant examples include sorbitan fatty acid ester, glycerin fatty acid ester, decaglycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene glycerin fatty acid ester, polyethylene glycol fatty acid ester, polyoxyethylene alkyl ether, Nonionic surfactants such as polyoxyethylene phytosterol, phytostanol, polyoxyethylene polyoxypropylene alkyl ether, polyoxyethylene alkylphenyl ether, polyoxyethylene castor oil, hydrogenated castor oil or polyoxyethylene lanolin; betaine acetate, etc.
  • the immunoassay reagent of the present invention can be used alone for measuring the HMGB1 degradation product contained in the sample. And it can be sold by itself.
  • the immunoassay reagent of the present invention can also be used for measurement of the HMGB1 degradation product contained in a sample in combination with other reagents. And it can also be sold in combination with other reagents.
  • the other reagents include buffers, sample diluents, reagent diluents, reagents containing labeling substances, reagents containing substances that generate signals such as color development, and signals related to color development.
  • a reagent containing a substance, a reagent containing a substance for performing calibration (calibration), a reagent containing a substance for performing accuracy control, and the like can be given.
  • the other reagent is the first reagent and the immunological measurement reagent of the present invention is the second reagent, or the immunological measurement reagent of the present invention is the first reagent, and the other reagent is the first reagent.
  • Two reagents can be used and sold in various combinations as appropriate.
  • the immunological measurement reagent of the present invention may be a measurement reagent kit comprising a plurality of constituent reagents such as the first reagent and the second reagent, or the other reagents described above.
  • Tris hydroxymethyl aminomethane
  • SDS sodium dodecyl sulfate
  • glycine 7.2 g were added to and mixed with pure water, and the mixture was adjusted to 500 mL, and the electrophoresis buffer solution [0.1 % SDS-192 mM glycine-25 mM Tris buffer].
  • Coomassie brilliant blue stain Quick-CBB (Wako Pure Chemical Industries [Japan]) was used.
  • molecular weight markers and thrombin were also used as samples.
  • A Molecular weight marker [Precision Plus Protein All Blue Standards marker; Marker molecular weight 10 KDa, 15 KDa, 20 KDa, 25 KDa, 37 KDa, 50 KDa, 75 KDa, 100 KDa, 150 KDa and 250 KDa;
  • B HMGB1
  • C HMGB1 degradation product
  • D HMGB2
  • E HMGB2 degradation product
  • F Thrombin
  • each of the 2 samples was subjected to electrophoresis by SDS-polyacrylamide gel electrophoresis by the following operation.
  • Each of the samples (b) to (f) in 2 was mixed with the sample processing solution in 1 (4) at a ratio of 1: 1 and treated at 100 ° C. for 5 minutes.
  • the buffer solution for electrophoresis tank of (1) above was placed in the lower electrophoresis tank.
  • the gel of (1) above was set in an electrophoresis tank.
  • the buffer solution for electrophoresis tank (1) described above was put in the upper electrophoresis tank.
  • FIG. 1 shows the gel stained in (3) above.
  • the band of “the HMGB1 degradation product” (lane denoted by “3”) is present on the lower molecular weight side than the band of “HMGB1” (lane denoted by “2”), and It can be seen that the band of “the degradation product of HMGB2” (lane denoted by “5”) is present on the lower molecular weight side than the band of “HMGB2” (lane denoted by “4”).
  • HMGB1 degradation product and “the HMGB2 degradation product” could be prepared, respectively.
  • Example 1 (Preparation of antibody binding to HMGB1 degradation product-1) An antibody that binds to the HMGB1 degradation product was prepared as follows. (1) HMGB1 (total length) prepared in [1] of Reference Example 1 was used as an immunogen. 1 volume of HMGB1 (total length) solution prepared in [1] of Reference Example 1 as the immunogen to 1 volume of FREUND complete adjuvant (DIFCO LABORATORIES) as a chemically synthesized adjuvant To prepare a mixture of HMGB1 solution and FREUND complete adjuvant.
  • HMGB1 total length
  • FREUND incomplete adjuvant DIFCO LABORATORIES
  • a mixture of HMGB1 solution and FREUND incomplete adjuvant was prepared.
  • a mixture of the above-mentioned HMGB1 solution and FREUND complete adjuvant was injected into the abdominal cavity of a mouse (BALB / c) as an immunogen at 300 to 500 ⁇ g / animal / dose, 2 weeks and 4 weeks later. Mice were injected intraperitoneally with a mixture of the HMGB1 solution and FREUND incomplete adjuvant.
  • HMGB1 total length
  • Reference Example 1 the stock solution of HMGB1 (total length) prepared in [1] of Reference Example 1 was boosted with 300 ⁇ g / mouse, and the next day, spleen cells of the immunized mouse were used.
  • Myeloma cells (P3U1) were mixed at a ratio of 1: 1 to 10: 1, and polyethylene glycol [PEG1500; Roche (Switzerland)] was added by a general method to cause cell fusion. Sorted. Specifically, cell fusion was performed as follows.
  • the mixed spleen cells and myeloma cells (P3U1) are centrifuged to remove the supernatant, suspended in 1 mL of polyethylene glycol [PEG 1500; Roche (Switzerland)] at room temperature for 1 minute, and stirred at 37 ° C. for 1 minute. did.
  • the operation of adding 1 mL of serum-free medium over 1 minute was performed twice, and then 7 mL of serum-free medium was added over 2 minutes.
  • the cells were washed several times, suspended in a medium containing hypoxanthine, aminopterin and thymidine, dispensed into a 96-well microtiter plate, and 5% CO at 37 ° C. 2 Cultured in the presence.
  • HMGB1 prepared in Reference Example 1 (whole length) was immobilized and fused cell culture.
  • the supernatant was used as a primary antibody in an ELISA method system. Specifically, this ELISA method was performed as follows.
  • a coloring solution consisting of 0.045% 3,3 ′, 5,5′-tetramethylbenzidine hydrochloride aqueous solution (pH 2.0) containing 0.2 mM EDTA ⁇ disodium, and 5 mM excess 100 ⁇ L of a chromogenic substrate solution prepared by mixing 1: 1 with a substrate solution consisting of 60 mM aqueous disodium phosphate solution (pH 4.3) containing hydrogen oxide, 41 mM citric acid, 0.2 mM EDTA ⁇ disodium The solution was injected into each well of the microtiter plate washed in (vi) and allowed to stand at room temperature for 5 to 30 minutes to cause the reaction to develop color.
  • This monoclonal antibody-producing cell line was transformed into CO using PFHM-II (GIBCO). 2 The cells were cultured at 37 ° C. in an incubator. After the culture, IgG in the supernatant was bound to a protein A column [GE Healthcare Bio-Sciences (Sweden)]. The bound IgG was eluted with a 100 mM aqueous citric acid solution (pH 3.0). 1 volume of 0.5 M phosphate buffer (pH 7.5) buffer was added to 1 volume of eluate, and the antibody that binds to the HMGB1 was obtained from the monoclonal antibody-producing cell line as purified IgG. . This antibody was an antibody that binds to the HMGB1 degradation product.
  • an antibody (monoclonal antibody) that binds to the HMGB1 degradation product (hereinafter referred to as “the present anti-HMGB1 degradation product antibody (2H6)”) could be obtained from the 2H6 monoclonal antibody-producing cell line.
  • the anti-HMGB1 degradation product antibody (2H6) had an affinity for the HMGB1 degradation product of 1.5 times or more compared with the affinity for HMGB1, as described later.
  • this anti-HMGB1 degradation product antibody (2H6) has an affinity for the HMGB1 degradation product of 10% each compared with the affinity for HMGB2 and the affinity for the HMGB2 degradation product. It was more than twice.
  • this anti-HMGB1 degradation product antibody (2H6) is the antibody (a) in the measurement method and measurement reagent of the HMGB1 degradation product of the present invention.
  • the 2H6 strain which is a monoclonal antibody-producing cell line of this “anti-HMGB1 degradation product antibody (2H6)”, is the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (Kazusa Kamashiji, Kisarazu City, Chiba Prefecture, Japan). No. 5-8) is received as “Receipt Number: NITE AP-1570” dated March 15, 2013.
  • Example 2 Preparation of antibody binding to HMGB1 degradation product-2
  • HMGB1 total length prepared in [1] of Reference Example 1 was used as an immunogen. Thereafter, the procedure described in Example 1 (1) to (4) was followed to prepare an antibody that binds to the HMGB1 degradation product. As a result, one clone was established from the grown fused cell lines and designated as 5D1 strain.
  • An antibody (monoclonal antibody) that binds to the HMGB1 degradation product hereinafter referred to as “anti-HMGB1 degradation product antibody (5D1)”) could be obtained from the 5D1 monoclonal antibody-producing cell line.
  • This anti-HMGB1 degradation product antibody (5D1) was an antibody having a high affinity for the HMGB1 degradation product, as will be described later. That is, this anti-HMGB1 degradation product antibody (5D1) is the antibody of (b) in the measurement method and measurement reagent of the HMGB1 degradation product of the present invention.
  • the 5D1 strain which is a monoclonal antibody-producing cell line of this “anti-HMGB1 degradation product antibody (5D1)”, is the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (Kazusa Kamashiji, Kisarazu City, Chiba Prefecture, Japan). No. 5-8) has been received as “Reception Number: NITE AP-1571” on March 15, 2013.
  • Example 3 Preparation of antibody binding to HMGB1 degradation product-3)
  • HMGB1 total length prepared in [1] of Reference Example 1 was used as an immunogen. Thereafter, the procedure described in Example 1 (1) to (4) was followed to prepare an antibody that binds to the HMGB1 degradation product.
  • 2A10 strain An antibody (monoclonal antibody) that binds to the HMGB1 degradation product (hereinafter referred to as “anti-HMGB1 degradation product antibody (2A10)”) could be obtained from the 2A10 monoclonal antibody-producing cell line.
  • This anti-HMGB1 degradation product antibody (2A10) was an antibody having a high affinity for the HMGB1 degradation product, as will be described later. That is, this anti-HMGB1 degradation product antibody (2A10) is the antibody (b) in the measurement method and measurement reagent of the HMGB1 degradation product of the present invention.
  • the 2A10 strain which is a monoclonal antibody-producing cell line of this “anti-HMGB1 degradation product antibody (2A10)”, is a patent microorganism deposit center of the National Institute of Technology and Evaluation (Kazusa Kamashiji, Kisarazu City, Chiba Prefecture, Japan). No. 5-8) is received as “Receipt Number: NITE AP-1572” on March 15, 2013.
  • Example 4 Preparation of antibody binding to HMGB1 degradation product-4.
  • HMGB1 total length prepared in [1] of Reference Example 1 was used as an immunogen. Thereafter, the procedure described in Example 1 (1) to (4) was followed to prepare an antibody that binds to the HMGB1 degradation product.
  • 6H3 line An antibody (monoclonal antibody) that binds to the HMGB1 degradation product (hereinafter referred to as “anti-HMGB1 degradation product antibody (6H3)”) could be obtained from the 6H3 monoclonal antibody-producing cell line.
  • This anti-HMGB1 degradation product antibody (6H3) was an antibody having a high affinity for the HMGB1 degradation product, as will be described later. That is, this anti-HMGB1 degradation product antibody (6H3) is the antibody of (b) in the measurement method and measurement reagent of the HMGB1 degradation product of the present invention.
  • the 6H3 strain which is a monoclonal antibody-producing cell line of the “anti-HMGB1 degradation product antibody (6H3)”, is a patent microorganism deposit center of the National Institute of Technology and Evaluation (Kazusa Kamashiji, Kisarazu City, Chiba Prefecture, Japan). No. 5-8) has been received as “Receipt Number: NITE AP-1573” on March 15, 2013.
  • Example 5 (Confirmation of reactivity of antibody binding to HMGB1 degradation product) About each of the antibody couple
  • Anti-HMGB1 degradation product antibody (5D1) Regarding the “anti-HMGB1 degradation product antibody (5D1)” obtained in Example 2, the reactivity with each of HMGB1, HMGB1 degradation product, HMGB2, and HMGB2 degradation product was confirmed as follows. 1. SDS-polyacrylamide gel electrophoresis (1) Reagent The following reagents (a) to (c) were prepared respectively.
  • molecular weight markers [Precision Plus Protein All Blue Standards markers; marker molecular weights 10 KDa, 15 KDa, 20 KDa, 25 KDa, 37 KDa, 50 KDa, 75 KDa, 100 KDa, 150 KDa and 250 KDa; (3) Electrophoresis Using the reagent prepared in (1), each sample of (2) was subjected to electrophoresis by SDS-polyacrylamide gel electrophoresis by the following operation. (A) About each sample of said (2), it prepared so that a sample density
  • the sample and the molecular weight marker injected into this gel are the following “1” “molecular weight marker”, “2” “HMGB1”, “3” “HMGB1” from the left lane. “Degradation product”, “4” “HMGB2”, and “5” “the HMGB2 degradation product” were injected in this order. “1”: “Molecular weight marker” “2”: “HMGB1” “3”: “degradation product of HMGB1” “4”: “HMGB2” “5”: “The HMGB2 degradation product” (D) Next, electrophoresis was performed at a current of 20 mA for 90 minutes. (E) After finishing the electrophoresis in (d), the gel was taken out of the glass plate.
  • a 9 cm ⁇ 9 cm polyvinyl difluoride membrane (BIO-RAD Laboratories [USA]) was layered on the gel, and 48 mM Tris (hydroxymethyl) aminomethane [Tris], 39 mM glycine, and 20% ( V / V) Using a transfer buffer composed of methanol, transfer was performed at an electric current of 100 mA for 1 hour, and the HMGB1, HMGB1 degradation product, HMGB2 and HMGB2 degradation product located in the gel according to the molecular weight. And the like were transferred from the gel to the polyvinyl difluoride film.
  • Tris hydroxymethyl aminomethane
  • each said HMGB1, said HMGB1 degradation product, HMGB2, and said HMGB2 degradation product transcribe
  • a POD-labeled anti-mouse IgG antibody [DakoCytomation (Denmark)] was added to a labeled antibody diluent [50 mM tris (hydroxymethyl) amino containing 0.5% casein and 100 mM sodium chloride.
  • the polyvinyl difluoride film of (5) was immersed in a solution prepared by diluting 1000 times with a methane buffer [Tris buffer] (pH 8.0)] at room temperature for 90 minutes to react.
  • the polyvinyl difluoride film subjected to the operation of (6) was washed by shaking in 20 mL of the cleaning solution for 5 minutes. This operation was performed three times.
  • HMGB2 lane (denoted “4”), and “the HMGB2 degradation product” lane (denoted “5”).
  • 5D1 anti-HMGB1 degradation product antibody
  • color development is observed at the positions indicating HMGB1, the HMGB1 degradation product, HMGB2, and the HMGB2 degradation product.
  • the “anti-HMGB1 degradation product antibody (5D1)” recognizes and binds to all of HMGB1, the HMGB1 degradation product, HMGB2, and the HMGB2 degradation product.
  • Anti-HMGB1 degradation product antibody (2H6) With respect to the “anti-HMGB1 degradation product antibody (2H6)” obtained in Example 1, the reactivity with HMGB1, the HMGB1 degradation product, HMGB2 and the HMGB2 degradation product was confirmed. That is, in place of the “anti-HMGB1 degradation product antibody (5D1)” instead of using the “anti-HMGB1 degradation product antibody (2H6)” obtained in Example 1, as described in [1] above. The operation was carried out to confirm the reactivity of the “anti-HMGB1 degradation product antibody (2H6)” with HMGB1, the HMGB1 degradation product, HMGB2, and the HMGB2 degradation product.
  • FIG. 3 shows a developed polyvinyl difluoride film as a result of confirming this reactivity.
  • the “molecular weight marker” lane (denoted as “1”), the “HMGB1” lane (denoted as “2”), and the “the HMGB1 degradation product” lane (denoted as “3”). Notation), “HMGB2” lane (denoted “4”), and “the HMGB2 degradation product” lane (denoted “5”).
  • the “anti-HMGB1 degradation product antibody (2H6) a deep color development is observed at the position showing the HMGB1 degradation product, but only an extremely light color development appears at the position showing HMGB1. .
  • TBS Tris buffered saline
  • POD-labeled anti-mouse IgG antibody solution POD-labeled anti-mouse IgG antibody [DakoCytomation (Denmark)] was diluted 1000-fold with 50 mM Tris-HCl buffer (pH 8.0) containing 0.5% sodium caseinate and 100 mM sodium chloride. This was used as a POD-labeled anti-mouse IgG antibody solution.
  • Diluent A 50 mM Tris-HCl buffer solution (pH 8.0) containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide was used as a diluent.
  • Coloring solution A 0.045% 3,3 ′, 5,5′-tetramethylbenzidine hydrochloride aqueous solution (pH 2.0) containing 0.2 mM EDTA ⁇ disodium was used as a color developing solution.
  • Substrate solution A 60 mM disodium phosphate aqueous solution (pH 4.3) containing 5 mM hydrogen peroxide, 41 mM citric acid, and 0.2 mM EDTA ⁇ disodium was used as a substrate solution.
  • Chromogenic substrate The chromogenic solution and the substrate solution were returned to room temperature before use, and mixed in equal amounts at the time of use to obtain a chromogenic substrate.
  • Reaction stop solution 0.7N sulfuric acid was used as a reaction stop solution. 2. sample An antibody solution for confirming the affinity with HMGB1, the HMGB1 degradation product, HMGB2, and the HMGB2 degradation product was prepared as follows and used as a sample.
  • Anti-HMGB1 antibody (2D4) An antibody (monoclonal antibody) that binds to HMGB1 produced from the 2D4 monoclonal antibody-producing cell line (hereinafter referred to as “anti-HMGB1 antibody (2D4)”) has a concentration of 0.625 ng / mL, 1
  • the antibody solution of each concentration of anti-HMGB1 antibody (2D4) was prepared by diluting with the dilution solution of (1) of (1) so as to be .25 ng / mL, 2.5 ng / mL, and 5 ng / mL.
  • Anti-HMGB1 antibody (4F12) An antibody (monoclonal antibody) that binds to HMGB1 produced from the 4F12 monoclonal antibody-producing cell line (hereinafter referred to as “anti-HMGB1 antibody (4F12)”) has a concentration of 0.625 ng / mL, 1
  • the antibody solution of each concentration of anti-HMGB1 antibody (4F12) was prepared by diluting with the diluent (1) of 1 above so as to be .25 ng / mL, 2.5 ng / mL, and 5 ng / mL.
  • Anti-HMGB1 antibody (8H4) An antibody (monoclonal antibody) that binds to HMGB1 and the like produced from the 8H4 monoclonal antibody-producing cell line (hereinafter referred to as “anti-HMGB1 antibody (8H4)”) has a concentration of 0.625 ng / mL, 1
  • the antibody solution of anti-HMGB1 antibody (8H4) of each concentration was prepared by diluting with the diluent (1) of 1 above so as to be .25 ng / mL, 2.5 ng / mL, and 5 ng / mL.
  • Anti-HMGB1 degradation product antibody (2H6) The anti-HMGB1 degradation product antibody (2H6) was diluted in the above (4) so that its concentrations were 0.625 ng / mL, 1.25 ng / mL, 2.5 ng / mL, and 5 ng / mL, respectively.
  • the antibody solution of this anti-HMGB1 degradation product antibody (2H6) of each concentration was prepared by diluting with a liquid.
  • Antibodies such as anti-HMGB1 degradation products The anti-HMGB1 degradation product or the like antibody (5D1) is diluted (1) in (1) so that the concentrations thereof are 0.625 ng / mL, 1.25 ng / mL, 2.5 ng / mL, and 5 ng / mL, respectively.
  • the antibody solution of anti-HMGB1 degradation product antibody (5D1) at each concentration was prepared by diluting with a solution.
  • Anti-HMGB1 degradation product antibody (2A10) The anti-HMGB1 degradation product or the like antibody (2A10) is diluted in (4) of 1 above so that the concentrations thereof are 0.625 ng / mL, 1.25 ng / mL, 2.5 ng / mL, and 5 ng / mL, respectively.
  • the antibody solution of antibodies (2A10) such as anti-HMGB1 degradation products at each concentration was prepared by diluting with a solution.
  • Anti-HMGB1 degradation product antibody (6H3) The anti-HMGB1 degradation product antibody (6H3) is diluted (4) in the above 1 so that the concentrations thereof are 0.625 ng / mL, 1.25 ng / mL, 2.5 ng / mL, and 5 ng / mL, respectively.
  • the antibody solution of antibody (6H3) such as anti-HMGB1 degradation product at each concentration was prepared by diluting with a solution.
  • Anti-HMGB1 antibody (MD78) Monoclonal antibody-producing cell line MD78 strain (incorporated administrative agency National Institute of Advanced Industrial Science and Technology patent biological deposit center (1st, 1st, 1st, 1st, 1st, 1st, 1st, Tsukuba, Ibaraki, Japan) as FERM P-18405, July 4, 2001
  • the antibody (monoclonal antibody) that binds to HMGB1 and the like produced from the above hereinafter referred to as “anti-HMGB1 antibody (MD78)” has a concentration of 0.625 ng / mL
  • the antibody solution of each concentration of anti-HMGB1 antibody (MD78) was prepared by diluting with the diluent (1) of (1) so as to be 1.25 ng / mL, 2.5 ng / mL, and 5 ng / mL.
  • Anti-HMGB1 antibody (9)
  • the monoclonal antibody-producing cell line MD77 strain (incorporated administrative agency National Institute of Advanced Industrial Science and Technology patent biological deposit center (1-6, 1st east, Tsukuba city, Ibaraki, Japan) FERM P-18404 July 4, 2001
  • the antibody (monoclonal antibody) that binds to HMGB1 and the like produced from the above (hereinafter referred to as “anti-HMGB1 antibody (MD77)”) has a concentration of 0.625 ng / mL
  • the antibody solution of each concentration of anti-HMGB1 antibody (MD77) was prepared by diluting with the diluent of (1) of (1) so as to be 1.25 ng / mL, 2.5 ng / mL, and 5 ng / mL.
  • Anti-HMGB1 antibody (4C3) Antibody that binds to HMGB1 and the like produced from the 4C3 monoclonal antibody-producing cell line (monoclonal antibody; trade name: Anti-HMGB1 antibody [4C3] (Abcam)) (hereinafter referred to as “anti-HMGB1 antibody (4C3)”) are diluted with the diluent (1) above so that the concentrations thereof are 0.625 ng / mL, 1.25 ng / mL, 2.5 ng / mL, and 5 ng / mL, respectively. An antibody solution of anti-HMGB1 antibody (4C3) was prepared.
  • Anti-HMGB1 antibody An antibody that binds to HMGB1 and the like produced from the J2E1 strain of a monoclonal antibody-producing cell line (monoclonal antibody; trade name: HMG-1 Antibody (J2E1): sc-135809 (Santa cruz biotechnology, Inc.)) [hereinafter, “ The anti-HMGB1 antibody (J2E1) ”), so that the concentrations thereof are 0.625 ng / mL, 1.25 ng / mL, 2.5 ng / mL, and 5 ng / mL, respectively. It diluted with the dilution liquid and prepared the antibody solution of anti-HMGB1 antibodies (J2E1) of each concentration.
  • Anti-HMGB1 antibody An antibody that binds to HMGB1 and the like produced from the monoclonal antibody-producing cell line HAP46.5 (monoclonal antibody; trade name: Mouse monoclonal [HAP46.5] to HMGB1 (Abcam)) [hereinafter referred to as “anti-HMGB1 antibody ( HAP46.5) ”) with the dilution of (1) in (1) above so that the concentrations thereof are 0.625 ng / mL, 1.25 ng / mL, 2.5 ng / mL, and 5 ng / mL, respectively. After dilution, an antibody solution of anti-HMGB1 antibody (HAP46.5) at each concentration was prepared. 3.
  • each well was washed three times with 400 ⁇ L of the washing liquid of (1) above.
  • 100 ⁇ L of the POD-labeled anti-mouse IgG antibody solution of (1) above was dispensed into each well and allowed to stand at 25 ° C. for 1 hour to cause antigen-antibody reaction.
  • each well was washed 3 times with 400 ⁇ L of the washing liquid of (1) above.
  • 100 ⁇ L of the chromogenic substrate of (7) above was dispensed into each well, and allowed to stand at room temperature for 20 minutes to cause a chromogenic reaction with peroxidase (POD) as a labeling enzyme.
  • POD peroxidase
  • Measurement result (1) The measurement results in 3 above, that is, the results of confirming the affinity of each of the antibodies with HMGB1, HMGB1 degradation product, HMGB2, and HMGB2 degradation product, are shown in FIGS.
  • the letters representing the antibody-producing cell lines whose affinity was confirmed were shown above the figures.
  • the horizontal axis indicates the antibody concentration (ng / mL) in the antibody solution used as the sample
  • the vertical axis indicates the absorbance obtained by the measurement [from the absorbance at the main wavelength (450 nm) to the sub-wavelength (550 nm). The value obtained by subtracting the absorbance in FIG.
  • ⁇ indicates a measured value (the above-described absorbance) in a well in which HMGB1 is immobilized
  • ⁇ indicates a measured value (in the above-described absorbance in a well on which the HMGB1 degradation product is immobilized).
  • ⁇ indicates the measured value (the above-mentioned absorbance) in the well in which HMGB2 is immobilized
  • ⁇ indicates the measured value (in the above-described absorbance) in the well on which the HMGB2 degradation product is immobilized.
  • Table 1 shows the measured values of the measurement results in 3 above. In these tables, letters representing antibody producing cell lines whose affinity was confirmed were shown above the tables.
  • the left column indicates the antibody concentration (ng / mL) in the antibody solution used as a sample, and the absorbance [primary wavelength ( 450 nm) minus the absorbance at the sub-wavelength (550 nm)].
  • the value obtained by dividing the measured value in the well in which the HMGB1 degradation product is immobilized (the absorbance described above) by the antibody concentration (ng / mL) value in the antibody solution used as a sample is shown.
  • the indicated column (the third column from the left for each solid-phased well) was also provided. 5. Summary (1) From FIG.
  • Antibody (a) in the immunological measurement method and reagent of the HMGB1 degradation product contained in the sample of the present invention] (Ii) an antibody that binds to the HMGB1 degradation product and has high affinity for the HMGB1 degradation product: “anti-HMGB1 degradation product antibody (5D1)”, “anti-HMGB1 degradation product antibody (2A10)”, And “Anti-HMGB1 degradation product antibody (6H3)” [* Note that the value obtained by dividing the measured value (the absorbance described above) in the well in which the HMGB1 degradation product is immobilized by the antibody concentration (ng / mL) value in the antibody solution used as a sample is 0.5 or more.
  • the present anti-HMGB1 degradation product antibody (2H6) is an antibody that binds to the HMGB1 degradation product, and the affinity for the HMGB1 degradation product is higher than the affinity for HMGB1. The antibody was confirmed to be at least 1.5 times. Further, “anti-HMGB1 degradation product antibody (5D1)”, “anti-HMGB1 degradation product antibody (2A10)” and “anti-HMGB1 degradation product antibody (6H3)” are antibodies that bind to the HMGB1 degradation product, respectively.
  • HMGB1 degradation product had a high affinity for the HMGB1 degradation product.
  • Example 7 (Confirmation of measurement of HMGB1 degradation product) It was confirmed that the HMGB1 degradation product according to the present invention can be measured using the antibodies that bind to the HMGB1 degradation products obtained in Examples 1 to 4 and other HMGB1 binding antibodies.
  • Reagent (1) Antibody-immobilized microplate Each of the following five types of antibodies (i) to (v) is purified using protein A, and the concentration of these antibodies is 2.5 ⁇ g / mL with phosphate buffered saline (PBS). Each was diluted as follows.
  • Anti-HMGB1 degradation product antibody (2H6) (Ii) Anti-HMGB1 degradation product antibody (5D1) (Iii) Antibody such as anti-HMGB1 degradation product (2A10) (Iv) Antibodies such as anti-HMGB1 degradation products (6H3) (V) Anti-HMGB1 antibody (MD77)
  • 100 ⁇ L of each of these antibody solutions was dispensed into each well of a microtiter plate (microplate) [Nunc, trade name: Maxisorp] and allowed to stand overnight at 25 ° C. Each was individually immobilized on a well of a microplate.
  • Anti-HMGB1 degradation product antibody (2H6) B) Anti-HMGB1 degradation product antibody (5D1)
  • C Anti-HMGB1 degradation product antibody (2A10)
  • D Antibody (6H3) such as anti-HMGB1 degradation product
  • E Anti-HMGB1 antibody (MD77)
  • F Anti-HMGB1 antibody (04) [Mouse antibody (monoclonal antibody) that binds to HMGB1 etc. produced from the 04 monoclonal antibody-producing cell line [hereinafter referred to as “anti-HMGB1 antibody (04)]].
  • Diluent A 50 mM Tris-HCl buffer solution (pH 8.0) containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide was used as a diluent.
  • Coloring solution A 0.045% 3,3 ′, 5,5′-tetramethylbenzidine hydrochloride aqueous solution (pH 2.0) containing 0.2 mM EDTA ⁇ disodium was used as a color developing solution.
  • Substrate solution A 60 mM disodium phosphate aqueous solution (pH 4.3) containing 5 mM hydrogen peroxide, 41 mM citric acid, and 0.2 mM EDTA ⁇ disodium was used as a substrate solution.
  • Measurement result (1) Measurement results in the above 3, ie, an enzyme immunoassay method (ELISA method) using an antibody that binds to the HMGB1 degradation product obtained in Examples 1 to 4 and an antibody that binds to other HMGB1
  • FIG. 5 shows the results of measuring the HMGB1 degradation product and HMGB1.
  • the upper part of the horizontal axis shows the letters representing the antibody production cell line of the POD-labeled antibody solution used for the measurement
  • the lower part of the horizontal axis shows the production of the antibody on the antibody-immobilized microplate used for the measurement.
  • each bar represents a measured value (measurement value described above) measured for the sample (HMGB1) in order from the left side for each POD-labeled antibody solution and antibody-immobilized microplate used for the measurement.
  • the measured value when measured for the sample (the HMGB1 degradation product) (the absorbance described above), and the measured value when measured for the sample (reagent blind) are shown.
  • Table 2 shows the measurement values of the measurement results in 3 above.
  • this anti-HMGB1 degradation product antibody (2H6) and “anti-HMGB1 degradation product antibody (5D1)”, “anti-HMGB1 degradation product antibody (2A10)” or “anti-HMGB1 degradation product antibody (6H3)”
  • the measured value (absorbance) of HMGB1 is low, while the measured value (absorbance) of the HMGB1 degradation product is high. Also, in this case, it can be seen that all of the reagent blind tests (reagent blanks) are low.
  • the antibody of (i) of (5) in Example 6 (the antibody of (a) in the immunological measurement method and immunological measurement reagent of the HMGB1 degradation product of the present invention), and “ Used in combination with the antibody of (ii) (ii) of Example 6 (the method for immunological measurement of the HMGB1 degradation product of the present invention and the antibody (b) in the immunological measurement reagent) of Example 6
  • the antibody of (i) of (5) in Example 6 the antibody of (a) in the immunological measurement method and immunological measurement reagent of the HMGB1 degradation product of the present invention
  • Example 8 (Method and reagent-1 for immunological measurement of degradation products of HMGB1)
  • the HMGB1 degradation product and the HMGB1 contained in the sample by the immunological measurement method and immunological measurement reagent of the HMGB1 degradation product of the present invention and the conventional immunological measurement method and immunoassay reagent was measured.
  • HMGB1 ELISA Kit II which is a measurement reagent (research reagent) for HMGB1 based on ELISA / sandwich method using solid-phased anti-HMGB1 polyclonal antibody and peroxidase (POD) -labeled anti-HMGB1,2 monoclonal antibody.
  • Sinotest Japan
  • Sample (1) Sample (the HMGB1 degradation product) The HMGB1 degradation product prepared in [2] of Reference Example 1 was added to each 2.5 ng / kg of phosphate buffered saline containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide.
  • Samples (the aforementioned HMGB1 degradation products) were prepared so as to have concentrations of mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, and 80 ng / mL, respectively.
  • a phosphate buffered saline containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide was used as a sample (the HMGB1 degradation product) having a concentration of the HMGB1 degradation product of 0 ng / mL. .
  • HMGB1 prepared in [1] of Reference Example 1 was added to each of 2.5 ng / mL and 5 ng with phosphate buffered saline containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide.
  • Samples (HMGB1) were prepared to have concentrations of / ng, 10 ng / mL, 20 ng / mL, 40 ng / mL, and 80 ng / mL.
  • a phosphate buffered saline containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide was used as a sample (HMGB1) having a HMGB1 concentration of 0 ng / mL. 3.
  • Measurement (1) For each of the sample of (2) (1) (the degradation product of HMGB1) [total 7 concentrations] and each of the sample of (2) (HMGB1) (total 7 concentrations), Using “HMGB1 ELISA Kit II” (Sinotest Inc. [Japan]), which is an immunological measurement reagent, measurement was performed as described in the package insert. (2) As a result of measurement of each sample, absorbance (450 nm) was obtained by the measurement of (1). 4). Measurement result (1) The measurement results in 3 above, that is, the results of measurement of the HMGB1 degradation product contained in the sample and HMGB1 contained in the sample by the conventional immunological measurement method and immunological measurement reagent are shown in FIG. It was shown to.
  • the horizontal axis represents the HMGB1 degradation product contained in the sample or the concentration (ng / mL) of HMGB1 contained in the sample
  • the vertical axis represents the absorbance (450 nm) obtained by the measurement.
  • “ ⁇ ” indicates a measured value (the absorbance described above) when measured on the sample of (2) (1) (decomposed product of HMGB1) [total 7 concentrations]
  • “ ⁇ ” indicates the above 2
  • the measured value (the above-mentioned absorbance) when measured for the sample (HMGB1) (total 7 concentrations) of (2).
  • Table 3 shows the measurement values of the measurement results in 3 above.
  • Antibody-immobilized microplate The antibody (5D1) such as anti-HMGB1 degradation product of Example 2 was purified using protein A, and diluted with phosphate buffered saline (PBS) to a concentration of 2.5 ⁇ g / mL. Next, 100 ⁇ L of this was dispensed into each well of a microtiter plate (microplate) [Nunc, trade name: Maxisorp] and allowed to stand at 25 ° C. overnight, and the above-mentioned anti-HMGB1 degradation product antibody ( 5D1) was immobilized on a well of a microplate.
  • PBS phosphate buffered saline
  • This biotin-labeled antibody was mixed with 50 mM Tris-HCl buffer (pH 7.8) containing 100 mM sodium chloride, 0.5% sodium caseinate, 2 mM EDTA.2 sodium, 0.1% sodium azide, and 10% mouse serum. was dissolved to a concentration of 2 ⁇ g / mL, and this was used as a biotin-labeled antibody solution (2H6).
  • Streptavidin-peroxidase conjugate solution Streptavidin-PolyHRP40 (Stereospecific Detection Technologies [Germany]; product code number: SP40C), 100 mM sodium chloride, 0.5% casein (vitamin free) and 100 mM Tris-HCl buffer (pH 7) containing 0.5 mM calcium chloride 8) was diluted 10,000 times. This was used as a streptavidin-peroxidase conjugate solution.
  • Cleaning fluid Phosphate buffered saline containing 0.05% Tween 20 was used as a washing solution.
  • Diluent A diluted solution was 100 mM CHES buffer (pH 9.5) containing 100 mM sodium chloride, 0.5% sodium caseinate, 2 mM EDTA.2 sodium, 0.1% sodium azide, and 10% mouse serum.
  • Coloring solution A 0.045% 3,3 ′, 5,5′-tetramethylbenzidine hydrochloride aqueous solution (pH 2.0) containing 0.2 mM EDTA ⁇ disodium was used as a color developing solution.
  • Reaction stop solution 0.7N sulfuric acid was used as a reaction stop solution. 2.
  • Sample (the HMGB1 degradation product)
  • concentration of the degradation product of HMGB1 is 0 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, and 40 ng / mL, respectively, as described in (1) of (1) -2 above.
  • 80 ng / mL sample (the HMGB1 degradation product) was prepared.
  • HMGB1 As described in (2) of [1] above, the concentrations of HMGB1 are 0 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, and 80 ng, respectively.
  • / ML sample (HMGB1) was prepared. 3. Measurement (1) Each well of the antibody-immobilized microplate (5D1) of (1) was washed 3 times with 250 ⁇ L of the washing solution of (4). (2) Next, 100 ⁇ L of the diluted solution (1) was dispensed into each well.
  • Antigen-antibody reaction between the degradation product and biotin-labeled antibody was performed.
  • each well was washed 5 times with 400 ⁇ L of the washing solution of (1) above.
  • 100 ⁇ L of the streptavidin-peroxidase conjugate solution of (1) above is dispensed into each well and left at 25 ° C. for 1 hour to perform the “biotin-streptavidin” binding reaction. I did it.
  • each well was washed 5 times with 400 ⁇ L of the washing solution of (1) above.
  • the horizontal axis represents the HMGB1 degradation product contained in the sample or the concentration (ng / mL) of HMGB1 contained in the sample
  • the vertical axis represents the absorbance (450 nm) obtained by the measurement.
  • “ ⁇ ” indicates a measured value (the absorbance described above) when measured on the sample of (2) (1) (decomposed product of HMGB1) [total 7 concentrations]
  • “ ⁇ ” indicates the above 2
  • the measured value (the above-mentioned absorbance) when measured for the sample (HMGB1) (total 7 concentrations) of (2).
  • Table 4 shows the measured values of the measurement results in 3 above.
  • the measured value (absorbance difference) of the HMGB1 degradation product is high, indicating that the HMGB1 degradation product contained in the sample can be measured with high specificity.
  • the calibration curve is a graph of the HMGB1 degradation product contained in the sample. It also shows that the HMGB1 degradation product contained in the sample can be quantitatively measured over a wide range from a low concentration range to a high concentration range, extending linearly in proportion to the concentration.
  • the sample (the HMGB1 degradation product) [2.5 to 80 ng / mL] in which the measurement was performed The value (absorbance difference) in (the HMGB1 degradation product) divided by the value (absorbance difference) in the sample (HMGB1) was in the range of about 0.72 to 0.79. That is, in the conventional immunological measurement method and immunological measurement reagent, the measured value (absorbance difference) of HMGB1 is higher than the measured value (absorbance difference) of the HMGB1 degradation product at the same concentration at any concentration. Is high.
  • the immunological measurement method and the immunological measurement reagent of the HMGB1 degradation product of the present invention have high specificity for the HMGB1 degradation product. Measurement was suppressed, that is, it was suppressed that a positive error derived from HMGB1 was generated, and it was confirmed that only the HMGB1 degradation product can be quantitatively measured with high specificity, accuracy and sensitivity. .
  • Example 9 (Method for immunological measurement of HMGB1 degradation product and measurement reagent-2)
  • the HMGB1 degradation product contained in the sample the HMGB1 contained in the sample, the HMGB2 degradation product contained in the sample, and the sample Measurement of HMGB2 was performed.
  • Reagent (1) Antibody-immobilized microplate (5D1) Prepared as described in 1 of (2) of Example 8 [1], this was used as an antibody-immobilized microplate (5D1).
  • Biotin-labeled antibody solution (2H6) Prepared as described in Example 8, [2], 1 (2), and this was used as a biotin-labeled antibody solution (2H6).
  • (3) Streptavidin-peroxidase conjugate solution This was prepared as described in Example 8, [2], 1 (3), and this was used as a streptavidin-peroxidase conjugate solution.
  • (4) Cleaning fluid Prepared as described in Example 8, [2], 1 (4), and this was used as a cleaning solution.
  • Diluent Prepared as described in Example 8, [2], 1 (5), and this was used as a diluent.
  • (6) Coloring solution It was prepared as described in Example 8, [2], 1 (6), and this was used as a color developing solution.
  • Sample (the HMGB1 degradation product)
  • the HMGB1 degradation product prepared in [2] of Reference Example 1 was mixed with a phosphate buffered saline containing 0.5% sodium caseinate, 100 mM sodium chloride, and 0.1% sodium azide, each 1.5 ng / Samples prepared with concentrations of mL, 3.1 ng / mL, 6.2 ng / mL, 12.5 ng / mL, 25 ng / mL, 50 ng / mL, and 100 ng / mL, respectively (the HMGB1 degradation product) It was.
  • a phosphate buffered saline containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide was used as a sample (the HMGB1 degradation product) having a concentration of the HMGB1 degradation product of 0 ng / mL. .
  • HMGB1 The HMGB1 prepared in [1] of Reference Example 1 was mixed with phosphate buffered saline containing 0.5% sodium caseinate, 100 mM sodium chloride, and 0.1% sodium azide, 1.5 ng / mL, 3 Samples (HMGB1) were prepared to have concentrations of 0.1 ng / mL, 6.2 ng / mL, 12.5 ng / mL, 25 ng / mL, 50 ng / mL, and 100 ng / mL, respectively.
  • HMGB1 A phosphate buffered saline containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide was used as a sample (HMGB1) having a HMGB1 concentration of 0 ng / mL.
  • Sample (said HMGB2 degradation product) The HMGB2 degradation product prepared in [3] of Reference Example 1 was added to a phosphate buffered saline containing 0.5% sodium caseinate, 100 mM sodium chloride, and 0.1% sodium azide, each 1.5 ng / Samples prepared with concentrations of mL, 3.1 ng / mL, 6.2 ng / mL, 12.5 ng / mL, 25 ng / mL, 50 ng / mL, and 100 ng / mL, respectively (the HMGB2 degradation product) It was.
  • a phosphate buffered saline containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide was used as a sample having the HMGB2 degradation product concentration of 0 ng / mL (the HMGB2 degradation product).
  • HMGB2 The HMGB2 prepared in [1] of Reference Example 1 was mixed with phosphate buffered saline containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide at 1.5 ng / mL, 3 Samples (HMGB2) were prepared to have concentrations of .1 ng / mL, 6.2 ng / mL, 12.5 ng / mL, 25 ng / mL, 50 ng / mL, and 100 ng / mL.
  • phosphate buffered saline containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide was used as a sample (HMGB2) having a concentration of HMGB2 of 0 ng / mL. 3. Measurement (1) Each well of the antibody-immobilized microplate (5D1) of (1) was washed 3 times with 250 ⁇ L of the washing solution of (4). (2) Next, 100 ⁇ L of the diluted solution (1) was dispensed into each well. (3) Next, 10 ⁇ L of each of the samples of 8 concentrations (2) (1) (the degradation product of HMGB1) of 2 above was dispensed into each well, and then the top of each well was sealed with a plate seal, and 5 ° C.
  • each well was washed 5 times with 400 ⁇ L of the washing solution of (1) above.
  • 100 ⁇ L of the biotin-labeled antibody solution (2H6) of (1) above is dispensed into each well, left at 25 ° C. for 2 hours, and the HMGB1 bound to the solid-phased antibody.
  • Antigen-antibody reaction between the degradation product and biotin-labeled antibody was performed.
  • each well was washed 5 times with 400 ⁇ L of the washing solution of (1) above.
  • Measurement result (1) The measurement result in 3 above, that is, the HMGB1 degradation product contained in the sample, the HMGB1 contained in the sample, contained in the sample by the immunological measurement method and the immunological measurement reagent of the HMGB1 degradation product of the present invention
  • FIG. 8 shows the measurement results of the HMGB2 degradation product and HMGB2 contained in the sample.
  • letters representing the production cell lines of the immobilized antibody and the labeled antibody used for the measurement are shown above the figure.
  • the horizontal axis represents the concentration (ng / mL) of the HMGB1 degradation product contained in the sample, the HMGB1 contained in the sample, the HMGB2 degradation product contained in the sample, or the HMGB2 contained in the sample.
  • the axis indicates the absorbance obtained by measurement [absorbance at the main wavelength (450 nm) minus the absorbance at the sub-wavelength (550 nm)].
  • ⁇ indicates a measured value (the absorbance described above) when measured for the sample of (2) (1) (the degradation product of HMGB1) [total 8 concentrations]
  • ⁇ indicates the above 2 (2) sample (HMGB1) [total 8 concentrations] measured value (absorbance described above)
  • ⁇ represents the sample (3) (decomposition product of HMGB2) [total 8
  • ⁇ indicates the measurement value (the absorbance described above) measured for the sample (HMGB2) (total 8 concentrations) of (2) above.
  • Table 5 shows the measured values of the measurement results in 3 above.
  • letters representing the production cell lines of the immobilized antibody and the labeled antibody used for the measurement are shown in the upper left of the table.
  • the measured value (absorbance difference) of HMGB1, the measured value (absorbance difference) of the HMGB2 degradation product, and the measured value of HMGB2 (Absorbance difference) are all very low, and it can be seen that the measurement of HMGB1, the HMGB2 degradation product, and HMGB2 contained in the sample is extremely suppressed.
  • the measured value (absorbance difference) of the HMGB1 degradation product is high, indicating that the HMGB1 degradation product contained in the sample can be measured with high specificity.
  • the calibration curve is a graph of the HMGB1 degradation product contained in the sample. It also shows that the HMGB1 degradation product contained in the sample can be quantitatively measured over a wide range from a low concentration range to a high concentration range, extending linearly in proportion to the concentration.
  • the HMGB1 degradation product immunological measurement method and immunological measurement reagent of the present invention have high specificity for the HMGB1 degradation product, and HMGB1 Measurement is suppressed, that is, the occurrence of a positive error derived from HMGB1 or the like is suppressed, and only the HMGB1 degradation product can be quantitatively measured with high specificity and accuracy with high sensitivity. It was confirmed.
  • Example 10 Method for immunological measurement of HMGB1 degradation product and measurement reagent-3
  • the HMGB1 degradation product contained in the sample the HMGB1 contained in the sample, the HMGB2 degradation product contained in the sample, and the sample Measurement of HMGB2 was performed.
  • Reagent (1) Antibody-immobilized microplate (I) Antibody-immobilized microplate (2A10) Example 1 except that the anti-HMGB1 degradation product antibody (2A10) of Example 3 is used instead of the anti-HMGB1 degradation product antibody (5D1) in 1 (1) of Example 8 [2].
  • Streptavidin-peroxidase conjugate solution This was prepared as described in Example 8, [2], 1 (3), and this was used as a streptavidin-peroxidase conjugate solution.
  • Cleaning fluid Prepared as described in Example 8, [2], 1 (4), and this was used as a cleaning solution.
  • Diluent Prepared as described in Example 8, [2], 1 (5), and this was used as a diluent.
  • Coloring solution It was prepared as described in Example 8, [2], 1 (6), and this was used as a color developing solution.
  • Substrate solution Prepared as described in Example 8 [2] 1 (7), this was used as the substrate solution.
  • Samples (the aforementioned HMGB1 degradation products) were prepared so as to have concentrations of mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, and 80 ng / mL, respectively.
  • a phosphate buffered saline containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide was used as a sample (the HMGB1 degradation product) having a concentration of the HMGB1 degradation product of 0 ng / mL. .
  • HMGB1 prepared in [1] of Reference Example 1 was added to each of 2.5 ng / mL and 5 ng with phosphate buffered saline containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide.
  • Samples (HMGB1) were prepared to have concentrations of / ng, 10 ng / mL, 20 ng / mL, 40 ng / mL, and 80 ng / mL.
  • a phosphate buffered saline containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide was used as a sample (HMGB1) having a HMGB1 concentration of 0 ng / mL.
  • Sample (said HMGB2 degradation product) The HMGB2 degradation product prepared in [3] of Reference Example 1 was added to each 2.5 ng / kg of phosphate buffered saline containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide. Samples (the aforementioned HMGB2 degradation products) were prepared to have concentrations of mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, and 80 ng / mL, respectively.
  • a phosphate buffered saline containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide was used as a sample having the HMGB2 degradation product concentration of 0 ng / mL (the HMGB2 degradation product). .
  • Sample (HMGB2) The HMGB2 prepared in [1] of Reference Example 1 was mixed with phosphate buffered saline containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide, 2.5 ng / mL and 5 ng, respectively.
  • HMGB2 phosphate buffered saline containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide was used as a sample (HMGB2) having a concentration of HMGB2 of 0 ng / mL. 3. Measurement (1) Each well of the antibody-immobilized microplate (2A10) of (1) (1) of (1) was washed three times with 400 ⁇ L of the washing solution of (1) (4). (2) Next, 100 ⁇ L of the diluted solution (1) was dispensed into each well.
  • Antigen-antibody reaction between the degradation product and biotin-labeled antibody was performed.
  • each well was washed 5 times with 400 ⁇ L of the washing solution of (1) above.
  • 100 ⁇ L of the streptavidin-peroxidase conjugate solution of (1) above is dispensed into each well and left at 25 ° C. for 1 hour to perform the “biotin-streptavidin” binding reaction. I did it.
  • each well was washed 5 times with 400 ⁇ L of the washing solution of (1) above.
  • Measurement result (1) The measurement result in 3 above, that is, the HMGB1 degradation product contained in the sample, the HMGB1 contained in the sample, contained in the sample by the immunological measurement method and the immunological measurement reagent of the HMGB1 degradation product of the present invention
  • FIG. 9 Results of measurement using an antibody-immobilized microplate (2A10) as the antibody-immobilized microplate] and the results of the measurement of the HMGB2 degradation product and the HMGB2 contained in the sample.
  • FIG. 10 Resultss of measurement when antibody-immobilized microplate (6H3) is used as the antibody-immobilized microplate].
  • letters representing the production cell lines of the immobilized antibody and the labeled antibody used for the measurement are shown above the figures.
  • the horizontal axis indicates the concentration (ng / mL) of the HMGB1 degradation product contained in the sample, the HMGB1 contained in the sample, the HMGB2 degradation product contained in the sample, or the HMGB2 contained in the sample.
  • the vertical axis represents the absorbance obtained by measurement [the absorbance at the main wavelength (450 nm) minus the absorbance at the sub-wavelength (550 nm)].
  • ⁇ indicates the measured value (the absorbance described above) when measured for the sample of (2) (1) (decomposition product of HMGB1) [total 7 concentrations], and “ ⁇ ” indicates the above 2 (2) sample (HMGB1) [total 7 concentrations] measured value (the absorbance described above), “ ⁇ ” indicates the sample of (3) (decomposition product of HMGB2) [total 7 (concentration) indicates the measured value (absorbance), and “ ⁇ ” indicates the measured value (absorbance) when measured for the sample (HMGB2) (total 7 concentrations) in (2) above. Indicates.
  • the value (absorbance difference) in (the HMGB1 degradation product) divided by the value (absorbance difference) in the sample (HMGB2) was in the range of about ⁇ 78 to 29.
  • the value (absorbance difference) in (the HMGB1 degradation product) divided by the value (absorbance difference) in the sample (the HMGB2 degradation product) was in the range of about ⁇ 5 to 29.
  • the measured value (absorbance difference) of HMGB1, the measured value (absorbance difference) of the HMGB2 degradation product, and the measured value of HMGB2 (Absorbance difference) are all very low, and it can be seen that the measurement of HMGB1, the HMGB2 degradation product, and HMGB2 contained in the sample is extremely suppressed.
  • the measured value (absorbance difference) of the HMGB1 degradation product is high, indicating that the HMGB1 degradation product contained in the sample can be measured with high specificity.
  • the calibration curve is a graph of the HMGB1 degradation product contained in the sample. It also shows that the HMGB1 degradation product contained in the sample can be quantitatively measured over a wide range from a low concentration range to a high concentration range, extending linearly in proportion to the concentration.
  • the immunology of the HMGB1 degradation product of the present invention is also based on the results of the examination in this example [results of measurement using the antibody-immobilized microplate (2A10) as the antibody-immobilized microplate].
  • the measurement method and the immunological measurement reagent have high specificity for the HMGB1 degradation product, and the measurement of HMGB1 and the like is suppressed, that is, the occurrence of a positive error derived from HMGB1 and the like is suppressed, It was confirmed that only the HMGB1 degradation product can be quantitatively measured with high specificity, accuracy and sensitivity.
  • the sample (the HMGB1 degradation product) [2.5 to 80 ng / mL] in which the measurement was performed The value (absorbance difference) in (the HMGB1 degradation product) divided by the value (absorbance difference) in the sample (HMGB1) was in the range of about 3.1 to 5.8.
  • the value (absorbance difference) in (the HMGB1 degradation product) divided by the value (absorbance difference) in the sample (HMGB2) was in the range of about ⁇ 47 to 227.
  • the value (absorbance difference) in (the HMGB1 degradation product) divided by the value (absorbance difference) in the sample (the HMGB2 degradation product) was in the range of about ⁇ 680 to 77.
  • the measured value (absorbance difference) of HMGB1, the measured value (absorbance difference) of the HMGB2 degradation product, and the measured value of HMGB2 (Absorbance difference) are all very low, and it can be seen that the measurement of HMGB1, the HMGB2 degradation product, and HMGB2 contained in the sample is extremely suppressed.
  • the measured value (absorbance difference) of the HMGB1 degradation product is high, indicating that the HMGB1 degradation product contained in the sample can be measured with high specificity.
  • the calibration curve is a graph of the HMGB1 degradation product contained in the sample. It also shows that the HMGB1 degradation product contained in the sample can be quantitatively measured over a wide range from a low concentration range to a high concentration range, extending linearly in proportion to the concentration.
  • the immunology of the HMGB1 degradation product of the present invention is also based on the results of the examination in this example [results of measurement using an antibody-immobilized microplate (6H3) as an antibody-immobilized microplate].
  • the measurement method and the immunological measurement reagent have high specificity for the HMGB1 degradation product, and the measurement of HMGB1 and the like is suppressed, that is, the occurrence of a positive error derived from HMGB1 and the like is suppressed, It was confirmed that only the HMGB1 degradation product can be quantitatively measured with high specificity, accuracy and sensitivity.

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Abstract

Provided are an antibody that is highly specific to an HMGB1 decomposition product, and an assay method and assay reagent capable of accurately performing quantitative assay of only an HMGB1 decomposition product. Provided are the following: an antibody that binds with the decomposition product of HMGB1 induced by thrombin or thrombin-thrombomodulin complex, and in which the affinity for the decomposition product of HMGB1 induced by thrombin or thrombin-thrombomodulin complex is at least 1.5-times the affinity for HMGB1; and an immunoassay method and immunoassay reagent for an HMGB1 decomposition product using the aforementioned antibody and an antibody that binds with the decomposition product of HMGB1 induced by thrombin or thrombin-thrombomodulin complex and that has a high affinity for the decomposition product of HMGB1 induced by thrombin or thrombin-thrombomodulin complex.

Description

ＨＭＧＢ１の分解産物と特異的に結合する抗体、並びにＨＭＧＢ１の分解産物の測定方法及び測定試薬Antibody specifically binding to degradation product of HMGB1, and method and reagent for measuring degradation product of HMGB1
　本発明は、ＨＭＧＢ１に比べ細胞障害性が低減したＨＭＧＢ１の分解産物と特異的に結合する抗体、並びにＨＭＧＢ１の分解産物の測定方法及び測定試薬に関するものである。
　本発明は、臨床検査、臨床病理学、免疫学及び医学などの生命科学分野、並びに分析化学などの化学分野等において有用なものである。 The present invention relates to an antibody that specifically binds to a degradation product of HMGB1 with reduced cytotoxicity compared to HMGB1, and a method and reagent for measuring the degradation product of HMGB1.
The present invention is useful in the field of life science such as clinical examination, clinical pathology, immunology and medicine, and in the field of chemistry such as analytical chemistry.
　ＨＭＧＢ（ハイ　モビリティー　グループ　ボックス　プロテイン；Ｈｉｇｈ　Ｍｏｂｉｌｉｔｙ　Ｇｒｏｕｐ　Ｂｏｘ　Ｐｒｏｔｅｉｎ）は、以前、ＨＭＧ（ハイ　モビリティー　グループ　プロテイン；Ｈｉｇｈ　Ｍｏｂｉｌｉｔｙ　Ｇｒｏｕｐ　Ｐｒｏｔｅｉｎ）と呼ばれていたが、これはクロマチン構造に含まれる大量の非ヒストンタンパク質として１９６４年に発見され、すべての高等動植物に普遍的に含まれるタンパク質であり、種族間で一次構造の保存性は極めて高い。
　また、核内ばかりではなく、細胞質内にも豊富に存在することが分かっている。
　生理作用ははっきりとは分かっていないが、ＨＭＧＢはＤＮＡと結合する際に二重らせん構造を緩めることから、転写反応の際にＤＮＡの高次構造を最適構造に変化させて転写活性を高めるという、極めて広範囲の転写促進因子及びヌクレオソーム弛緩因子として機能すると考えられている。
　ＨＭＧＢには、いくつかの種類が存在する。
　例えば、ＨＭＧＢ１（ハイ　モビリティー　グループ　ボックス　プロテイン１）、ＨＭＧＢ２（ハイモビリティー　グループ　ボックス　プロテイン２）、ＨＭＧＢ３（ハイモビリティー　グループ　ボックス　プロテイン３）、ＨＭＧＢ８（ハイモビリティー　グループ　ボックス　プロテイン８）、ＨＭＧＢ１７（ハイモビリティー　グループ　ボックス　プロテイン１７）、ＨＭＧＢ　Ｉ（ハイモビリティー　グループ　ボックス　プロテイン　Ｉ）、ＨＭＧＢ　Ｙ（ハイモビリティー　グループ　ボックス　プロテイン　Ｙ）、ＨＭＧＢ　Ｉ（Ｙ）（ハイモビリティー　グループ　ボックス　プロテイン　Ｉ（Ｙ））、ＨＭＧＢ　Ｉ−Ｃ（ハイモビリティー　グループ　ボックス　プロテイン　Ｉ−Ｃ）等を挙げることができる。
　なお、本発明者らが、遺伝情報処理ソフトウェア「ＧＥＮＥＴＹＸ」（ＳＯＦＴＷＡＲＥ　ＤＥＶＥＬＯＰＭＥＮＴ社）を使用してアミノ酸配列の相同性の解析を行ったところ、ヒトのＨＭＧＢ１に対して、ウシＨＭＧＢ１の相同性は９８．６％であり、ブタＨＭＧＢ１の相同性は９９．１％であった。
　また、同様に、ヒトのＨＭＧＢ１に対して、ヒトのＨＭＧＢ２の相同性は８１．２％であり、ウシＨＭＧＢ２との相同性は７２．３％であり、ブタのＨＭＧＢ２との相同性は７９．４％であった。
　ワング（Ｗａｎｇ）らは１９９９年に、ＨＭＧＢ１自体を免疫原として調製したポリクローナル抗体を使用したウエスタンブロット法により、初めて血清中（血液中）のＨＭＧＢ１の定量測定を行った。
　その結果、ワングらは、ＨＭＧＢ１が敗血症のマーカーとなりうることを示した。
　そして、敗血症の患者において、生き残る患者と、死に至る患者を判別することが、精密に血液中のＨＭＧＢ１を測定することによって可能であることを示した（非特許文献１参照。）。
　なお、先に、ＨＭＧＢ１の測定に用いる抗体、即ちＨＭＧＢ１に結合する抗体については、パーキネン（Ｐａｒｋｋｉｎｅｎ）らや、レップ（Ｌｅｐｐ）らによって調製可能なことが示されていた（非特許文献２及び非特許文献３参照。）。
　この抗体を用いてレップらはＨＭＧＢ１に関して固相酵素免疫測定法（Ｓｏｌｉｄ−ｐｈａｓｅ　Ｅｎｚｙｍｅ　Ｉｍｍｕｎｏａｓｓａｙ）が可能であることを述べている。
　また、カバート（Ｃａｂａｒｔ）らにより、ヒトＨＭＧＢ１及びヒトＨＭＧＢ２の調製方法が示されていた（非特許文献４参照。）。
　また、ＨＭＧＢ１は、炎症でも誘導され、このことが各種サイトカインの大量分泌の原因と考えられるという文献も発表されている（非特許文献５参照、非特許文献６参照及び非特許文献７参照。）。
　なお、本発明者らは、先に、ヒトＨＭＧＢ１には結合するが、ヒトＨＭＧＢ２には結合しない抗体、及びＨＭＧＢ２を測り込むことなく、誤差を含まない正確な測定値を得ることができるヒトＨＭＧＢ１の免疫学的測定試薬及び免疫学的測定方法を開発した（特許文献１参照。）。
　また、本発明者らは、先に、ヒトＨＭＧＢ１との結合能力が高い高力価の抗体であって、かつ当該抗体を高い確率で取得することができる抗ヒトＨＭＧＢ１抗体、試料中にごく微量に含まれるヒトＨＭＧＢ１までも正確に測定することができる高感度な試料中のヒトＨＭＧＢ１の免疫学的測定方法及び免疫学的測定試薬等を開発した（特許文献２参照。）。
　ところで、本発明者らは、トロンビン又はトロンビン・トロンボモジュリン複合体により、ヒトＨＭＧＢ１の１０番目のアルギニン（Ｒ１０）−１１番目のグリシン（Ｇ１１）が切断され、このＨＭＧＢ１のＮ末端の「ＭＧＫＧＤＰＫＫＰＲ」の１０のアミノ酸残基よりなるペプチドが分離して、新たに露呈したＮ末端「ＧＫＭＳＳ・・・・・」を有するＨＭＧＢ１の分解産物が生じることを見出した。（本発明者らは、このヒトＨＭＧＢ１の分解産物を「ｄｅｓ−ＨＭＧＢ１」と名付けた。）
　本発明者らの検討の結果、このＨＭＧＢ１の分解産物は、ＨＭＧＢ１に比べその細胞障害性が低いものであることが分かった。
　このことより、ＨＭＧＢ１の細胞障害性を減じるため、トロンビンやトロンビン・トロンボモジュリン複合体を用いて、ＨＭＧＢ１を前記の通り分解（切断）して、より細胞障害性の低いＨＭＧＢ１の分解産物に変換することが発想された。
　そして、このトロンビン又はトロンビン・トロンボモジュリン複合体によるＨＭＧＢ１の分解の効果を把握するため、このＨＭＧＢ１の分解産物の定量測定が求められ、測定方法が検討されたが、従来の方法は、このＨＭＧＢ１の分解産物と共にＨＭＧＢ１等も測り込んでしまうものであって、ＨＭＧＢ１の分解産物だけを特異的に測定することはできないものであった（非特許文献８参照。）。 HMGB (High Mobility Group Box Protein), formerly called HMG (High Mobility Group Protein), is a large amount of non-histone protein in the chromatin structure It was discovered in 1964 and is a protein that is universally included in all higher animals and plants, and has a very high conservation of primary structure among species.
It has also been found that it exists not only in the nucleus but also in the cytoplasm.
Physiological effects are not clearly understood, but HMGB relaxes the double helix structure when it binds to DNA, so that the transcriptional activity is increased by changing the higher order structure of DNA to the optimal structure during the transcription reaction. It is thought to function as a very wide range of transcriptional promoters and nucleosome relaxing factors.
There are several types of HMGB.
For example, HMGB1 (high mobility group box protein 1), HMGB2 (high mobility group box protein 2), HMGB3 (high mobility group box protein 3), HMGB8 (high mobility group box protein 8), HMGB17 (high mobility group box protein 17) ), HMGB I (High Mobility Group Box Protein I), HMGB Y (High Mobility Group Box Protein Y), HMGB I (Y) (High Mobility Group Box Protein I (Y)), HMGB I-C (High Mobility Group Box) Protein I-C) and the like.
In addition, when the present inventors analyzed the homology of the amino acid sequence using genetic information processing software “GENETYX” (SOFTWARE DEVELOPMENT), the homology of bovine HMGB1 was 98 with respect to human HMGB1. The homology of porcine HMGB1 was 99.1%.
Similarly, human HMGB2 has a homology of 81.2% with respect to human HMGB1, a homology with bovine HMGB2 of 72.3%, and a homology with porcine HMGB2 of 79. 4%.
In 1999, Wang et al. Quantitatively measured HMGB1 in serum (in blood) for the first time by Western blotting using a polyclonal antibody prepared using HMGB1 itself as an immunogen.
As a result, Wang et al. Showed that HMGB1 can be a marker for sepsis.
Then, it was shown that it is possible to accurately determine the patient who survives and the patient who will die in sepsis patients by measuring HMGB1 in blood (see Non-Patent Document 1).
It has been previously shown that antibodies used for HMGB1 measurement, that is, antibodies that bind to HMGB1, can be prepared by Parkinen et al., Lepp et al. (See Patent Document 3).
Using this antibody, Rep et al. Stated that a solid-phase enzyme immunoassay is possible for HMGB1.
In addition, a method for preparing human HMGB1 and human HMGB2 has been shown by Cabart et al. (See Non-Patent Document 4).
Further, HMGB1 is also induced by inflammation, and literatures that this is considered to be a cause of mass secretion of various cytokines have been published (see Non-Patent Document 5, Non-Patent Document 6 and Non-Patent Document 7). .
In addition, the present inventors previously obtained an antibody that binds to human HMGB1 but does not bind to human HMGB2, and human HMGB1 that can obtain an accurate measurement value without error without measuring HMGB2. An immunological measurement reagent and an immunological measurement method were developed (see Patent Document 1).
In addition, the present inventors previously described an anti-human HMGB1 antibody that is a high-titer antibody that has a high binding ability to human HMGB1 and that can obtain the antibody with a high probability, Has developed an immunological measurement method and an immunological measurement reagent for human HMGB1 in a highly sensitive sample capable of accurately measuring even human HMGB1 contained in the above (see Patent Document 2).
By the way, the present inventors cleave the 10th arginine (R10) -11th glycine (G11) of human HMGB1 by thrombin or thrombin thrombomodulin complex, It was found that a peptide consisting of the amino acid residues of HMGB1 was separated to produce a degradation product of HMGB1 having a newly exposed N-terminal “GKMSS...”. (The inventors named this degradation product of human HMGB1 as “des-HMGB1”.)
As a result of the study by the present inventors, it was found that the degradation product of HMGB1 has a lower cytotoxicity than HMGB1.
From this, in order to reduce the cytotoxicity of HMGB1, HMGB1 is decomposed (cleaved) as described above using thrombin or thrombin / thrombomodulin complex, and converted to a degradation product of HMGB1 having a lower cytotoxicity. Was conceived.
Then, in order to grasp the effect of degradation of HMGB1 by this thrombin or thrombin-thrombomodulin complex, quantitative measurement of the degradation product of HMGB1 was required and a measurement method was examined. Conventional methods are based on degradation of HMGB1. HMGB1 and the like are also measured together with the product, and only the degradation product of HMGB1 cannot be specifically measured (see Non-Patent Document 8).
特開２００３−９６０９９号公報JP 2003-96099 A 国際公開第２００８／０７５７８８号International Publication No. 2008/0775788
　前述のように、ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物を正確に定量測定することが求められた。
　しかしながら、従来の抗体は、前記のＨＭＧＢ１の分解産物に対する特異性が高いものではなく、前記ＨＭＧＢ１分解産物に対する親和性とＨＭＧＢ１に対する親和性が同程度のものであった。
　そして、従来の測定方法及び測定試薬も、前記ＨＭＧＢ１分解産物に対する特異性が高いものではなく、前記ＨＭＧＢ１分解産物を測定するのみならず、ＨＭＧＢ１等も測り込んでしまうものであり、すなわちその測定値がＨＭＧＢ１等に由来する正の誤差を含んでしまうものであった。
　また、ＨＭＧＢ１の分解産物の全アミノ酸配列は、ＨＭＧＢ１中に存在するため、当該分解産物に対する親和性がＨＭＧＢ１に対する親和性よりも高い抗体を得ることは困難であると予想された。
　しかしながら、本発明者らは、前記課題の解決を目指して検討を重ねた結果、目的とする抗体を得ることに成功し、更に当該抗体と、特定の抗体を組み合わせることにより、前記ＨＭＧＢ１分解産物のみを正確に定量測定することができることを見出し、本発明を完成するに至った。 As described above, it was required to accurately and quantitatively measure the degradation products of HMGB1 by thrombin or thrombin / thrombomodulin complex.
However, the conventional antibody is not highly specific for the degradation product of HMGB1, and the affinity for the degradation product of HMGB1 is similar to that for HMGB1.
The conventional measurement method and reagent are not highly specific for the HMGB1 degradation product, and not only measure the HMGB1 degradation product but also measure HMGB1 and the like, that is, the measured value Included a positive error derived from HMGB1 and the like.
Further, since the entire amino acid sequence of the degradation product of HMGB1 is present in HMGB1, it was expected that it is difficult to obtain an antibody having higher affinity for the degradation product than that for HMGB1.
However, as a result of repeated studies aimed at solving the above problems, the present inventors have succeeded in obtaining the target antibody, and further combining the antibody with a specific antibody, so that only the HMGB1 degradation product is obtained. Has been found to be capable of accurate quantitative measurement, and the present invention has been completed.
　本発明の要旨は以下のとおりである。
（１）　ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物に対する親和性が、ＨＭＧＢ１に対する親和性と比較して、少なくとも１．５倍である、ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物に結合する抗体。
（２）　ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物に対する親和性が、ＨＭＧＢ２に対する親和性及びＨＭＧＢ２のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物に対する親和性とそれぞれ比較して、各々少なくとも１０倍である、前記（１）記載のＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物に結合する抗体。
（３）　ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物に結合する抗体がモノクローナル抗体である、前記（１）又は（２）記載のＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物に結合する抗体。
（４）　試料に含まれるＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物の免疫学的測定方法であって、次の（ａ）の抗体及び（ｂ）の抗体を使用する測定方法。
　（ａ）　前記（１）~（３）のいずれかに記載の抗体。
　（ｂ）　ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物に結合する抗体であって、ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物に対する親和性が高い抗体。
（５）　前記（４）における（ｂ）の抗体が、当該抗体の濃度が０．６２５~２．５ｎｇ／ｍＬの範囲の少なくともいずれかの濃度において、ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物を固相化したＥＬＩＳＡ法で、
（イ）当該分解産物に結合する当該抗体の量を測定するときに得られる吸光度の値を当該抗体の濃度の値で除したときの値が０．５以上となる抗体、又は
（ロ）当該分解産物に結合する当該抗体の量を測定するときに得られる吸光度の値が、基準とする抗体産生細胞であるハイブリドーマＭＤ７８（ＦＥＲＭ　Ｐ−１８４０５）より産生される抗体における当該吸光度の値の６倍以上となる抗体
である、前記（４）記載の測定方法。
（６）　（ａ）の抗体が、前記（２）記載の抗体である、前記（４）又は（５）記載の測定方法。
（７）　（ａ）の抗体及び（ｂ）の抗体がモノクローナル抗体である、前記（４）~（６）のいずれかに記載の測定方法。
（８）　固相化抗体及び標識抗体を用いるものであって、（ａ）の抗体及び（ｂ）の抗体のうち、いずれか一方の抗体が固相化抗体として使用され、他の一方の抗体が標識抗体として使用される、前記（４）~（７）のいずれかに記載の測定方法。
（９）　試料に含まれるＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物の免疫学的測定試薬であって、次の（ａ）の抗体及び（ｂ）の抗体を含有する測定試薬。
　（ａ）　前記（１）~（３）のいずれかに記載の抗体。
　（ｂ）　ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物に結合する抗体であって、ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物に対する親和性が高い抗体。
（１０）　前記（９）における（ｂ）の抗体が、当該抗体の濃度が０．６２５~２．５ｎｇ／ｍＬの範囲の少なくともいずれかの濃度において、ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物を固相化したＥＬＩＳＡ法で、
（イ）当該分解産物に結合する当該抗体の量を測定するときに得られる吸光度の値を当該抗体の濃度の値で除したときの値が０．５以上となる抗体、又は
（ロ）当該分解産物に結合する当該抗体の量を測定するときに得られる吸光度の値が、基準とする抗体産生細胞であるハイブリドーマＭＤ７８（ＦＥＲＭ　Ｐ−１８４０５）より産生される抗体における当該吸光度の値の６倍以上となる抗体
である、前記（９）記載の測定試薬。
（１１）　（ａ）の抗体が、前記（２）記載の抗体である、前記（９）又は（１０）記載の測定試薬。
（１２）　（ａ）の抗体及び（ｂ）の抗体がモノクローナル抗体である、前記（９）~（１１）のいずれかに記載の測定試薬。
（１３）　固相化抗体及び標識抗体を含有するものであって、（ａ）の抗体及び（ｂ）の抗体のうち、いずれか一方の抗体が固相化抗体として使用され、他の一方の抗体が標識抗体として使用される、前記（９）~（１２）のいずれかに記載の測定試薬。 The gist of the present invention is as follows.
(1) The degradation product of HMGB1 by the thrombin or thrombin / thrombomodulin complex is at least 1.5 times the affinity for the degradation product by the thrombin or thrombin / thrombomodulin complex by comparison with the affinity for HMGB1. An antibody that binds.
(2) The affinity of HMGB1 for the degradation product by thrombin or thrombin-thrombomodulin complex is at least 10 times greater than the affinity for HMGB2 and the affinity of HMGB2 for the degradation product by thrombin or thrombin-thrombomodulin complex, respectively. An antibody that binds to a degradation product of HMGB1 by thrombin or thrombin-thrombomodulin complex according to (1).
(3) The antibody that binds to the degradation product of HMGB1 by thrombin or thrombin / thrombomodulin complex is a monoclonal antibody, and binds to the degradation product of HMGB1 by thrombin or thrombin / thrombomodulin complex according to (1) or (2) above antibody.
(4) A method for immunologically measuring a degradation product of HMGB1 contained in a sample by thrombin or thrombin / thrombomodulin complex, wherein the following antibody (a) and antibody (b) are used.
(A) The antibody according to any one of (1) to (3).
(B) An antibody that binds to a degradation product of HMGB1 by a thrombin or thrombin-thrombomodulin complex, and has a high affinity for a degradation product of HMGB1 by a thrombin or thrombin-thrombomodulin complex.
(5) Degradation of HMGB1 by thrombin or thrombin-thrombomodulin complex in the antibody (b) in (4) above at least in the concentration range of 0.625 to 2.5 ng / mL. In ELISA method in which the product is immobilized,
(B) an antibody having a value of 0.5 or more when the absorbance value obtained by measuring the amount of the antibody bound to the degradation product is divided by the concentration value of the antibody, or (b) the The absorbance value obtained when measuring the amount of the antibody bound to the degradation product is 6 times the absorbance value of the antibody produced from the hybridoma MD78 (FERM P-18405), which is the standard antibody-producing cell. The measuring method according to (4) above, which is an antibody as described above.
(6) The measuring method according to (4) or (5), wherein the antibody of (a) is the antibody according to (2).
(7) The measurement method according to any one of (4) to (6), wherein the antibody (a) and the antibody (b) are monoclonal antibodies.
(8) A solid-phase antibody and a labeled antibody, wherein either one of the antibody (a) and the antibody (b) is used as a solid-phase antibody, and the other antibody The method according to any one of (4) to (7), wherein is used as a labeled antibody.
(9) A reagent for immunological measurement of degradation products of thrombin or thrombin / thrombomodulin complex of HMGB1 contained in a sample, comprising the following antibodies (a) and (b):
(A) The antibody according to any one of (1) to (3).
(B) An antibody that binds to a degradation product of HMGB1 by a thrombin or thrombin-thrombomodulin complex, and has a high affinity for a degradation product of HMGB1 by a thrombin or thrombin-thrombomodulin complex.
(10) Degradation of HMGB1 by thrombin or thrombin-thrombomodulin complex in the antibody (b) in the above (9) at least in the concentration range of 0.625 to 2.5 ng / mL. In ELISA method in which the product is immobilized,
(B) an antibody having a value of 0.5 or more when the absorbance value obtained by measuring the amount of the antibody bound to the degradation product is divided by the concentration value of the antibody, or (b) the The absorbance value obtained when measuring the amount of the antibody bound to the degradation product is 6 times the absorbance value of the antibody produced from the hybridoma MD78 (FERM P-18405), which is the standard antibody-producing cell. The measurement reagent according to (9), which is an antibody as described above.
(11) The measuring reagent according to (9) or (10), wherein the antibody of (a) is the antibody according to (2).
(12) The measuring reagent according to any one of (9) to (11), wherein the antibody of (a) and the antibody of (b) are monoclonal antibodies.
(13) An antibody comprising a solid-phased antibody and a labeled antibody, wherein either one of the antibody (a) and the antibody (b) is used as the solid-phased antibody, The measurement reagent according to any one of (9) to (12), wherein the antibody is used as a labeled antibody.
　本発明の抗体は、ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物に対する親和性が、ＨＭＧＢ１に対する親和性と比較して、少なくとも１．５倍高い抗体であり、すなわち、前記ＨＭＧＢ１分解産物に対する特異性が高い抗体である。
　また、本発明の測定方法及び測定試薬は、ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物に対する特異性が高いものであって、ＨＭＧＢ１等の測り込みが抑制され、すなわちＨＭＧＢ１等に由来する正の誤差が生じることが抑制され、前記ＨＭＧＢ１分解産物のみを正確に定量測定することができるものである。 The antibody of the present invention is an antibody whose affinity for degradation products of thrombin or thrombin-thrombomodulin complex of HMGB1 is at least 1.5 times higher than the affinity for HMGB1, ie, specific for the HMGB1 degradation product It is a highly potent antibody.
The measurement method and reagent of the present invention have high specificity for the degradation product of HMGB1 by thrombin or thrombin / thrombomodulin complex, and the measurement of HMGB1 and the like is suppressed, that is, positively derived from HMGB1 and the like. Is suppressed, and only the HMGB1 degradation product can be accurately quantitatively measured.
図１はＨＭＧＢ１、前記ＨＭＧＢ１分解産物、ＨＭＧＢ２、前記ＨＭＧＢ２分解産物をそれぞれ泳動したときのゲルの写真である。レーン１は分子量マーカー、レーン２はＨＭＧＢ１、レーン３は前記ＨＭＧＢ１分解産物、レーン４はＨＭＧＢ２、レーン５は前記ＨＭＧＢ２分解産物、レーン６はトロンビンを示す。FIG. 1 is a photograph of a gel when HMGB1, the HMGB1 degradation product, HMGB2, and the HMGB2 degradation product were respectively migrated. Lane 1 is a molecular weight marker, lane 2 is HMGB1, lane 3 is the HMGB1 degradation product, lane 4 is HMGB2, lane 5 is the HMGB2 degradation product, and lane 6 is thrombin. 図２は抗ＨＭＧＢ１分解産物等抗体（５Ｄ１）のＨＭＧＢ１、前記ＨＭＧＢ１分解産物、ＨＭＧＢ２、前記ＨＭＧＢ２分解産物それぞれとの反応性（親和性）を確かめたウエスタンブロット法の写真である。レーン１は分子量マーカー、レーン２はＨＭＧＢ１、レーン３は前記ＨＭＧＢ１分解産物、レーン４はＨＭＧＢ２、レーン５は前記ＨＭＧＢ２分解産物を示す。FIG. 2 is a photograph of Western blotting confirming the reactivity (affinity) of the anti-HMGB1 degradation product antibody (5D1) with HMGB1, the HMGB1 degradation product, HMGB2, and the HMGB2 degradation product. Lane 1 shows a molecular weight marker, lane 2 shows HMGB1, lane 3 shows the HMGB1 degradation product, lane 4 shows HMGB2, and lane 5 shows the HMGB2 degradation product. 図３は本抗ＨＭＧＢ１分解産物抗体（２Ｈ６）のＨＭＧＢ１、前記ＨＭＧＢ１分解産物、ＨＭＧＢ２、前記ＨＭＧＢ２分解産物それぞれとの反応性（親和性）を確かめたウエスタンブロット法の写真である。レーン１は分子量マーカー、レーン２はＨＭＧＢ１、レーン３は前記ＨＭＧＢ１分解産物、レーン４はＨＭＧＢ２、レーン５は前記ＨＭＧＢ２分解産物を示す。FIG. 3 is a photograph of Western blotting confirming the reactivity (affinity) of the anti-HMGB1 degradation product antibody (2H6) with HMGB1, the HMGB1 degradation product, HMGB2, and the HMGB2 degradation product. Lane 1 shows a molecular weight marker, lane 2 shows HMGB1, lane 3 shows the HMGB1 degradation product, lane 4 shows HMGB2, and lane 5 shows the HMGB2 degradation product. 図４Ａはマイクロタイタープレートに固定化した前記ＨＭＧＢ１分解産物等への抗体（２Ｄ４）の反応性を確認した図である。横軸は試料として用いた各抗体溶液中の抗体の濃度、縦軸はＥＬＩＳＡ法で測定したときの吸光度の値を示す。FIG. 4A shows the reactivity of the antibody (2D4) to the HMGB1 degradation product and the like immobilized on a microtiter plate. The horizontal axis represents the concentration of antibody in each antibody solution used as a sample, and the vertical axis represents the absorbance value when measured by ELISA. 図４Ｂはマイクロタイタープレートに固定化した前記ＨＭＧＢ１分解産物等への抗体（４Ｆ１２）の反応性を確認した図である。横軸は試料として用いた各抗体溶液中の抗体の濃度、縦軸はＥＬＩＳＡ法で測定したときの吸光度の値を示す。FIG. 4B is a diagram showing the reactivity of the antibody (4F12) to the HMGB1 degradation product and the like immobilized on a microtiter plate. The horizontal axis represents the concentration of antibody in each antibody solution used as a sample, and the vertical axis represents the absorbance value when measured by ELISA. 図４Ｃはマイクロタイタープレートに固定化した前記ＨＭＧＢ１分解産物等への抗体（８Ｈ４）の反応性を確認した図である。横軸は試料として用いた各抗体溶液中の抗体の濃度、縦軸はＥＬＩＳＡ法で測定したときの吸光度の値を示す。FIG. 4C is a view showing the reactivity of the antibody (8H4) to the HMGB1 degradation product and the like immobilized on a microtiter plate. The horizontal axis represents the concentration of antibody in each antibody solution used as a sample, and the vertical axis represents the absorbance value when measured by ELISA. 図４Ｄはマイクロタイタープレートに固定化した前記ＨＭＧＢ１分解産物等への抗体（２Ｈ６）の反応性を確認した図である。横軸は試料として用いた各抗体溶液中の抗体の濃度、縦軸はＥＬＩＳＡ法で測定したときの吸光度の値を示す。FIG. 4D is a view showing the reactivity of the antibody (2H6) to the HMGB1 degradation product and the like immobilized on a microtiter plate. The horizontal axis represents the concentration of antibody in each antibody solution used as a sample, and the vertical axis represents the absorbance value when measured by ELISA. 図４Ｅはマイクロタイタープレートに固定化した前記ＨＭＧＢ１分解産物等への抗体（５Ｄ１）の反応性を確認した図である。横軸は試料として用いた各抗体溶液中の抗体の濃度、縦軸はＥＬＩＳＡ法で測定したときの吸光度の値を示す。FIG. 4E shows the reactivity of the antibody (5D1) to the HMGB1 degradation product and the like immobilized on a microtiter plate. The horizontal axis represents the concentration of antibody in each antibody solution used as a sample, and the vertical axis represents the absorbance value when measured by ELISA. 図４Ｆはマイクロタイタープレートに固定化した前記ＨＭＧＢ１分解産物等への抗体（２Ａ１０）の反応性を確認した図である。横軸は試料として用いた各抗体溶液中の抗体の濃度、縦軸はＥＬＩＳＡ法で測定したときの吸光度の値を示す。FIG. 4F is a view showing the reactivity of the antibody (2A10) to the HMGB1 degradation product and the like immobilized on a microtiter plate. The horizontal axis represents the concentration of antibody in each antibody solution used as a sample, and the vertical axis represents the absorbance value when measured by ELISA. 図４Ｇはマイクロタイタープレートに固定化した前記ＨＭＧＢ１分解産物等への抗体（６Ｈ３）の反応性を確認した図である。横軸は試料として用いた各抗体溶液中の抗体の濃度、縦軸はＥＬＩＳＡ法で測定したときの吸光度の値を示す。FIG. 4G is a view showing the reactivity of the antibody (6H3) to the HMGB1 degradation product and the like immobilized on a microtiter plate. The horizontal axis represents the concentration of antibody in each antibody solution used as a sample, and the vertical axis represents the absorbance value when measured by ELISA. 図４Ｈはマイクロタイタープレートに固定化した前記ＨＭＧＢ１分解産物等への抗体（ＭＤ７８）の反応性を確認した図である。横軸は試料として用いた各抗体溶液中の抗体の濃度、縦軸はＥＬＩＳＡ法で測定したときの吸光度の値を示す。FIG. 4H is a view showing the reactivity of the antibody (MD78) to the HMGB1 degradation product and the like immobilized on a microtiter plate. The horizontal axis represents the concentration of antibody in each antibody solution used as a sample, and the vertical axis represents the absorbance value when measured by ELISA. 図４Ｉはマイクロタイタープレートに固定化した前記ＨＭＧＢ１分解産物等への抗体（ＭＤ７７）の反応性を確認した図である。横軸は試料として用いた各抗体溶液中の抗体の濃度、縦軸はＥＬＩＳＡ法で測定したときの吸光度の値を示す。FIG. 4I shows the reactivity of the antibody (MD77) to the HMGB1 degradation product and the like immobilized on a microtiter plate. The horizontal axis represents the concentration of antibody in each antibody solution used as a sample, and the vertical axis represents the absorbance value when measured by ELISA. 図４Ｊはマイクロタイタープレートに固定化した前記ＨＭＧＢ１分解産物等への抗体（４Ｃ３）の反応性を確認した図である。横軸は試料として用いた各抗体溶液中の抗体の濃度、縦軸はＥＬＩＳＡ法で測定したときの吸光度の値を示す。FIG. 4J is a view in which the reactivity of the antibody (4C3) to the HMGB1 degradation product and the like immobilized on a microtiter plate is confirmed. The horizontal axis represents the concentration of antibody in each antibody solution used as a sample, and the vertical axis represents the absorbance value when measured by ELISA. 図４Ｋはマイクロタイタープレートに固定化した前記ＨＭＧＢ１分解産物等への抗体（Ｊ２Ｅ１）の反応性を確認した図である。横軸は試料として用いた各抗体溶液中の抗体の濃度、縦軸はＥＬＩＳＡ法で測定したときの吸光度の値を示す。FIG. 4K shows the reactivity of the antibody (J2E1) to the HMGB1 degradation product and the like immobilized on a microtiter plate. The horizontal axis represents the concentration of antibody in each antibody solution used as a sample, and the vertical axis represents the absorbance value when measured by ELISA. 図４Ｌはマイクロタイタープレートに固定化した前記ＨＭＧＢ１分解産物等への抗体（ＨＡＰ４６．５）の反応性を確認した図である。横軸は試料として用いた各抗体溶液中の抗体の濃度、縦軸はＥＬＩＳＡ法で測定したときの吸光度の値を示す。FIG. 4L shows the reactivity of the antibody (HAP46.5) to the HMGB1 degradation product and the like immobilized on a microtiter plate. The horizontal axis represents the concentration of antibody in each antibody solution used as a sample, and the vertical axis represents the absorbance value when measured by ELISA. 図５は、前記ＨＭＧＢ１分解産物に結合する抗体及び他のＨＭＧＢ１に結合する抗体等を用いＥＬＩＳＡ法（サンドイッチ法）により、ＨＭＧＢ１、前記ＨＭＧＢ１分解産物をそれぞれ測定したときの結果を示す図である。横軸の上段は当該測定に使用したＰＯＤ標識抗体液の抗体の産生細胞株を表す文字を示し、横軸の下段は当該測定に使用した抗体固相化マイクロプレートの抗体の産生細胞株を表す文字を示し、縦軸はＥＬＩＳＡ法で測定したときの吸光度の値を示す。FIG. 5 is a diagram showing the results of measuring HMGB1 and the HMGB1 degradation product by ELISA (sandwich method) using an antibody that binds to the HMGB1 degradation product and an antibody that binds to other HMGB1. The upper part of the abscissa indicates the letters indicating the antibody producing cell line of the POD-labeled antibody solution used in the measurement, and the lower part of the abscissa indicates the antibody producing cell line of the antibody-immobilized microplate used for the measurement. Letters are shown, and the vertical axis represents absorbance values measured by ELISA. 図６は従来試薬・方法のＥＬＩＳＡ法（サンドイッチ法）により、ＨＭＧＢ１、前記ＨＭＧＢ１分解産物をそれぞれ測定したときの検量線を示す図である。横軸は試料中のＨＭＧＢ１、又は前記ＨＭＧＢ１分解産物の濃度（０~８０ｎｇ／ｍＬ）、縦軸はＥＬＩＳＡ法で測定したときの吸光度の値を示す。FIG. 6 is a diagram showing calibration curves when HMGB1 and the HMGB1 degradation product are measured by ELISA method (sandwich method) as a conventional reagent / method. The horizontal axis represents the concentration of HMGB1 or the degradation product of HMGB1 in the sample (0 to 80 ng / mL), and the vertical axis represents the absorbance value when measured by the ELISA method. 図７は本発明にしたがってＥＬＩＳＡ法（サンドイッチ法）により、ＨＭＧＢ１、前記ＨＭＧＢ１分解産物をそれぞれ測定したときの検量線を示す図である。横軸は試料中のＨＭＧＢ１、又は前記ＨＭＧＢ１分解産物の濃度（０~８０ｎｇ／ｍＬ）、縦軸はＥＬＩＳＡ法で測定したときの吸光度の値を示す。FIG. 7 is a diagram showing a calibration curve when HMGB1 and the HMGB1 degradation product are measured by ELISA method (sandwich method) according to the present invention. The horizontal axis represents the concentration of HMGB1 or the degradation product of HMGB1 in the sample (0 to 80 ng / mL), and the vertical axis represents the absorbance value when measured by the ELISA method. 図８は本発明にしたがって抗体（ａ）として本抗ＨＭＧＢ１分解産物抗体（２Ｈ６）、抗体（ｂ）として抗ＨＭＧＢ１分解産物等抗体（５Ｄ１）を用いてＥＬＩＳＡ法（サンドイッチ法）により、ＨＭＧＢ１、前記ＨＭＧＢ１分解産物、ＨＭＧＢ２、前記ＨＭＧＢ２分解産物をそれぞれ測定したときの検量線を示す図である。横軸は試料中のＨＭＧＢ１、前記ＨＭＧＢ１分解産物、ＨＭＧＢ２、又は前記ＨＭＧＢ２分解産物の濃度（０~１００ｎｇ／ｍＬ）、縦軸はＥＬＩＳＡ法で測定したときの吸光度の値を示す。FIG. 8 shows the anti-HMGB1 degradation product antibody (2H6) as the antibody (a) according to the present invention and the anti-HMGB1 degradation product antibody (5D1) as the antibody (b) by ELISA (sandwich method). It is a figure which shows a calibration curve when each of HMGB1 degradation product, HMGB2, and the said HMGB2 degradation product is measured. The horizontal axis represents the concentration (0 to 100 ng / mL) of HMGB1, HMGB1 degradation product, HMGB2, or HMGB2 degradation product in the sample, and the vertical axis represents the absorbance value measured by the ELISA method. 図９は本発明にしたがって抗体（ａ）として本抗ＨＭＧＢ１分解産物抗体（２Ｈ６）、抗体（ｂ）として抗ＨＭＧＢ１分解産物等抗体（２Ａ１０）を用いてＥＬＩＳＡ法（サンドイッチ法）により、ＨＭＧＢ１、前記ＨＭＧＢ１分解産物、ＨＭＧＢ２、前記ＨＭＧＢ２分解産物をそれぞれ測定したときの検量線を示す図である。横軸は試料中のＨＭＧＢ１、前記ＨＭＧＢ１分解産物、ＨＭＧＢ２、又は前記ＨＭＧＢ２分解産物の濃度（０~８０ｎｇ／ｍＬ）、縦軸はＥＬＩＳＡ法で測定したときの吸光度差の値を示す。FIG. 9 shows the anti-HMGB1 degradation product antibody (2H6) as the antibody (a) according to the present invention and the anti-HMGB1 degradation product antibody (2A10) as the antibody (b) by ELISA (sandwich method). It is a figure which shows a calibration curve when each of HMGB1 degradation product, HMGB2, and the said HMGB2 degradation product is measured. The horizontal axis represents the concentration (0 to 80 ng / mL) of HMGB1, HMGB1 degradation product, HMGB2, or HMGB2 degradation product in the sample, and the vertical axis represents the value of the absorbance difference when measured by the ELISA method. 図１０は本発明にしたがって抗体（ａ）として本抗ＨＭＧＢ１分解産物抗体（２Ｈ６）、抗体（ｂ）として抗ＨＭＧＢ１分解産物等抗体（６Ｈ３）を用いてＥＬＩＳＡ法（サンドイッチ法）により、ＨＭＧＢ１、前記ＨＭＧＢ１分解産物、ＨＭＧＢ２、前記ＨＭＧＢ２分解産物をそれぞれ測定したときの検量線を示す図である。横軸は試料中のＨＭＧＢ１、前記ＨＭＧＢ１分解産物、ＨＭＧＢ２、又は前記ＨＭＧＢ２分解産物の濃度（０~８０ｎｇ／ｍＬ）、縦軸はＥＬＩＳＡ法で測定したときの吸光度差の値を示す。FIG. 10 shows the anti-HMGB1 degradation product antibody (2H6) as the antibody (a) according to the present invention and the anti-HMGB1 degradation product antibody (6H3) as the antibody (b) by ELISA (sandwich method). It is a figure which shows a calibration curve when each of HMGB1 degradation product, HMGB2, and the said HMGB2 degradation product is measured. The horizontal axis represents the concentration (0 to 80 ng / mL) of HMGB1, HMGB1 degradation product, HMGB2, or HMGB2 degradation product in the sample, and the vertical axis represents the value of the absorbance difference when measured by the ELISA method.
〔Ｉ〕抗ＨＭＧＢ１分解産物抗体
（１）本発明の抗ＨＭＧＢ１分解産物抗体
　本発明の抗体は、「ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物に対する親和性が、ＨＭＧＢ１に対する親和性と比較して、少なくとも１．５倍である、ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物に結合する抗体」である。（なお、この本発明の抗体を、以下、「本抗ＨＭＧＢ１分解産物抗体」ということがある。）
　なお、ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物に対する親和性が、ＨＭＧＢ１に対する親和性と比較して、「少なくとも１．５倍」とは、当該抗体の濃度が０．６２５~５ｎｇ／ｍＬの範囲の少なくともいずれかの濃度（好ましくは当該範囲のすべての濃度）において、後述の「親和性の測定方法」で測定した時の親和性が少なくとも１．５倍であることをいう。
　この本抗ＨＭＧＢ１分解産物抗体としては、ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物に対する親和性が、ＨＭＧＢ２に対する親和性及びＨＭＧＢ２のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物に対する親和性とそれぞれ比較して、各々少なくとも１０倍であるものが好ましい。（抗体濃度：０．６２５~５ｎｇ／ｍＬの範囲の少なくともいずれかの濃度（好ましくは当該範囲のすべての濃度））
　また、この本抗ＨＭＧＢ１分解産物抗体としては、モノクローナル抗体が好ましい。
　なお、本抗ＨＭＧＢ１分解産物抗体は、ポリクローナル抗体、ポリクローナル抗体を含む抗血清、モノクローナル抗体、又はこれらの抗体のフラグメント（Ｆａｂ、Ｆ（ａｂ’）２又はＦａｂ’など）等のいずれのものであってもよい。
（２）ＨＭＧＢ１分解産物
　本発明における「ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物」はトロンビン又はトロンビン・トロンボモジュリン複合体によりヒトＨＭＧＢ１が加水分解されることによって生じたタンパク質又はペプチドであるが、本発明においては、特に、トロンビン又はトロンビン・トロンボモジュリン複合体により、ヒトＨＭＧＢ１の１０番目のアルギニン（Ｒ１０）−１１番目のグリシン（Ｇ１１）が切断され、このＨＭＧＢ１のＮ末端の「ＭＧＫＧＤＰＫＫＰＲ」の１０のアミノ酸残基よりなるペプチドが分離することにより生じる、新たに露呈したＮ末端「ＧＫＭＳＳ・・・・・」を有するＨＭＧＢ１の分解産物のことをいう。
　ヒトＨＭＧＢ１のアミノ酸配列を配列番号１として、また、前記のヒトＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物のアミノ酸配列を配列番号２として、配列表に示した。
　なお、本発明において、前記のヒトＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物としては、前記の配列番号２として示したアミノ酸配列に１ないし数個のアミノ酸残基の欠失、置換、挿入、付加若しくは修飾を施すことにより得られるアミノ酸配列よりなるタンパク質、あるいはこのタンパク質に糖類又は脂質などが結合したものであってもよい。
　なお、前記のアミノ酸残基の欠失、置換、挿入、付加又は修飾におけるアミノ酸残基数は、通常１~４個、好ましくは１~３個、更に好ましくは１~２個、特に好ましくは１個である。
　また、本発明において、前記のトロンビン又はトロンビン・トロンボモジュリン複合体としては、ヒトの体内に存在するトロンビン又はトロンビン・トロンボモジュリン複合体はもちろんのこと、遺伝子組換え体などの人工的に調製されたもの又は人工的に単離されたもの等も含むものである。
（なお、以上述べた「ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物」を、以下、「前記ＨＭＧＢ１分解産物」ということがある。）
（３）　親和性の測定方法
　本抗ＨＭＧＢ１分解産物抗体は、前記の通り、前記ＨＭＧＢ１分解産物に対する親和性が、ＨＭＧＢ１に対する親和性と比較して、少なくとも１．５倍であるものである。
　この抗体の親和性の測定方法は、特に限定されるものではないが、例えば、次のように行うことができる。
〔Ａ〕測定試薬の調製
（ｉ）ＨＭＧＢ１分解産物等固相化マイクロプレート
　ＨＭＧＢ１、前記ＨＭＧＢ１分解産物、ＨＭＧＢ２、及びＨＭＧＢ２のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物（以下、「前記ＨＭＧＢ２分解産物」ということがある。）のそれぞれをリン酸緩衝生理食塩水により１μｇ／ｍＬの濃度となるように調製したものを、各々９６穴マイクロタイタープレート〔Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ　Ｉｎｃ．社（米国・イリノイ州）〕のウェルに１００μＬ注入し、２５℃で１８時間静置し、前記のＨＭＧＢ１、前記ＨＭＧＢ１分解産物、ＨＭＧＢ２、及び前記ＨＭＧＢ２分解産物のそれぞれを、前記マイクロタイタープレートのウェルに固相化した。
　次に、このマイクロタイタープレートのウェル中の液を除去した後、０．５％カゼイン及び０．１％アジ化ナトリウムを含むトリス緩衝生理食塩水（ＴＢＳ）〔ｐＨ８．０〕の２５０μＬずつを各ウェルに分注し、ブロッキング処理を行った。
　この後、各ウェルの上をプレートシールで封をし、蒸発しないようにして、使用時まで冷蔵保存した。
　これを、ＨＭＧＢ１分解産物等固相化マイクロプレートとした。
（ｉｉ）ＰＯＤ標識抗マウスＩｇＧ抗体液
　ＰＯＤ標識抗マウスＩｇＧ抗体〔ＤａｋｏＣｙｔｏｍａｔｉｏｎ社（デンマーク国）〕を、０．５％カゼインナトリウム、及び１００ｍＭ塩化ナトリウムを含む５０ｍＭトリス−塩酸緩衝液（ｐＨ８．０）によって１０００倍希釈した。
　これをＰＯＤ標識抗マウスＩｇＧ抗体液とした。
（ｉｉｉ）洗浄液
　０．０５％のＴｗｅｅｎ２０を含むリン酸緩衝生理食塩水を、洗浄液とした。
（ｉｖ）希釈液
　０．５％カゼインナトリウム、１００ｍＭ塩化ナトリウム及び０．１％アジ化ナトリウムを含む５０ｍＭトリス−塩酸緩衝液（ｐＨ８．０）を、希釈液とした。
（ｖ）発色液
　０．２ｍＭのＥＤＴＡ・２ナトリウムを含む０．０４５％の３，３’，５，５’−テトラメチルベンジジン塩酸塩水溶液（ｐＨ２．０）を、発色液とした。
（ｖｉ）基質液
　５ｍＭ過酸化水素、４１ｍＭクエン酸、０．２ｍＭのＥＤＴＡ・２ナトリウムを含む６０ｍＭリン酸二ナトリウム水溶液（ｐＨ４．３）を、基質液とした。
（ｖｉｉ）発色基質
　前記の発色液と基質液を使用前に室温に戻した上で、使用時に等量混合し、発色基質とした。
（ｖｉｉｉ）反応停止液
　０．７Ｎ硫酸を、反応停止液とした。
〔Ｂ〕試料
　ＨＭＧＢ１、前記ＨＭＧＢ１分解産物、ＨＭＧＢ２、及び前記ＨＭＧＢ２分解産物のそれぞれとの親和性を測定しようとする抗体を、その濃度が各々、０．６２５ｎｇ／ｍＬ、１．２５ｎｇ／ｍＬ、２．５ｎｇ／ｍＬ、及び５ｎｇ／ｍＬとなるように前記〔Ａ〕の（ｉｖ）の希釈液により希釈して、前記４濃度の抗体溶液をそれぞれ調製した。
〔Ｃ〕測定
（ｉ）　前記〔Ａ〕の（ｉ）のＨＭＧＢ１分解産物等固相化マイクロプレートの各ウェルを、前記〔Ａ〕の（ｉｉｉ）の洗浄液の４００μＬで３回洗浄した。
（ｉｉ）　次に、前記〔Ｂ〕の４濃度の抗体溶液それぞれの１００μＬを試料として各ウェルに分注した後、２５℃で２時間静置し、これらの各濃度の抗体溶液に含まれていた抗体を、このＨＭＧＢ１分解産物等固相化マイクロプレートのウェルに固相化された前記のＨＭＧＢ１、前記ＨＭＧＢ１分解産物、ＨＭＧＢ２、及び前記ＨＭＧＢ２分解産物の各々と、それぞれ抗原抗体反応を行わせた。
（ｉｉｉ）　次に、各ウェルを、前記〔Ａ〕の（ｉｉｉ）の洗浄液の４００μＬで３回洗浄した。
（ｉｖ）　次に、前記〔Ａ〕の（ｉｉ）のＰＯＤ標識抗マウスＩｇＧ抗体液の１００μＬを各ウェルに分注し、２５℃で１時間静置し、それぞれ抗原抗体反応を行わせた。
（ｖ）　次に、各ウェルを、前記〔Ａ〕の（ｉｉｉ）の洗浄液の４００μＬで３回洗浄した。
（ｖｉ）　次に、各ウェルに、前記〔Ａ〕の（ｖｉｉ）の発色基質を１００μＬ分注し、室温で２０分間静置し、標識酵素であるパーオキシダーゼ（ＰＯＤ）による発色反応を行わせた。
（ｖｉｉ）　次に、各ウェルに、前記〔Ａ〕の（ｖｉｉｉ）の反応停止液を１００μＬ分注し、発色反応を停止させた。
（ｖｉｉｉ）　次に、各ウェルの液の吸光度（主波長：４５０ｎｍ、副波長：５５０ｎｍ）を測定し、前記の４濃度の抗体溶液それぞれを試料として測定した場合の吸光度を得た。
　そして、この吸光度の値を前記の親和性を測定しようとする抗体の前記のＨＭＧＢ１、前記ＨＭＧＢ１分解産物、ＨＭＧＢ２、及び前記ＨＭＧＢ２分解産物のそれぞれとの親和性を示す値とした。
　例えば、親和性を測定しようとする抗体の溶液（例えば、２．５ｎｇ／ｍＬの濃度）を試料として、前記の通り測定を行ったときに、前記ＨＭＧＢ１分解産物を固相化したウェルで得られた前記吸光度差の値が１．３５であり、前記のＨＭＧＢ１を固相化したウェルで得られた前記吸光度差の値が０．９であった場合には、この抗体は、前記ＨＭＧＢ１分解産物に対する親和性が、ＨＭＧＢ１に対する親和性と比較して、少なくとも１．５倍である、といえる。
　すなわち、この場合、この抗体は、本抗ＨＭＧＢ１分解産物抗体である。
（４）免疫原
　本抗ＨＭＧＢ１分解産物抗体を取得するための免疫原について、以下説明を行う。
　本抗ＨＭＧＢ１分解産物抗体を取得するための免疫原としては、例えば、ヒトＨＭＧＢ１、ヒトＨＭＧＢ１のアミノ酸配列との相同性が高い動物（例えば、ウシ又はブタ等）のＨＭＧＢ１、又は前記ＨＭＧＢ１分解産物の全体又は一部等を挙げることができる。
　なお、前記ＨＭＧＢ１分解産物の一部としては、例えば、この前記ＨＭＧＢ１分解産物のＮ末端の「ＧＫＭＳＳＹＡＦＦＶＱＴＣＲＥＥＨＫＫＫ」の２０のアミノ酸残基よりなるペプチド等を挙げることができる。
　また、本抗ＨＭＧＢ１分解産物抗体を取得するための免疫原としては、これまで述べた免疫原としてのタンパク質又はペプチドについて、１ないし数個のアミノ酸残基の欠失、置換、挿入、付加又は修飾を施すことにより得られるタンパク質又はペプチドであってもよい。
　なお、前記のアミノ酸残基の欠失、置換、挿入、付加又は修飾におけるアミノ酸残基数は、通常１~４個、好ましくは１~３個、更に好ましくは１~２個、特に好ましくは１個である。
　また、本抗ＨＭＧＢ１分解産物抗体を取得するために動物等に免疫するものとしては、これまで述べた免疫原としてのタンパク質又はペプチドについて、担体（キャリア）を結合させたものであってもよい。
　特に、前記の免疫原としてのタンパク質又はペプチドが低分子量のものである場合には、担体との結合により抗体産生能が向上するので、担体を結合させたものを免疫原として用いることが好ましい。
　なお、この担体としては、例えば、スカシガイのヘモシアニン（ＫＬＨ）、ウシ血清アルブミン（ＢＳＡ）、ヒト血清アルブミン（ＨＳＡ）、ニワトリ血清アルブミン、ポリ−Ｌ−リシン、ポリアラニルリシン、ジパルミチルリシン、破傷風トキソイド又は多糖類等の担体として公知なものを用いることができる。
（５）本抗ＨＭＧＢ１分解産物抗体の免疫原等の取得方法
　本抗ＨＭＧＢ１分解産物抗体を取得するために動物等に免疫する免疫原等の取得方法について、以下説明を行う。
　本抗ＨＭＧＢ１分解産物抗体を取得するための免疫原であるが、前記（４）に記載したヒトＨＭＧＢ１、又はヒトＨＭＧＢ１のアミノ酸配列との相同性が高い動物のＨＭＧＢ１は、例えば、ヒト又はヒトのＨＭＧＢ１のアミノ酸配列と相同性が高いヒト以外の哺乳類動物（ブタ、ウシ、ウサギ、ヤギ、ヒツジ、マウス又はラットなど）等の体液、細胞、組織又は臓器等より、公知の方法等により抽出、精製等して、取得することができる。
　また、前記ＨＭＧＢ１分解産物は、例えば、ヒトＨＭＧＢ１、又はヒトＨＭＧＢ１のアミノ酸配列との相同性が高い動物（例えば、ウシ又はブタ等）のＨＭＧＢ１を、前記のトロンビン又はトロンビン・トロンボモジュリン複合体に接触させて、加水分解し、そして、これについて、公知の方法等により抽出、精製等して、取得することができる。
　また、前記の免疫原は、液相法及び固相法等のペプチド合成の方法により合成することができ、更にペプチド自動合成装置を用いてもよい。
　例えば、日本生化学会編「生化学実験講座１　タンパク質の化学ＩＶ」，東京化学同人，１９７５年；泉屋ら「ペプチド合成の基礎と実験」，丸善，１９８５年；又は日本生化学会編「続生化学実験講座２　タンパク質の化学　下」，東京化学同人，１９８７年等に記載された方法に従い合成することができる。
　更に、前記の免疫原は、対応する核酸塩基配列を持つＤＮＡ又はＲＮＡより遺伝子工学技術を用いて調製してもよく、日本生化学会編「続生化学実験講座１　遺伝子研究法Ｉ」，東京化学同人，１９８６年；日本生化学会編「続生化学実験講座１　遺伝子研究法ＩＩ」，東京化学同人，１９８６年；又は日本生化学会編「続生化学実験講座１　遺伝子研究法ＩＩＩ」，東京化学同人，１９８７年等を参照して調製すればよい。
　例えば、前記の配列番号１で示されるヒトＨＭＧＢ１のアミノ酸配列、若しくは前記の配列番号２で示される前記ＨＭＧＢ１分解産物のアミノ酸配列、又はこれらのアミノ酸配列の一部に対応する遺伝子をクローニングし、得られた遺伝子をプラスミド等の発現ベクターへ組み込む。
　次に、この発現ベクターを大腸菌等の宿主細胞に導入し、得られた形質転換体を培養することにより、前記のアミノ酸配列又はその一部よりなるタンパク質又はペプチドを発現させることができる。
　なお、遺伝子の塩基配列をクローニングする方法としては、例えば、ＰＣＲ法、リコンビナントＰＣＲ法、ライゲーション法、又はリンカーライゲーション法等を挙げることができる。
　ところで、免疫原が低分子物質の場合には、免疫原に担体（キャリア）を結合させたものを動物等に免疫するのが一般的ではあるが、アミノ酸数５のペプチドを免疫原としてこれに対する特異抗体を産生させたとの報告（木山ら，「日本薬学会第１１２回年会講演要旨集３」，１２２頁，１９９２年発行）もあるので、担体を使用することは必須ではない。
　なお、前記のタンパク質又はペプチドに担体を結合させたものを動物等に免疫する場合の担体としては、スカシガイのヘモシアニン（ＫＬＨ）、ウシ血清アルブミン（ＢＳＡ）、ヒト血清アルブミン（ＨＳＡ）、ニワトリ血清アルブミン、ポリ−Ｌ−リシン、ポリアラニルリシン、ジパルミチルリシン、破傷風トキソイド又は多糖類等の担体として公知なものを用いることができる。
　前記のタンパク質又はペプチドと担体との結合法は、グルタルアルデヒド法、１−エチル−３−（３−ジメチルアミノプロピル）カルボジイミド法、マレイミドベンゾイル−Ｎ−ヒドロキシサクシニミドエステル法、ビスジアゾ化ベンジジン法又はＮ−サクシミジル−３−（２−ピリジルジチオ）プロピオン酸法等の公知の結合法を用いることができる。
　また、ニトロセルロース粒子、ポリビニルピロリドン又はリポソーム等の担体に前記のタンパク質又はペプチドを吸着させたものも、本抗ＨＭＧＢ１分解産物抗体の取得のため、前記の動物等への免疫に用いることができる。
（６）本抗ＨＭＧＢ１分解産物抗体（ポリクローナル抗体）の取得方法
　本抗ＨＭＧＢ１分解産物抗体において、ポリクローナル抗体又は抗血清は、以下の操作により取得することができる。
（ｉ）　まず、前記の免疫原、又は前記の免疫原と担体の結合物を哺乳動物（マウス、ウサギ、ラット、ヒツジ、ヤギ、ウマ等）又は鳥類（ニワトリ等）等に免疫する。
　この前記の免疫原、又は前記の免疫原と担体の結合物の免疫量は、免疫動物の種類、免疫注射部位等により決められるものであるが、例えば、ウサギの場合にはウサギ一匹当り一回につき１０μｇ~数十ｍｇの前記免疫原、又は前記免疫原と担体の結合物を免疫注射する。
　なお、この前記の免疫原、又は前記の免疫原と担体の結合物は、アジュバントと添加混合して免疫注射することが好ましい。
　アジュバントとしては、フロイント完全アジュバント、フロイント不完全アジュバント、水酸化アルミニウムアジュバント又は百日咳菌アジュバント等の公知のものを用いることができる。
　免疫注射は、皮下、静脈内、腹腔内又は背部等の部位に行えばよい。
　初回免疫後、２~３週間間隔で皮下、静脈内、腹腔内又は背部等の部位に、前記の免疫原、又は前記の免疫原と担体の結合物を追加免疫注射する。この場合も、前記の免疫原、又は前記の免疫原と担体の結合物は、アジュバントを添加混合して追加免疫注射することが好ましい。
　初回免疫の後、免疫動物の血清中の抗体価の測定をＥＬＩＳＡ法等により繰り返し行い、抗体価がプラトーに達したら全採血を行い、血清を分離して本抗ＨＭＧＢ１分解産物抗体を含む抗血清を得る。
　この抗血清を、硫酸アンモニウム、硫酸ナトリウム等による塩析法、イオン交換クロマトグラフィー、ゲル濾過法又はアフィニティークロマトグラフィー等の方法、あるいはこれらの方法を組み合わせて抗体の精製を行い、ポリクローナル抗体を得る。
（ｉｉ）　ここで得られたポリクローナル抗体は、前記ＨＭＧＢ１分解産物に対する親和性がＨＭＧＢ１に対する親和性と比較して少なくとも１．５倍である抗体（本抗ＨＭＧＢ１分解産物抗体）と、前記ＨＭＧＢ１分解産物に対する親和性がＨＭＧＢ１に対する親和性と比較して１．５倍未満である抗体（従来の抗体）の両方を含むものであるので、更に、これをＨＭＧＢ１をリガンドとして固相に固定化したアフィニティークロマトグラフィーのカラムに通し接触させる。
　前記の従来の抗体は、このカラムのリガンド（ＨＭＧＢ１）を介して固相に結合し、捕集される。
　これに対して、本抗ＨＭＧＢ１分解産物抗体は、このカラムのリガンド（ＨＭＧＢ１）に結合し難く、このカラムを素通りするものが多いので、素通りした画分を得ることにより、本抗ＨＭＧＢ１分解産物抗体を取得することができる。
　なお、これを、前記ＨＭＧＢ１分解産物をリガンドとして固相に固定化したアフィニティークロマトグラフィーのカラムに通し接触させ、本抗ＨＭＧＢ１分解産物抗体をこのカラムのリガンド（前記ＨＭＧＢ１分解産物）を介して固相に結合させるとともに、他の抗体等はこのカラムを素通りさせて分離し、その後、この固相に結合させていた本抗ＨＭＧＢ１分解産物抗体をリガンド（前記ＨＭＧＢ１分解産物）から分離し、このカラムから流出する画分を得ることにより、更に純度の高い本抗ＨＭＧＢ１分解産物抗体を取得することができる。
（ｉｉｉ）　なお、免疫原と担体の結合物を用いて動物等に免疫した場合には、得られた抗血清又はポリクローナル抗体中に、この担体に対する抗体が存在するので、このような担体に対する抗体の除去処理を行うことが好ましい。
　この除去処理方法としては、担体を、得られたポリクローナル抗体又は抗血清の溶液中に添加して生成した凝集物を取り除くか、担体を不溶化固相に固定化してアフィニティークロマトグラフィーにより除去する方法等を用いることができる。
（７）本抗ＨＭＧＢ１分解産物抗体（モノクローナル抗体）の取得方法
　本抗ＨＭＧＢ１分解産物抗体において、モノクローナル抗体は、以下の操作により取得することができる。
　モノクローナル抗体は、ケラーらの細胞融合法（Ｇ．Ｋｏｅｈｌｅｒら，Ｎａｔｕｒｅ，２５６巻，４９５~４９７頁，１９７５年発行）によるハイブリドーマ、又はエプスタン−バーウイルス等のウイルスによる腫瘍化細胞等の抗体産生細胞により得ることができる。
　細胞融合法によるモノクローナル抗体の調製は、以下の操作により行うことができる。
　まず、前記の免疫原、又は前記の免疫原と担体の結合物を、哺乳動物（マウス、ヌードマウス、ラットなど、例えば近交系マウスのＢＡＬＢ／ｃ）又は鳥類（ニワトリなど）等に免疫する。
　この前記の免疫原、又は前記の免疫原と担体の結合物の免疫量は、免疫動物の種類、免疫注射部位等により適宜決められるものであるが、例えば、マウスの場合には一匹当り一回につき０．１μｇ~５ｍｇの前記の免疫原、又は前記の免疫原と担体の結合物を免疫注射するのが好ましい。
　なお、前記の免疫原、又は前記の免疫原と担体の結合物は、アジュバントを添加混合して免疫注射することが好ましい。
　アジュバントとしては、フロイント完全アジュバント、フロイント不完全アジュバント、水酸化アルミニウムアジュバント又は百日咳菌アジュバント等の公知なものを用いることができる。
　免疫注射は、皮下、静脈内、腹腔内又は背部等の部位に行えばよい。
　初回免疫後、１~２週間間隔で皮下、静脈内、腹腔内又は背部等の部位に、前記の免疫原、又は前記の免疫原と担体の結合物を追加免疫注射する。
　この追加免疫注射の回数としては２~６回が一般的である。
　この場合も前記の免疫原、又は前記の免疫原と担体の結合物は、アジュバントを添加混合して追加免疫注射することが好ましい。
　初回免疫の後、免疫動物の血清中の抗体価の測定をＥＬＩＳＡ法等により繰り返し行い、抗体価がプラトーに達したら、前記の免疫原、又は前記の免疫原と担体の結合物を生理食塩水（０．９％塩化ナトリウム水溶液）に溶解したものを静脈内又は腹腔内に注射し、最終免疫とする。
　この最終免疫の３~５日後に、免疫動物の脾細胞、リンパ節細胞又は末梢リンパ球等の抗体産生能を有する細胞を取得する。
　この免疫動物より得られた抗体産生能を有する細胞と哺乳動物等（マウス、ヌードマウス、ラットなど）の骨髄腫細胞（ミエローマ細胞）とを細胞融合させるのであるが、ミエローマ細胞としてはヒポキサンチン・グアニン・ホスホリボシル・トランスフェラーゼ（ＨＧＰＲＴ）又はチミジンキナーゼ（ＴＫ）等の酵素を欠損した細胞株のものが好ましく、例えば、ＢＡＬＢ／ｃマウス由来のＨＧＰＲＴ欠損細胞株である、Ｐ３−Ｘ６３−Ａｇ８株（ＡＴＣＣ　ＴＩＢ９）、Ｐ３−Ｘ６３−Ａｇ８−Ｕ１株（癌研究リサーチソースバンク（ＪＣＲＢ）９０８５）、Ｐ３−ＮＳ１−１−Ａｇ４−１株（ＪＣＲＢ　０００９）、Ｐ３−Ｘ６３−Ａｇ８・６５３株（ＪＣＲＢ　００２８）又はＳＰ２／Ｏ−Ａｇ−１４株（ＪＣＲＢ　００２９）等を用いることができる。
　細胞融合は、各種分子量のポリエチレングリコール（ＰＥＧ）、リポソームもしくはセンダイウイルス（ＨＶＪ）等の融合促進剤を用いて行うか、又は電気融合法により行うことができる。
　ミエローマ細胞がＨＧＰＲＴ欠損株又はＴＫ欠損株のものである場合には、ヒポキサンチン・アミノプテリン・チミジンを含む選別用培地（ＨＡＴ培地）を用いることにより、抗体産生能を有する細胞とミエローマ細胞の融合細胞（ハイブリドーマ）のみを選択的に培養し、増殖させることができる。
　このようにして得られたハイブリドーマの培養上清を、前記の免疫原、前記の免疫原と担体の結合物、又は前記ＨＭＧＢ１分解産物等を用いてＥＬＩＳＡ法やウエスタンブロット法等の免疫学的測定法により測定することにより、本抗ＨＭＧＢ１分解産物抗体を産生するハイブリドーマを選択することができる。
　また、前記のハイブリドーマの培養上清を、ＨＭＧＢ１等を用いてＥＬＩＳＡ法やウエスタンブロット法等の免疫学的測定法により測定することにより、ＨＭＧＢ１等には結合しない抗体を産生するハイブリドーマを選択することができる。
　この２種類のハイブリドーマ選択方法と限界希釈法等の公知のクローニングの方法を組み合わせて行うことにより、本抗ＨＭＧＢ１分解産物抗体（モノクローナル抗体）、即ち「ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物に対する親和性が、ＨＭＧＢ１に対する親和性と比較して、少なくとも１．５倍である、ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物に結合する抗体」（モノクローナル抗体）の産生細胞株を単離して得ることができる。
　このモノクローナル抗体産生細胞株を適当な培地で培養して、その培養上清から本抗ＨＭＧＢ１分解産物抗体（モノクローナル抗体）を得ることができるが、培地としては無血清培地又は低濃度血清培地等を用いてもよく、この場合は抗体の精製が容易となる点で好ましく、ＤＭＥＭ培地、ＲＰＭＩ１６４０培地又はＡＳＦ培地１０３等の培地を用いることができる。
　また、モノクローナル抗体産生細胞株を、これに適合性がありプリスタン等であらかじめ刺激した哺乳動物の腹腔内に注入し、一定期間の後、腹腔にたまった腹水より本抗ＨＭＧＢ１分解産物抗体（モノクローナル抗体）を得ることもできる。
　このようにして得られたモノクローナル抗体は、硫酸アンモニウム、硫酸ナトリウムなどによる塩析法、イオン交換クロマトグラフィー、ゲル濾過法又はアフィニティークロマトグラフィーなどの方法、あるいはこれらの方法を組み合わせること等により、精製された本抗ＨＭＧＢ１分解産物抗体（モノクローナル抗体）を得ることができる。
〔ＩＩ〕試料に含まれるＨＭＧＢ１分解産物の免疫学的測定方法
（１）総論
　本発明の試料に含まれるＨＭＧＢ１分解産物の免疫学的測定方法（以下、「本発明の免疫学的測定方法」又は「本発明の測定方法」ということがある。）は、次の（ａ）の抗体及び（ｂ）の抗体を使用する測定方法である。
　（ａ）　本抗ＨＭＧＢ１分解産物抗体。
　（ｂ）　前記ＨＭＧＢ１分解産物に結合する抗体であって、前記ＨＭＧＢ１分解産物に対する親和性が高い抗体。
　前記の（ｂ）の抗体としては、当該抗体の濃度が０．６２５~２．５ｎｇ／ｍＬの範囲の少なくともいずれかの濃度（好ましくは当該範囲のすべての濃度）において、ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物を固相化したＥＬＩＳＡ法で、
（イ）当該分解産物に結合する当該抗体の量を測定するときに得られる吸光度の値を当該抗体の濃度の値で除したときの値が０．５以上となる抗体、又は
（ロ）当該分解産物に結合する当該抗体の量を測定するときに得られる吸光度の値が、基準とする抗体産生細胞であるハイブリドーマＭＤ７８（ＦＥＲＭ　Ｐ−１８４０５）より産生される抗体における当該吸光度の値の６倍以上となる抗体
が好ましい。
　前記の（ａ）の抗体（本抗ＨＭＧＢ１分解産物抗体）としては、ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物に対する親和性が、ＨＭＧＢ２に対する親和性及びＨＭＧＢ２のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物に対する親和性とそれぞれ比較して、各々少なくとも１０倍であるものが好ましい。（抗体濃度：０．６２５~５ｎｇ／ｍＬの範囲の少なくともいずれかの濃度（好ましくは当該範囲のすべての濃度））
　また、前記の（ａ）の抗体（本抗ＨＭＧＢ１分解産物抗体）、及び／又は前記の（ｂ）の抗体が、モノクローナル抗体であることが好ましい。
　なお、前記の（ａ）の抗体（本抗ＨＭＧＢ１分解産物抗体）、及び／又は前記の（ｂ）の抗体は、ポリクローナル抗体、ポリクローナル抗体を含む抗血清、モノクローナル抗体、又はこれらの抗体のフラグメント（Ｆａｂ、Ｆ（ａｂ’）２又はＦａｂ’など）等のいずれのものであってもよい。
　また、本発明の測定方法は、固相化抗体及び標識抗体を用いるものであって、前記の（ａ）の抗体及び前記の（ｂ）の抗体のうち、いずれか一方の抗体が固相化抗体として使用され、他の一方の抗体が標識抗体として使用されるものであってもよい。
　本発明の測定方法は、前記の（ａ）の抗体及び前記の（ｂ）の抗体を使用することにより、前記ＨＭＧＢ１分解産物に対する特異性が高く、前記ＨＭＧＢ１分解産物のみを正確に定量測定することができる。
　なお、前記の（ａ）の抗体は、前記の通りの抗体であれば特に制限なく使用することができる。
　また、前記の（ｂ）の抗体は、前記の通りの抗体であれば特に制限なく使用することができる。
　なお、前記の（ａ）の抗体及び前記の（ｂ）の抗体はそれぞれ、１種類のものだけではなく、複数種類のものを同時に使用してもよい。
（２）前記の（ａ）の抗体
　前記の（ａ）の抗体、すなわち本抗ＨＭＧＢ１分解産物抗体の詳細については、前記の「〔Ｉ〕抗ＨＭＧＢ１分解産物抗体」の項などに記載した通りである。
（３）前記の（ｂ）の抗体
（ｉ）　前記の（ｂ）の抗体は、「前記ＨＭＧＢ１分解産物に結合する抗体であって、前記ＨＭＧＢ１分解産物に対する親和性が高い抗体」である。
　なお、前記の（ｂ）の抗体としては、当該抗体の濃度が０．６２５~２．５ｎｇ／ｍＬの範囲の少なくともいずれかの濃度（好ましくは当該範囲のすべての濃度）において、ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物を固相化したＥＬＩＳＡ法で、
（イ）当該分解産物に結合する当該抗体の量を測定するときに得られる吸光度の値を当該抗体の濃度の値で除したときの値が０．５以上となる抗体、又は
（ロ）当該分解産物に結合する当該抗体の量を測定するときに得られる吸光度の値が、基準とする抗体産生細胞であるハイブリドーマＭＤ７８（ＦＥＲＭ　Ｐ−１８４０５）より産生される抗体における当該吸光度の値の６倍以上となる抗体
が好ましい。
（ｉｉ）　親和性の測定方法
　前記の（ｂ）の抗体は、前記の通り、前記ＨＭＧＢ１分解産物に結合する抗体であって、前記ＨＭＧＢ１分解産物に対する親和性が高い抗体である。
　なお、前記の（ｂ）の抗体としては、当該抗体の濃度が０．６２５~２．５ｎｇ／ｍＬの範囲の少なくともいずれかの濃度（好ましくは当該範囲のすべての濃度）において、ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物を固相化したＥＬＩＳＡ法で、
（イ）当該分解産物に結合する当該抗体の量を測定するときに得られる吸光度の値を当該抗体の濃度の値で除したときの値が０．５以上となる抗体、又は
（ロ）当該分解産物に結合する当該抗体の量を測定するときに得られる吸光度の値が、基準とする抗体産生細胞であるハイブリドーマＭＤ７８（ＦＥＲＭ　Ｐ−１８４０５）より産生される抗体における当該吸光度の値の６倍以上となる抗体
が好ましい。
（ｉｉ−１）　前記抗体（イ）における吸光度の測定方法
　前記抗体（イ）における吸光度の測定方法は、特に限定されるものではないが、例えば、次のように行うことができる。〔本発明における「抗体の親和性の測定方法」と同じ。〕
〔Ａ〕測定試薬の調製
〔ｉ〕ＨＭＧＢ１分解産物固相化マイクロプレート
　前記ＨＭＧＢ１分解産物をリン酸緩衝生理食塩水により１μｇ／ｍＬの濃度となるように調製したものを、各々９６穴マイクロタイタープレート〔Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ　Ｉｎｃ．社（米国・イリノイ州）〕のウェルに１００μＬ注入し、２５℃で１８時間静置し、前記ＨＭＧＢ１分解産物を、前記マイクロタイタープレートのウェルに固相化した。
　次に、このマイクロタイタープレートのウェル中の液を除去した後、０．５％カゼイン及び０．１％アジ化ナトリウムを含むトリス緩衝生理食塩水（ＴＢＳ）〔ｐＨ８．０〕の２５０μＬずつを各ウェルに分注し、ブロッキング処理を行った。この後、各ウェルの上をプレートシールで封をし、蒸発しないようにして、使用時まで冷蔵保存した。これを、ＨＭＧＢ１分解産物固相化マイクロプレートとした。
〔ｉｉ〕ＰＯＤ標識抗マウスＩｇＧ抗体液
　ＰＯＤ標識抗マウスＩｇＧ抗体〔ＤａｋｏＣｙｔｏｍａｔｉｏｎ社（デンマーク国）〕を、０．５％カゼインナトリウム、及び１００ｍＭ塩化ナトリウムを含む５０ｍＭトリス−塩酸緩衝液（ｐＨ８．０）によって１０００倍希釈した。これをＰＯＤ標識抗マウスＩｇＧ抗体液とした。
〔ｉｉｉ〕洗浄液
　０．０５％のＴｗｅｅｎ２０を含むリン酸緩衝生理食塩水を、洗浄液とした。
〔ｉｖ〕希釈液
　０．５％カゼインナトリウム、１００ｍＭ塩化ナトリウム及び０．１％アジ化ナトリウムを含む５０ｍＭトリス−塩酸緩衝液（ｐＨ８．０）を、希釈液とした。
〔ｖ〕発色液
　０．２ｍＭのＥＤＴＡ・２ナトリウムを含む０．０４５％の３，３’，５，５’−テトラメチルベンジジン塩酸塩水溶液（ｐＨ２．０）を、発色液とした。
〔ｖｉ〕基質液
　５ｍＭ過酸化水素、４１ｍＭクエン酸、０．２ｍＭのＥＤＴＡ・２ナトリウムを含む６０ｍＭリン酸二ナトリウム水溶液（ｐＨ４．３）を、基質液とした。
〔ｖｉｉ〕発色基質
　前記の発色液と基質液を使用前に室温に戻した上で、使用時に等量混合し、発色基質とした。
〔ｖｉｉｉ〕反応停止液
　０．７Ｎ硫酸を、反応停止液とした。
〔Ｂ〕試料
　前記抗体（ｂ）のそれぞれを、その濃度が各々、０．６２５ｎｇ／ｍＬ、１．２５ｎｇ／ｍＬ、及び／又は２．５ｎｇ／ｍＬとなるように前記〔Ａ〕の〔ｉｖ〕の希釈液により希釈して、前記３濃度の抗体溶液をそれぞれ調製した。
〔Ｃ〕測定
〔ｉ〕　前記〔Ａ〕の〔ｉ〕のＨＭＧＢ１分解産物固相化マイクロプレートの各ウェルを、前記〔Ａ〕の〔ｉｉｉ〕の洗浄液の４００μＬで３回洗浄した。
〔ｉｉ〕　次に、前記〔Ｂ〕の３濃度の抗体溶液それぞれの１００μＬを試料として各ウェルに分注した後、２５℃で２時間静置し、これらの各濃度の抗体溶液に含まれていた前記の抗体（ｂ）を、このＨＭＧＢ１分解産物固相化マイクロプレートのウェルに固相化された前記ＨＭＧＢ１分解産物と、それぞれ抗原抗体反応を行わせた。
〔ｉｉｉ〕　次に、各ウェルを、前記〔Ａ〕の〔ｉｉｉ〕の洗浄液の４００μＬで３回洗浄した。
〔ｉｖ〕　次に、前記〔Ａ〕の〔ｉｉ〕のＰＯＤ標識抗マウスＩｇＧ抗体液の１００μＬを各ウェルに分注し、２５℃で１時間静置し、それぞれ抗原抗体反応を行わせた。
〔ｖ〕　次に、各ウェルを、前記〔Ａ〕の〔ｉｉｉ〕の洗浄液の４００μＬで３回洗浄した。
〔ｖｉ〕　次に、各ウェルに、前記〔Ａ〕の〔ｖｉｉ〕の発色基質を１００μＬ分注し、室温で２０分間静置し、標識酵素であるパーオキシダーゼ（ＰＯＤ）による発色反応を行わせた。
〔ｖｉｉ〕　次に、各ウェルに、前記〔Ａ〕の〔ｖｉｉｉ〕の反応停止液を１００μＬ分注し、発色反応を停止させた。
〔ｖｉｉｉ〕　次に、各ウェルの液の吸光度（主波長：４５０ｎｍ、副波長：５５０ｎｍ）を測定し、前記の３濃度の前記の抗体（ｂ）の抗体溶液それぞれを試料として測定した場合の吸光度を得た。
　そして、この吸光度の値を前記の抗体（ｂ）の前記ＨＭＧＢ１分解産物との親和性を示す値とした。
　例えば、親和性を測定しようとする抗体の溶液（例えば、２．５ｎｇ／ｍＬの濃度）を試料として、前記の通り測定を行ったときに、前記ＨＭＧＢ１分解産物を固相化したウェルで得られた前記吸光度差の値が１．２５であった場合には、１．２５÷２．５ｎｇ／ｍＬ＝０．５（ｎｇ／ｍＬ）−１となり、この抗体は、『当該抗体の濃度が０．６２５~２．５ｎｇ／ｍＬの範囲において、ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物を固相化したＥＬＩＳＡ法で、当該分解産物に結合する当該抗体の量を測定するときに得られる吸光度の値を当該抗体の濃度の値で除したときの値が０．５以上となる抗体である』、といえる。
　すなわち、この場合、この抗体は、前記の（ｂ）の抗体である。
（ｉｉ−２）　前記抗体（ロ）における吸光度の測定方法
　前記抗体（ロ）における吸光度の測定方法は、特に限定されるものではないが、例えば、前記「（ｉｉ−１）　前記抗体（イ）における吸光度の測定方法」における〔Ａ〕~〔Ｃ〕と同様にして行うことができる。〔本発明における「抗体の親和性の測定方法」と同じ。〕
　例えば、親和性を測定しようとする抗体の濃度が０．６２５~２．５ｎｇ／ｍＬの範囲において、前記の通り測定を行ったときに、前記ＨＭＧＢ１分解産物を固相化したウェルで得られた当該分解産物に結合する当該抗体の量を測定するときに得られた前記吸光度差の値が１．２であり、基準とする抗体産生細胞であるハイブリドーマＭＤ７８（ＦＥＲＭ　Ｐ−１８４０５）より産生される抗体における当該吸光度の値が０．２あった場合には、１．２÷０．２＝６となり、この抗体は、『当該抗体の濃度が０．６２５~２．５ｎｇ／ｍＬの範囲において、ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物を固相化したＥＬＩＳＡ法で、当該分解産物に結合する当該抗体の量を測定するときに得られる吸光度の値が、前記の基準とする抗体産生細胞より産生される抗体における当該吸光度の値の６倍以上となる抗体である』、といえる。
　すなわち、この場合、この抗体は、前記の（ｂ）の抗体である。
（ｉｉｉ）免疫原
　前記の（ｂ）の抗体を取得するための免疫原としては、例えば、前記の「〔Ｉ〕抗ＨＭＧＢ１分解産物抗体」の「（４）免疫原」に記載の免疫原等を用いることができる。
（ｉｖ）前記の（ｂ）の抗体の免疫原等の取得方法
　前記の（ｂ）の抗体を取得するために動物等に免疫する免疫原等の取得方法については、例えば、前記の「〔Ｉ〕抗ＨＭＧＢ１分解産物抗体」の「（５）本抗ＨＭＧＢ１分解産物抗体の免疫原等の取得方法」に記載の取得方法等を用いることができる。
（ｖ）前記の（ｂ）の抗体（ポリクローナル抗体）の取得方法
　前記の（ｂ）の抗体において、ポリクローナル抗体又は抗血清は、以下の操作により取得することができる。
（Ａ）　まず、前記の免疫原、又は前記の免疫原と担体の結合物を哺乳動物（マウス、ウサギ、ラット、ヒツジ、ヤギ、ウマ等）又は鳥類（ニワトリ等）等に免疫する。
　この前記の免疫原、又は前記の免疫原と担体の結合物の免疫量は、免疫動物の種類、免疫注射部位等により決められるものであるが、例えば、ウサギの場合にはウサギ一匹当り一回につき１０μｇ~数十ｍｇの前記免疫原、又は前記免疫原と担体の結合物を免疫注射する。
　なお、この前記の免疫原、又は前記の免疫原と担体の結合物は、アジュバントと添加混合して免疫注射することが好ましい。
　アジュバントとしては、フロイント完全アジュバント、フロイント不完全アジュバント、水酸化アルミニウムアジュバント又は百日咳菌アジュバント等の公知のものを用いることができる。
　免疫注射は、皮下、静脈内、腹腔内又は背部等の部位に行えばよい。
　初回免疫後、２~３週間間隔で皮下、静脈内、腹腔内又は背部等の部位に、前記の免疫原、又は前記の免疫原と担体の結合物を追加免疫注射する。この場合も、前記の免疫原、又は前記の免疫原と担体の結合物は、アジュバントを添加混合して追加免疫注射することが好ましい。
　初回免疫の後、免疫動物の血清中の抗体価の測定をＥＬＩＳＡ法等により繰り返し行い、抗体価がプラトーに達したら全採血を行い、血清を分離して前記の（ｂ）の抗体を含む抗血清を得る。
　この抗血清を、硫酸アンモニウム、硫酸ナトリウム等による塩析法、イオン交換クロマトグラフィー、ゲル濾過法又はアフィニティークロマトグラフィー等の方法、あるいはこれらの方法を組み合わせて抗体の精製を行い、ポリクローナル抗体を得る。
（Ｂ）　ここで得られたポリクローナル抗体について、前記（ｉｉ）の記載のように抗体の親和性を測定し、前記ＨＭＧＢ１分解産物に対する親和性が高い抗体を選択することにより、前記の（ｂ）の抗体を取得することができる。
　例えば、前記（ｉｉ）の記載のようにして、この抗体の親和性の測定を行ったときに、親和性を測定しようとする抗体の濃度が０．６２５~２．５ｎｇ／ｍＬの範囲の少なくともいずれかの濃度（好ましくは当該範囲のすべての濃度）において、ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物を固相化したＥＬＩＳＡ法で、
（イ）当該分解産物に結合する当該抗体の量を測定するときに得られる吸光度の値を当該抗体の濃度の値で除したときの値が０．５以上となる抗体、又は
（ロ）当該分解産物に結合する当該抗体の量を測定するときに得られる吸光度の値が、基準とする抗体産生細胞であるハイブリドーマＭＤ７８（ＦＥＲＭ　Ｐ−１８４０５）より産生される抗体における当該吸光度の値の６倍以上となる抗体
は、前記ＨＭＧＢ１分解産物に対する親和性が高い、といえる。
　なお、これを、前記ＨＭＧＢ１分解産物をリガンドとして固相に固定化したアフィニティークロマトグラフィーのカラムに通し接触させ、前記の（ｂ）の抗体をこのカラムのリガンド（前記ＨＭＧＢ１分解産物）を介して固相に結合させるとともに、他の抗体等はこのカラムを素通りさせて分離し、その後、この固相に結合させていた前記の（ｂ）の抗体をリガンド（前記ＨＭＧＢ１分解産物）から分離し、このカラムから流出する画分を得ることにより、更に純度の高い前記の（ｂ）の抗体を取得することができる。
（Ｃ）　なお、免疫原と担体の結合物を用いて動物等に免疫した場合には、得られた抗血清又はポリクローナル抗体中に、この担体に対する抗体が存在するので、このような担体に対する抗体の除去処理を行うことが好ましい。
　この除去処理方法としては、担体を、得られたポリクローナル抗体又は抗血清の溶液中に添加して生成した凝集物を取り除くか、担体を不溶化固相に固定化してアフィニティークロマトグラフィーにより除去する方法等を用いることができる。
（ｖｉ）前記の（ｂ）の抗体（モノクローナル抗体）の取得方法
　前記の（ｂ）の抗体において、モノクローナル抗体は、以下の操作により取得することができる。
　モノクローナル抗体は、ケラーらの細胞融合法（Ｇ．Ｋｏｅｈｌｅｒら，Ｎａｔｕｒｅ，２５６巻，４９５~４９７頁，１９７５年発行）によるハイブリドーマ、又はエプスタン−バーウイルス等のウイルスによる腫瘍化細胞等の抗体産生細胞により得ることができる。
　細胞融合法によるモノクローナル抗体の調製は、以下の操作により行うことができる。
　まず、前記の免疫原、又は前記の免疫原と担体の結合物を、哺乳動物（マウス、ヌードマウス、ラットなど、例えば近交系マウスのＢＡＬＢ／ｃ）又は鳥類（ニワトリなど）等に免疫する。
　この前記の免疫原、又は前記の免疫原と担体の結合物の免疫量は、免疫動物の種類、免疫注射部位等により適宜決められるものであるが、例えば、マウスの場合には一匹当り一回につき０．１μｇ~５ｍｇの前記の免疫原、又は前記の免疫原と担体の結合物を免疫注射するのが好ましい。
　なお、前記の免疫原、又は前記の免疫原と担体の結合物は、アジュバントを添加混合して免疫注射することが好ましい。
　アジュバントとしては、フロイント完全アジュバント、フロイント不完全アジュバント、水酸化アルミニウムアジュバント又は百日咳菌アジュバント等の公知なものを用いることができる。
　免疫注射は、皮下、静脈内、腹腔内又は背部等の部位に行えばよい。
　初回免疫後、１~２週間間隔で皮下、静脈内、腹腔内又は背部等の部位に、前記の免疫原、又は前記の免疫原と担体の結合物を追加免疫注射する。
　この追加免疫注射の回数としては２~６回が一般的である。
　この場合も前記の免疫原、又は前記の免疫原と担体の結合物は、アジュバントを添加混合して追加免疫注射することが好ましい。
　初回免疫の後、免疫動物の血清中の抗体価の測定をＥＬＩＳＡ法等により繰り返し行い、抗体価がプラトーに達したら、前記の免疫原、又は前記の免疫原と担体の結合物を生理食塩水（０．９％塩化ナトリウム水溶液）に溶解したものを静脈内又は腹腔内に注射し、最終免疫とする。
　この最終免疫の３~５日後に、免疫動物の脾細胞、リンパ節細胞又は末梢リンパ球等の抗体産生能を有する細胞を取得する。
　この免疫動物より得られた抗体産生能を有する細胞と哺乳動物等（マウス、ヌードマウス、ラットなど）の骨髄腫細胞（ミエローマ細胞）とを細胞融合させるのであるが、ミエローマ細胞としてはヒポキサンチン・グアニン・ホスホリボシル・トランスフェラーゼ（ＨＧＰＲＴ）又はチミジンキナーゼ（ＴＫ）等の酵素を欠損した細胞株のものが好ましく、例えば、ＢＡＬＢ／ｃマウス由来のＨＧＰＲＴ欠損細胞株である、Ｐ３−Ｘ６３−Ａｇ８株（ＡＴＣＣ　ＴＩＢ９）、Ｐ３−Ｘ６３−Ａｇ８−Ｕ１株（癌研究リサーチソースバンク（ＪＣＲＢ）９０８５）、Ｐ３−ＮＳ１−１−Ａｇ４−１株（ＪＣＲＢ　０００９）、Ｐ３−Ｘ６３−Ａｇ８・６５３株（ＪＣＲＢ　００２８）又はＳＰ２／Ｏ−Ａｇ−１４株（ＪＣＲＢ　００２９）等を用いることができる。
　細胞融合は、各種分子量のポリエチレングリコール（ＰＥＧ）、リポソームもしくはセンダイウイルス（ＨＶＪ）等の融合促進剤を用いて行うか、又は電気融合法により行うことができる。
　ミエローマ細胞がＨＧＰＲＴ欠損株又はＴＫ欠損株のものである場合には、ヒポキサンチン・アミノプテリン・チミジンを含む選別用培地（ＨＡＴ培地）を用いることにより、抗体産生能を有する細胞とミエローマ細胞の融合細胞（ハイブリドーマ）のみを選択的に培養し、増殖させることができる。
　このようにして得られたハイブリドーマの培養上清を、前記の免疫原、前記の免疫原と担体の結合物、又は前記ＨＭＧＢ１分解産物等を用いてＥＬＩＳＡ法やウエスタンブロット法等の免疫学的測定法により測定することにより、前記ＨＭＧＢ１分解産物に対する親和性が高い抗体を産生するハイブリドーマを選択することができる。
　そして、このようにして選択したハイブリドーマの培養上清について、前記（ｉｉ）の記載のように抗体の親和性を測定し、前記ＨＭＧＢ１分解産物に対する親和性が高い抗体を選択することにより、前記の（ｂ）の抗体を産生するハイブリドーマを選択することができる。
　これらのハイブリドーマ選択方法と限界希釈法等の公知のクローニングの方法を組み合わせて行うことにより、前記の（ｂ）の抗体（モノクローナル抗体）、即ち「前記ＨＭＧＢ１分解産物に結合する抗体であって、前記ＨＭＧＢ１分解産物に対する親和性が高い抗体」（モノクローナル抗体）の産生細胞株を単離して得ることができる。
　このモノクローナル抗体産生細胞株を適当な培地で培養して、その培養上清から前記の（ｂ）の抗体（モノクローナル抗体）を得ることができるが、培地としては無血清培地又は低濃度血清培地等を用いてもよく、この場合は抗体の精製が容易となる点で好ましく、ＤＭＥＭ培地、ＲＰＭＩ１６４０培地又はＡＳＦ培地１０３等の培地を用いることができる。
　また、モノクローナル抗体産生細胞株を、これに適合性がありプリスタン等であらかじめ刺激した哺乳動物の腹腔内に注入し、一定期間の後、腹腔にたまった腹水より前記の（ｂ）の抗体（モノクローナル抗体）を得ることもできる。
　このようにして得られたモノクローナル抗体は、硫酸アンモニウム、硫酸ナトリウムなどによる塩析法、イオン交換クロマトグラフィー、ゲル濾過法又はアフィニティークロマトグラフィーなどの方法、あるいはこれらの方法を組み合わせること等により、精製された前記の（ｂ）の抗体（モノクローナル抗体）を得ることができる。
（４）免疫学的測定方法
　本発明の免疫学的測定方法は、試料に含まれる前記ＨＭＧＢ１分解産物の免疫学的測定方法であって、前記の（ａ）の抗体〔本抗ＨＭＧＢ１分解産物抗体〕及び前記の（ｂ）の抗体を使用するものであれば、特にその測定原理は限定されるものではなく、所期の効果を奏するものである。
　この免疫学的測定方法としては、例えば、酵素免疫測定法（ＥＬＩＳＡ、ＥＩＡ）、蛍光免疫測定法（ＦＩＡ）、放射免疫測定法（ＲＩＡ）、発光免疫測定法（ＬＩＡ）、酵素抗体法、蛍光抗体法、イムノクロマトグラフィー法、免疫比濁法、ラテックス比濁法、ラテックス凝集反応測定法、赤血球凝集反応法、粒子凝集反応法、特開平９−２２９９３６号公報及び特開平１０−１３２８１９号公報などに記載された測定対象物質（被検物質）に対する特異的結合物質が固定され、これで被覆された面を有する担体、及び測定対象物質（被検物質）に対する特異的結合物質が固定された粒子を用いる測定法、又はＤａｈｌｂｅａｃｋらが示したＥＬＳＡ法（Ｅｎｚｙｍｅ−ｌｉｎｋｅｄ　Ｌｉｇａｎｄｓｏｒｂｅｎｔ　Ａｓｓａｙ）（Ｔｈｒｏｍｂ．Ｈａｅｍｏｓｔ．，７９巻，７６７~７７２頁，１９９８年発行；ＷＯ９８／２３９６３）等を挙げることができる。
　そして、前記の免疫学的測定方法においては、サンドイッチ法、競合法又は均一系法（ホモジニアス系法）等のいずれの手法においても、本発明の免疫学的測定方法を適用することができる。
　また、本発明の免疫学的測定方法における測定は、用手法により行ってもよいし、又は分析装置等の装置を用いて行ってもよい。
（５）試料
　本発明の免疫学的測定方法における試料としては、ヒトの血液、血清、血漿、尿、***、髄液、唾液、汗、涙、腹水若しくは羊水などの体液；大便；血管若しくは肝臓などの臓器；組織；細胞；又は大便、臓器、組織若しくは細胞などの抽出液等、前記ＨＭＧＢ１分解産物が含まれる可能性のある生体試料等の試料であれば対象となる。
（６）標識抗体を用いた免疫学的測定方法
　本発明の免疫学的測定方法を、標識物質を抗体（若しくは抗原）に結合した標識抗体（若しくは標識抗原）、及び抗体（若しくは抗原）を固相担体に固相化した固相化抗体（若しくは固相化抗原）を用いる、酵素免疫測定法、蛍光免疫測定法、放射免疫測定法又は発光免疫測定法等の免疫学的測定方法により実施する場合には、サンドイッチ法又は競合法等により行うことができるが、サンドイッチ法により実施することが好ましい。
　本発明の免疫学的測定方法を、前記のサンドイッチ法により実施する時には、前記の（ａ）の抗体及び前記の（ｂ）の抗体のうち、いずれか一方の抗体が固相化抗体として使用され、他の一方の抗体が標識抗体として使用されるものであってもよい。
　前記の免疫学的測定方法に用いる固相化抗体（若しくは固相化抗原）に使用する固相担体としては、ポリスチレン、ポリカーボネート、ポリビニルトルエン、ポリプロピレン、ポリエチレン、ポリ塩化ビニル、ナイロン、ポリメタクリレート、ポリアクリルアミド、ラテックス、リポソーム、ゼラチン、アガロース、セルロース、セファロース、ガラス、セラミックス、金属又は磁性体等の材質よりなる、マイクロカプセル、ビーズ、マイクロプレート（マイクロタイタープレート）、試験管、スティック又は試験片等の形状の固相担体を使用することができる。
　固相化抗体（若しくは固相化抗原）は、前記の（ａ）の抗体若しくは前記の（ｂ）の抗体等の抗体又は抗原と固相担体とを物理的吸着法、化学的結合法又はこれらの併用等の公知の方法により吸着、結合させて調製することができる。
　物理的吸着法による場合は、公知の方法に従い、抗体（若しくは抗原）と固相担体を緩衝液などの溶液中で混合し接触させたり、又は緩衝液などに溶解した抗体（若しくは抗原）と固相担体を接触させること等により行うことができる。
　また、化学的結合法により行う場合は、日本臨床病理学会編「臨床病理臨時増刊特集第５３号　臨床検査のためのイムノアッセイ−技術と応用−」，臨床病理刊行会，１９８３年発行；日本生化学会編「新生化学実験講座１　タンパク質ＩＶ」，東京化学同人，１９９１年発行等に記載の公知の方法に従い、抗体（若しくは抗原）と固相担体をグルタルアルデヒド、カルボジイミド、イミドエステル又はマレイミド等の二価性の架橋試薬と混合、接触させ、抗体（若しくは抗原）と固相担体のそれぞれのアミノ基、カルボキシル基、チオール基、アルデヒド基又は水酸基などと反応させること等により行うことができる。
　また、更に非特異的反応や固相担体の自然凝集等を抑制するために処理を行う必要があれば、抗体（若しくは抗原）を固相化させた固相担体の表面又は内壁面に、例えば、ウシ血清アルブミン（ＢＳＡ）、ヒト血清アルブミン（ＨＳＡ）、卵白アルブミン、カゼイン、ゼラチン若しくはその塩などのタンパク質、界面活性剤又は脱脂粉乳等を接触させ被覆させること等の公知の方法により処理して、固相担体のブロッキング処理（マスキング処理）を行ってもよい。
　標識物質としては、酵素免疫測定法の場合には、例えば、パーオキシダーゼ（ＰＯＤ）、アルカリホスファターゼ（ＡＬＰ）、β−ガラクトシダーゼ、ウレアーゼ、カタラーゼ、グルコースオキシダーゼ、乳酸脱水素酵素又はアミラーゼ等を用いることができる。
　また、蛍光免疫測定法の場合には、例えば、フルオレセインイソチオシアネート、テトラメチルローダミンイソチオシアネート、置換ローダミンイソチオシアネート又はジクロロトリアジンイソチオシアネート等を用いることができる。
　そして、放射免疫測定法の場合には、例えば、トリチウム、ヨウ素１２５又はヨウ素１３１等を用いることができる。
　また、発光免疫測定法においては、例えば、ＮＡＤＨ−ＦＭＮＨ２−ルシフェラーゼ反応系、ルミノール−過酸化水素−ＰＯＤ反応系、アクリジニウムエステル反応系、又はジオキセタン化合物反応系などの反応系に係わる物質等を用いることができる。
　前記の（ａ）の抗体又は前記の（ｂ）の抗体等の抗体（若しくは抗原）と酵素等の標識物質との結合法は、日本臨床病理学会編「臨床病理臨時増刊特集第５３号　臨床検査のためのイムノアッセイ−技術と応用−」，臨床病理刊行会，１９８３年発行；日本生化学会編「新生化学実験講座１　タンパク質ＩＶ」，東京化学同人，１９９１年発行等に記載の公知の方法に従い、抗体（若しくは抗原）と標識物質をグルタルアルデヒド、カルボジイミド、イミドエステル又はマレイミド等の二価性の架橋試薬と混合、接触させ、抗体（若しくは抗原）と標識物質のそれぞれのアミノ基、カルボキシル基、チオール基、アルデヒド基又は水酸基等と反応させることにより結合を行うことができる。
　前記の酵素免疫測定法、蛍光免疫測定法、放射免疫測定法又は発光免疫測定法等の免疫学的測定方法における測定の操作法は公知の方法（日本臨床病理学会編「臨床病理臨時増刊特集第５３号　臨床検査のためのイムノアッセイ−技術と応用−」，臨床病理刊行会，１９８３年発行；石川榮治ら編「酵素免疫測定法」，第３版，医学書院，１９８７年発行；北川常廣ら編「蛋白質核酸酵素別冊Ｎｏ．３１　酵素免疫測定法」，共立出版，１９８７年発行）等により行うことができる。
　例えば、固相化抗体（「固相担体−抗体」）と試料を反応させ、同時に標識抗体（「抗体−標識物質」）を反応させるか、又は洗浄の後に標識抗体を反応させることにより、「固相担体−抗体」＝「前記ＨＭＧＢ１分解産物」＝「抗体−標識物質」の複合体を形成させる。
　そして、未結合の標識抗体を洗浄分離して、「固相担体−抗体」＝「前記ＨＭＧＢ１分解産物」＝「抗体−標識物質」の結合により固相担体に間接的に結合した標識抗体の量又は未結合の標識抗体の量を測ることにより試料に含まれていた前記ＨＭＧＢ１分解産物の量（濃度）を測定することができる。
　具体的には、酵素免疫測定法の場合は、抗体に標識した酵素に、その至適条件下等で基質を反応させ、その酵素反応生成物の量を光学的方法等により測定する。
また、蛍光免疫測定法の場合には蛍光物質標識による蛍光強度を測定する。
　そして、放射免疫測定法の場合には放射性物質標識による放射線量を測定する。
　更に、発光免疫測定法の場合は発光反応系による発光量を測定する。
（７）凝集反応法による免疫学的測定方法
　本発明の免疫学的測定方法を、免疫複合体凝集物の生成を、その透過光や散乱光を光学的方法により測るか、又は目視的に測ることにより、試料に含まれていた前記ＨＭＧＢ１分解産物の量（濃度）を測定する、免疫比濁法、ラテックス比濁法、ラテックス凝集反応法、赤血球凝集反応法又は粒子凝集反応法等の免疫学的測定方法によっても実施することができる。
　前記の（ａ）の抗体及び／又は前記の（ｂ）の抗体を固相担体に固相化させて用いる場合には、固相担体としては、例えば、ポリスチレン、スチレン−スチレンスルホン酸塩共重合体、アクリロニトリル−ブタジエン−スチレン共重合体、塩化ビニル−アクリル酸エステル共重合体、酢酸ビニル−アクリル酸共重合体、ポリアクロレイン、スチレン−メタクリル酸共重合体、スチレン−グリシジル（メタ）アクリル酸共重合体、スチレン−ブタジエン共重合体、メタクリル酸重合体、アクリル酸重合体、ラテックス、ゼラチン、リポソーム、マイクロカプセル、赤血球、シリカ、アルミナ、カーボンブラック、金属化合物、セラミックス、金属又は磁性体等の材質よりなる粒子を使用することができる。
　前記の（ａ）の抗体及び／又は前記の（ｂ）の抗体を固相担体に固相化させる方法としては、物理的吸着法、化学的結合法又はこれらの併用等の公知の方法により行うことができる。
　物理的吸着法による場合は、公知の方法に従い、抗体と固相担体を緩衝液等の溶液中で混合し接触させたり、又は緩衝液等に溶解した抗体と固相担体を接触させること等により行うことができる。
　また、化学的結合法により行う場合は、日本臨床病理学会編「臨床病理臨時増刊特集第５３号　臨床検査のためのイムノアッセイ−技術と応用−」，臨床病理刊行会，１９８３年発行；日本生化学会編「新生化学実験講座１　タンパク質ＩＶ」，東京化学同人，１９９１年発行等に記載の公知の方法に従い、抗体と固相担体をグルタルアルデヒド、カルボジイミド、イミドエステル又はマレイミド等の二価性の架橋試薬と混合、接触させ、抗体と固相担体のそれぞれのアミノ基、カルボキシル基、チオール基、アルデヒド基又は水酸基等と反応させること等により行うことができる。
　また、更に非特異的反応や固相担体の自然凝集等を抑制するために処理を行う必要があれば、前記の（ａ）の抗体及び／又は前記の（ｂ）の抗体を固相化させた固相担体の表面又は内壁面に、ウシ血清アルブミン（ＢＳＡ）、ヒト血清アルブミン（ＨＳＡ）、卵白アルブミン、カゼイン、ゼラチン若しくはその塩などのタンパク質、界面活性剤又は脱脂粉乳等を接触させ被覆させること等の公知の方法により処理して、固相担体のブロッキング処理（マスキング処理）を行ってもよい。
　なお、ラテックス比濁法を測定原理とする場合、固相担体として用いるラテックス粒子の粒径については、特に制限はないものの、ラテックス粒子が前記の（ａ）の抗体又は前記の（ｂ）の抗体及び測定対象物質（前記ＨＭＧＢ１分解産物）を介して結合し、凝集塊を生成する程度、及びこの生成した凝集塊の測定の容易さ等の理由より、ラテックス粒子の粒径は、その平均粒径が、０．０４~１μｍであることが好ましい。
　また、ラテックス比濁法を測定原理とする場合、前記の（ａ）の抗体及び／又は前記の（ｂ）の抗体を固相化させたラテックス粒子を含ませる濃度については、試料中に含まれる前記ＨＭＧＢ１分解産物の濃度、前記の（ａ）の抗体及び／又は前記の（ｂ）の抗体のラテックス粒子表面上での分布密度、ラテックス粒子の粒径、試料と測定試薬の混合比率等の各種条件により最適な濃度は異なるので一概にいうことはできない。
　しかし、通常は、試料と測定試薬が混合され、ラテックス粒子に固相化された前記の（ａ）の抗体及び／又は前記の（ｂ）の抗体と試料中に含まれていた「前記ＨＭＧＢ１分解産物」との抗原抗体反応が行われる測定反応時に、前記の（ａ）の抗体及び／又は前記の（ｂ）の抗体を固相化させたラテックス粒子の濃度が、反応混合液中において０．００５~１％（ｗ／ｖ）となるようにするのが一般的であり、この場合、反応混合液中においてこのような濃度になるような濃度の「前記の（ａ）の抗体及び／又は前記の（ｂ）の抗体を固相化させたラテックス粒子」を測定試薬に含ませる。
　また、ラテックス凝集反応法、赤血球凝集反応法又は粒子凝集反応法等の間接凝集反応法を測定原理とする場合、固相担体として用いる粒子の粒径については、特に制限はないものの、その平均粒子径が０．０１~１００μｍの範囲内にあることが好ましく、０．５~１０μｍの範囲内にあることがより好ましい。
　そして、これらの粒子の比重は、１~１０の範囲内にあることが好ましく、１~２の範囲内にあることがより好ましい。
　なお、ラテックス凝集反応法、赤血球凝集反応法又は粒子凝集反応法等の間接凝集反応法を測定原理とする場合の測定に使用する容器としては、例えば、ガラス、ポリスチレン、ポリ塩化ビニル又はポリメタクリレートなどからなる、試験管、マイクロプレート（マイクロタイタープレート）又はトレイ等を挙げることができる。
　これらの容器の溶液収容部分（マイクロプレートのウェル等）の底面は、Ｕ型、Ｖ型又はＵＶ型等の底面中央から周辺にかけて傾斜を持つ形状であることが好ましい。
　本発明の免疫学的測定方法を、免疫比濁法、ラテックス比濁法、ラテックス凝集反応法、赤血球凝集反応法又は粒子凝集反応法等の免疫学的測定方法により実施する場合には、溶媒として、リン酸緩衝液、グリシン緩衝液、トリス緩衝液又はグッド緩衝液等を用いることができ、更にポリエチレングリコール等の反応促進剤や非特異的反応抑制剤を含ませてもよい。
　前記の免疫比濁法、ラテックス比濁法、ラテックス凝集反応法、赤血球凝集反応法又は粒子凝集反応法等の免疫学的測定方法における測定の操作法は、公知の方法等により行うことができるが、例えば、光学的方法により測定する場合には、試料と前記の「前記の（ａ）の抗体及び／又は前記の（ｂ）の抗体」、又は試料と固相担体に固相化させた「前記の（ａ）の抗体及び／又は前記の（ｂ）の抗体」を反応させ、エンドポイント法又はレート法により、透過光や散乱光を測定する。
　また、目視的に測定する場合には、プレートやマイクロプレート等の前記容器中で、試料と固相担体に固相化させた「前記の（ａ）の抗体及び／又は前記の（ｂ）の抗体」を反応させ、凝集の状態を目視的に判定する。
　なお、この目視的に測定する代わりにマイクロプレートリーダー等の機器を用いて測定を行ってもよい。
〔ＩＩＩ〕試料に含まれるＨＭＧＢ１分解産物の免疫学的測定試薬
（１）総論
　本発明の試料に含まれるＨＭＧＢ１分解産物の免疫学的測定試薬（以下「本発明の免疫学的測定試薬」又は「本発明の測定試薬」ということがある。）は、次の（ａ）の抗体及び（ｂ）の抗体を含有する測定試薬である。
　（ａ）　本抗ＨＭＧＢ１分解産物抗体。
　（ｂ）　前記ＨＭＧＢ１分解産物に結合する抗体であって、前記ＨＭＧＢ１分解産物に対する親和性が高い抗体。
　前記の（ｂ）の抗体としては、当該抗体の濃度が０．６２５~２．５ｎｇ／ｍＬの範囲の少なくともいずれかの濃度（好ましくは当該範囲のすべての濃度）において、ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物を固相化したＥＬＩＳＡ法で、
（イ）当該分解産物に結合する当該抗体の量を測定するときに得られる吸光度の値を当該抗体の濃度の値で除したときの値が０．５以上となる抗体、又は
（ロ）当該分解産物に結合する当該抗体の量を測定するときに得られる吸光度の値が、基準とする抗体産生細胞であるハイブリドーマＭＤ７８（ＦＥＲＭ　Ｐ−１８４０５）より産生される抗体における当該吸光度の値の６倍以上となる抗体
が好ましい。
　前記の（ａ）の抗体（本抗ＨＭＧＢ１分解産物抗体）としては、ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物に対する親和性が、ＨＭＧＢ２に対する親和性及びＨＭＧＢ２のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物に対する親和性とそれぞれ比較して、各々少なくとも１０倍であるものが好ましい。（抗体濃度：０．６２５~５ｎｇ／ｍＬの範囲の少なくともいずれかの濃度（好ましくは当該範囲のすべての濃度））
　また、前記の（ａ）の抗体（本抗ＨＭＧＢ１分解産物抗体）、及び／又は前記の（ｂ）の抗体が、モノクローナル抗体であることが好ましい。
　なお、前記の（ａ）の抗体（本抗ＨＭＧＢ１分解産物抗体）、及び／又は前記の（ｂ）の抗体は、ポリクローナル抗体、ポリクローナル抗体を含む抗血清、モノクローナル抗体、又はこれらの抗体のフラグメント（Ｆａｂ、Ｆ（ａｂ’）２又はＦａｂ’など）等のいずれのものであってもよい。
　また、本発明の測定試薬は、固相化抗体及び標識抗体を用いるものであって、前記の（ａ）の抗体及び前記の（ｂ）の抗体のうち、いずれか一方の抗体が固相化抗体として使用され、他の一方の抗体が標識抗体として使用されるものであってもよい。
　本発明の測定試薬は、前記の（ａ）の抗体及び前記の（ｂ）の抗体を使用することにより、前記ＨＭＧＢ１分解産物に対する特異性が高く、前記ＨＭＧＢ１分解産物のみを正確に定量測定することができる。
　なお、前記の（ａ）の抗体は、前記の通りの抗体であれば特に制限なく使用することができる。
　また、前記の（ｂ）の抗体は、前記の通りの抗体であれば特に制限なく使用することができる。
　なお、前記の（ａ）の抗体及び前記の（ｂ）の抗体はそれぞれ、１種類のものだけではなく、複数種類のものを同時に使用してもよい。
（２）前記の（ａ）の抗体
　前記の（ａ）の抗体、すなわち本抗ＨＭＧＢ１分解産物抗体の詳細については、前記の「〔Ｉ〕抗ＨＭＧＢ１分解産物抗体」の項などに記載した通りである。
（３）前記の（ｂ）の抗体
　前記の（ｂ）の抗体の詳細については、前記の「〔ＩＩ〕試料に含まれるＨＭＧＢ１分解産物の免疫学的測定方法」の「（３）前記の（ｂ）の抗体」の項などに記載した通りである。
（４）免疫学的測定試薬
　本発明の免疫学的測定試薬においては、その測定原理として、酵素免疫測定法、蛍光免疫測定法、放射免疫測定法若しくは発光免疫測定法などの標識物質を用いる免疫学的測定方法（サンドイッチ法又は競合法等）のもの、又は免疫比濁法、ラテックス比濁法、ラテックス凝集反応法、赤血球凝集反応法若しくは粒子凝集反応法などの免疫複合体凝集物の生成を測定する免疫学的測定方法のもの等、特に制限なく適用することができる。
　例えば、酵素免疫測定法（ＥＬＩＳＡ法）、蛍光免疫測定法又は発光免疫測定法等におけるサンドイッチ法を測定原理とする免疫学的測定試薬においては、前記の（ａ）の抗体及び前記の（ｂ）の抗体のうち、いずれか一方の抗体が固相化抗体として使用され、他の一方の抗体が標識抗体として使用されるものであってもよい。
　また、例えば、ラテックス比濁法、ラテックス凝集反応法、赤血球凝集反応法又は粒子凝集反応法等を測定原理とする免疫学的測定試薬においては、ラテックス粒子等の固相担体に固相化させる抗体が前記の（ａ）の抗体及び／又は前記の（ｂ）の抗体であればよく、また、免疫比濁法を測定原理とする測定試薬においては、抗体として前記の（ａ）の抗体及び前記の（ｂ）の抗体を用いればよい。
　なお、本発明の免疫学的測定試薬は、前記の（ａ）の抗体及び前記の（ｂ）の抗体を含有することを特徴とするものであるので、本発明の免疫学的測定試薬に含有させる「前記の（ａ）の抗体」の詳細は前記の「〔Ｉ〕抗ＨＭＧＢ１分解産物抗体」の項などに記載した通りであり、また、本発明の免疫学的測定試薬に含有させる「前記の（ｂ）の抗体」の詳細は前記の「〔ＩＩ〕試料に含まれるＨＭＧＢ１分解産物の免疫学的測定方法」の「（３）前記の（ｂ）の抗体」の項などに記載した通りであり、本発明の免疫学的測定試薬の測定原理などの詳細は前記の「〔ＩＩ〕試料に含まれるＨＭＧＢ１分解産物の免疫学的測定方法」の項などに記載した通りである。
（５）その他の試薬成分
　本発明の免疫学的測定試薬において、溶媒としては、各種の水系溶媒を用いることができる。
　この水系溶媒としては、例えば、精製水、生理食塩水、又は、トリス緩衝液、リン酸緩衝液若しくはリン酸緩衝生理食塩水などの各種緩衝液等を挙げることができる。
　この緩衝液のｐＨについては、適宜適当なｐＨを選択して用いればよく、特に制限はないものの、通常は、ｐＨ３~１２の範囲内のｐＨを選択して用いることが一般的である。
　また、本発明の免疫学的測定試薬には、前記の（ａ）の抗体若しくは前記の（ｂ）の抗体などの抗体を固相担体に固相化した「固相化抗体」、及び／又は前記の（ｂ）の抗体若しくは前記の（ａ）の抗体などの抗体と酵素などの標識物質を結合させた「標識抗体」等の試薬成分の他に、ウシ血清アルブミン（ＢＳＡ）、ヒト血清アルブミン（ＨＳＡ）、卵白アルブミン、カゼイン、ゼラチン若しくはその塩などのタンパク質；各種塩類；各種糖類；脱脂粉乳；正常ウサギ血清などの各種動物血清；アジ化ナトリウム若しくは抗生物質などの各種防腐剤；活性化物質；反応促進物質；ポリエチレングリコールなどの感度増加物質；非特異的反応抑制物質；又は、非イオン性界面活性剤、両性界面活性剤もしくは陰イオン性界面活性剤などの各種界面活性剤等の１種又は２種以上を適宜含有させてもよい。
　そして、これらを測定試薬に含有させる際の濃度は特に限定されるものではないが、０．００１~１０％（ｗ／ｖ）が好ましく、特に０．０１~５％（ｗ／ｖ）が好ましい。
　なお、前記の界面活性剤としては、例えば、ソルビタン脂肪酸エステル、グリセリン脂肪酸エステル、デカグリセリン脂肪酸エステル、ポリオキシエチレンソルビタン脂肪酸エステル、ポリオキシエチレングリセリン脂肪酸エステル、ポリエチレングリコール脂肪酸エステル、ポリオキシエチレンアルキルエーテル、ポリオキシエチレンフィトステロール、フィトスタノール、ポリオキシエチレンポリオキシプロピレンアルキルエーテル、ポリオキシエチレンアルキルフェニルエーテル、ポリオキシエチレンヒマシ油、硬化ヒマシ油若しくはポリオキシエチレンラノリンなどの非イオン性界面活性剤；酢酸ベタインなどの両性界面活性剤；アミン塩若しくは第４級アンモニウム塩などの陽イオン性界面活性剤；又は、ポリオキシエチレンアルキルエーテル硫酸塩若しくはポリオキシエチレンアルキルエーテル酢酸塩などの陰イオン性界面活性剤等を挙げることができる。
（６）測定試薬の構成
　本発明の免疫学的測定試薬は、そのもの単独にて、試料に含まれる前記ＨＭＧＢ１分解産物の測定に使用することができる。そして、そのもの単独にて、販売することができる。
　また、本発明の免疫学的測定試薬は、他の試薬と組み合わせて、試料に含まれる前記ＨＭＧＢ１分解産物の測定に使用することもできる。そして、他の試薬と組み合わせて、販売することもできる。
　前記の他の試薬としては、例えば、緩衝液、試料希釈液、試薬希釈液、標識物質を含有する試薬、発色などのシグナルを生成する物質を含有する試薬、発色などのシグナルの生成に関与する物質を含有する試薬、校正（キャリブレーション）を行うための物質を含有する試薬、又は精度管理を行うための物質を含有する試薬等を挙げることができる。
　そして、前記の他の試薬を第１試薬とし、本発明の免疫学的測定試薬を第２試薬としたり、又は本発明の免疫学的測定試薬を第１試薬とし、前記の他の試薬を第２試薬としたりして、適宜様々な組合せにて使用、及び販売を行うことができる。
　また、本発明の免疫学的測定試薬は、第１試薬及び第２試薬、又は前記のその他の試薬などの複数の構成試薬等からなる測定試薬キットであってもよい。 [I] Anti-HMGB1 degradation product antibody
(1) Anti-HMGB1 degradation product antibody of the present invention
The antibody of the present invention is a thrombin or thrombin-thrombomodulin complex of HMGB1, wherein the affinity for the degradation product of thrombin or thrombin-thrombomodulin complex of HMGB1 is at least 1.5 times compared to the affinity for HMGB1. An antibody that binds to a degradation product by (Hereinafter, this antibody of the present invention may also be referred to as “the present anti-HMGB1 degradation product antibody”.)
The affinity of HMGB1 for the degradation product by thrombin or thrombin / thrombomodulin complex is “at least 1.5 times” compared to the affinity for HMGB1, and the concentration of the antibody is 0.625 to 5 ng / mL. At least one concentration in the range (preferably all concentrations in the range) means that the affinity is at least 1.5 times as measured by the “method for measuring affinity” described later.
In this anti-HMGB1 degradation product antibody, the affinity of HMGB1 for the degradation product of thrombin or thrombin / thrombomodulin complex is compared with the affinity for HMGB2 and the affinity of HMGB2 for the degradation product of thrombin or thrombin / thrombomodulin complex, respectively. Thus, those that are at least 10 times each are preferred. (Antibody concentration: at least one concentration in the range of 0.625 to 5 ng / mL (preferably all concentrations in the range))
The anti-HMGB1 degradation product antibody is preferably a monoclonal antibody.
The anti-HMGB1 degradation product antibody is a polyclonal antibody, an antiserum containing a polyclonal antibody, a monoclonal antibody, or a fragment of these antibodies (Fab, F (ab ′)). 2 Or Fab ′ or the like.
(2) HMGB1 degradation products
The “degradation product of HMGB1 by thrombin or thrombin-thrombomodulin complex” in the present invention is a protein or peptide produced by hydrolysis of human HMGB1 by thrombin or thrombin-thrombomodulin complex. , The 10th arginine (R10) -11th glycine (G11) of human HMGB1 is cleaved by thrombin or thrombin-thrombomodulin complex, and a peptide comprising 10 amino acid residues of “MGKGDPKKPR” at the N-terminus of HMGB1 Refers to a degradation product of HMGB1 having a newly exposed N-terminal “GKMSS...” Generated by separation.
The amino acid sequence of human HMGB1 is shown in the sequence listing as SEQ ID NO: 1, and the amino acid sequence of the degradation product of human HMGB1 by thrombin or thrombin-thrombomodulin complex is shown as SEQ ID NO: 2.
In the present invention, as a degradation product of human HMGB1 by thrombin or thrombin / thrombomodulin complex, deletion, substitution, or insertion of one to several amino acid residues in the amino acid sequence shown as SEQ ID NO: 2 is used. Alternatively, it may be a protein comprising an amino acid sequence obtained by addition or modification, or a saccharide or lipid bound to this protein.
The number of amino acid residues in the deletion, substitution, insertion, addition or modification of the amino acid residues is usually 1 to 4, preferably 1 to 3, more preferably 1 to 2, particularly preferably 1. It is a piece.
In the present invention, the thrombin or thrombin / thrombomodulin complex is not only a thrombin or thrombin / thrombomodulin complex present in the human body, but also an artificially prepared one such as a gene recombinant or the like It includes those that have been artificially isolated.
(Hereinafter, “degradation product of HMGB1 by thrombin or thrombin-thrombomodulin complex” may be referred to as “the HMGB1 degradation product” hereinafter.)
(3) Affinity measurement method
As described above, the anti-HMGB1 degradation product antibody has an affinity for the HMGB1 degradation product that is at least 1.5 times the affinity for HMGB1.
The method for measuring the affinity of this antibody is not particularly limited, and can be performed, for example, as follows.
[A] Preparation of measurement reagent
(I) Solid-phase microplate such as HMGB1 degradation products
Each of HMGB1, HMGB1 degradation product, HMGB2 and HMGB2 degradation product by thrombin or thrombin-thrombomodulin complex (hereinafter sometimes referred to as “the HMGB2 degradation product”) is 1 μg / mL with phosphate buffered saline. Each of the 96-well microtiter plates [Thermo Fisher Scientific Inc. (Ind., Illinois, USA)] was injected into a well of 100 μL, allowed to stand at 25 ° C. for 18 hours, and the HMGB1, HMGB1 degradation product, HMGB2 and HMGB2 degradation product were respectively added to the wells of the microtiter plate. To solid phase.
Next, after removing the liquid in the well of the microtiter plate, 250 μL each of Tris buffered saline (TBS) [pH 8.0] containing 0.5% casein and 0.1% sodium azide was added to each microtiter plate. The well was dispensed and subjected to blocking treatment.
Thereafter, each well was sealed with a plate seal and kept refrigerated until use so as not to evaporate.
This was used as a solid-phased microplate such as HMGB1 degradation product.
(Ii) POD-labeled anti-mouse IgG antibody solution
POD-labeled anti-mouse IgG antibody [DakoCytomation (Denmark)] was diluted 1000-fold with 50 mM Tris-HCl buffer (pH 8.0) containing 0.5% sodium caseinate and 100 mM sodium chloride.
This was used as a POD-labeled anti-mouse IgG antibody solution.
(Iii) Cleaning solution
Phosphate buffered saline containing 0.05% Tween 20 was used as a washing solution.
(Iv) Diluent
A 50 mM Tris-HCl buffer solution (pH 8.0) containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide was used as a diluent.
(V) Color developing solution
A 0.045% 3,3 ′, 5,5′-tetramethylbenzidine hydrochloride aqueous solution (pH 2.0) containing 0.2 mM EDTA · disodium was used as a color developing solution.
(Vi) Substrate solution
A 60 mM disodium phosphate aqueous solution (pH 4.3) containing 5 mM hydrogen peroxide, 41 mM citric acid, and 0.2 mM EDTA · disodium was used as a substrate solution.
(Vii) Chromogenic substrate
The chromogenic solution and the substrate solution were returned to room temperature before use, and mixed in equal amounts at the time of use to obtain a chromogenic substrate.
(Viii) Reaction stop solution
0.7N sulfuric acid was used as a reaction stop solution.
[B] Sample
Antibodies to be measured for affinity with HMGB1, the HMGB1 degradation product, HMGB2 and the HMGB2 degradation product have concentrations of 0.625 ng / mL, 1.25 ng / mL, and 2.5 ng / mL, respectively. The antibody solutions of the above-mentioned four concentrations were prepared by diluting with the diluent (iv) of [A] so as to be 5 ng / mL.
[C] Measurement
(I) Each well of the [A] (i) HMGB1 degradation product solid-phased microplate was washed 3 times with 400 μL of the washing solution of [A] (iii).
(Ii) Next, 100 μL of each of the 4 concentrations of the antibody solution of [B] above was dispensed to each well as a sample, and then allowed to stand at 25 ° C. for 2 hours, and contained in these concentrations of the antibody solution. The antibody was allowed to undergo an antigen-antibody reaction with each of the HMGB1, HMGB1 degradation product, HMGB2, and HMGB2 degradation product immobilized on the wells of the immobilized HMGB1 degradation product microplate. .
(Iii) Next, each well was washed 3 times with 400 μL of the washing solution of (iii) of [A].
(Iv) Next, 100 μL of the POD-labeled anti-mouse IgG antibody solution of (ii) in [A] above was dispensed into each well and allowed to stand at 25 ° C. for 1 hour to cause an antigen-antibody reaction.
(V) Next, each well was washed three times with 400 μL of the washing solution of (iii) of [A].
(Vi) Next, 100 μL of the chromogenic substrate of (vii) of [A] above is dispensed into each well and allowed to stand at room temperature for 20 minutes to cause a color reaction with peroxidase (POD), which is a labeling enzyme. It was.
(Vii) Next, 100 μL of the reaction stop solution of (viii) in [A] was dispensed into each well to stop the color reaction.
(Viii) Next, the absorbance (primary wavelength: 450 nm, subwavelength: 550 nm) of the liquid in each well was measured to obtain the absorbance when each of the above-described four concentrations of the antibody solution was measured as a sample.
The absorbance value was set to a value indicating the affinity of the antibody to be measured for affinity with each of the HMGB1, HMGB1 degradation product, HMGB2, and HMGB2 degradation product.
For example, when the measurement is performed as described above using a solution of an antibody whose affinity is to be measured (for example, a concentration of 2.5 ng / mL) as a sample, it is obtained in a well in which the HMGB1 degradation product is immobilized. When the absorbance difference value is 1.35 and the absorbance difference value obtained in the well in which HMGB1 is immobilized is 0.9, this antibody is a product of degradation of HMGB1. It can be said that the affinity for is at least 1.5 times that of HMGB1.
That is, in this case, this antibody is the present anti-HMGB1 degradation product antibody.
(4) Immunogen
The immunogen for obtaining the anti-HMGB1 degradation product antibody will be described below.
Examples of the immunogen for obtaining this anti-HMGB1 degradation product antibody include, for example, human HMGB1, HMGB1 of animals having high homology with the amino acid sequence of human HMGB1 (for example, bovine or pig), or the HMGB1 degradation product The whole or a part can be mentioned.
Examples of the HMGB1 degradation product include a peptide comprising 20 amino acid residues of “GKMSYAFFVQTCREEHKK” at the N-terminus of the HMGB1 degradation product.
The immunogen for obtaining the anti-HMGB1 degradation product antibody is a deletion, substitution, insertion, addition or modification of one to several amino acid residues in the protein or peptide as the immunogen described so far. It may be a protein or peptide obtained by applying.
The number of amino acid residues in the deletion, substitution, insertion, addition or modification of the amino acid residues is usually 1 to 4, preferably 1 to 3, more preferably 1 to 2, particularly preferably 1. It is a piece.
Moreover, as what immunizes an animal etc. in order to acquire this anti- HMGB1 degradation product antibody, what combined the support | carrier (carrier) about the protein or peptide as an immunogen described so far may be sufficient.
In particular, when the protein or peptide as the immunogen has a low molecular weight, the antibody production ability is improved by binding to the carrier, and therefore, it is preferable to use the one bound to the carrier as the immunogen.
Examples of the carrier include mussel hemocyanin (KLH), bovine serum albumin (BSA), human serum albumin (HSA), chicken serum albumin, poly-L-lysine, polyalanyl lysine, dipalmityl lysine, Known carriers such as tetanus toxoid or polysaccharide can be used.
(5) Method for obtaining immunogen etc. of this anti-HMGB1 degradation product antibody
A method for obtaining an immunogen or the like for immunizing an animal or the like in order to obtain the present anti-HMGB1 degradation product antibody will be described below.
The anti-HMGB1 degradation product antibody is an immunogen for obtaining the human HMGB1 described in (4) above, or an animal HMGB1 having a high homology with the amino acid sequence of human HMGB1. Extraction and purification from body fluids, cells, tissues, organs, etc. of mammals other than humans (pigs, cows, rabbits, goats, sheep, mice, rats, etc.) having high homology with the amino acid sequence of HMGB1 by known methods And so on.
In addition, the HMGB1 degradation product may be obtained by, for example, bringing human HMGB1 or HMGB1 of an animal having high homology with the amino acid sequence of human HMGB1 (for example, bovine or pig) into contact with the thrombin or thrombin / thrombomodulin complex. It can be obtained by hydrolysis, and extraction and purification by a known method.
The immunogen can be synthesized by a peptide synthesis method such as a liquid phase method or a solid phase method, and an automatic peptide synthesizer may be used.
For example, “Biochemistry Experiment Course 1 Protein Chemistry IV”, Tokyo Kagaku Dojin, 1975; Izumiya et al. “Basics and Experiments of Peptide Synthesis”, Maruzen, 1985; It can be synthesized according to the method described in "Experimental Lecture 2 Protein Chemistry", Tokyo Kagaku Doujin, 1987, etc.
Further, the immunogen may be prepared from DNA or RNA having a corresponding nucleobase sequence by using genetic engineering technology, edited by the Japanese Biochemical Society, “Second Biochemistry Experiment Course 1, Gene Research Method I”, Tokyo Chemical. Doujin, 1986; “Sequential Biochemistry Experiment Course 1 Genetic Research Method II” edited by the Japanese Biochemical Society, Tokyo Chemical Doujin, 1986; or “The Biochemistry Experimental Course 1 Genetic Research Method III” edited by the Japanese Biochemical Society, Tokyo Chemical Doujin 1987 and the like.
For example, the amino acid sequence of human HMGB1 represented by SEQ ID NO: 1 or the amino acid sequence of the degradation product of HMGB1 represented by SEQ ID NO: 2, or a gene corresponding to a part of these amino acid sequences, The obtained gene is incorporated into an expression vector such as a plasmid.
Next, by introducing this expression vector into a host cell such as Escherichia coli and culturing the resulting transformant, a protein or peptide comprising the above amino acid sequence or a part thereof can be expressed.
Examples of a method for cloning a gene base sequence include a PCR method, a recombinant PCR method, a ligation method, and a linker ligation method.
By the way, when the immunogen is a low-molecular substance, it is common to immunize an animal or the like with a carrier (carrier) bound to the immunogen, but a peptide having 5 amino acids is used as an immunogen. Since there is also a report (Kiyama et al., “Abstract 3 of the 112th Annual Meeting of the Japanese Pharmaceutical Society”, page 122, published in 1992) that a specific antibody was produced, it is not essential to use a carrier.
In addition, as a carrier when immunizing an animal or the like with the above protein or peptide bound to a carrier, mussel hemocyanin (KLH), bovine serum albumin (BSA), human serum albumin (HSA), chicken serum albumin Any known carrier such as poly-L-lysine, polyalanyl lysine, dipalmityl lysine, tetanus toxoid or polysaccharide can be used.
The protein or peptide and the carrier may be bound by the glutaraldehyde method, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide method, maleimidobenzoyl-N-hydroxysuccinimide ester method, bisdiazotized benzidine method or Known coupling methods such as the N-succimidyl-3- (2-pyridyldithio) propionic acid method can be used.
Moreover, what adsorb | sucked the said protein or peptide to carriers, such as a nitrocellulose particle | grain, polyvinylpyrrolidone, or a liposome, can be used for the immunization to the said animal etc. for acquisition of this anti- HMGB1 degradation product antibody.
(6) Method for obtaining this anti-HMGB1 degradation product antibody (polyclonal antibody)
In this anti-HMGB1 degradation product antibody, the polyclonal antibody or antiserum can be obtained by the following operation.
(I) First, a mammal (a mouse, a rabbit, a rat, a sheep, a goat, a horse, etc.) or a bird (a chicken, etc.) is immunized with the immunogen or a conjugate of the immunogen and a carrier.
The immunity of the immunogen or the conjugate of the immunogen and the carrier is determined by the type of animal to be immunized, the site of immunization, and the like. Each time, 10 μg to several tens of mg of the immunogen or a conjugate of the immunogen and a carrier is immunized.
The immunogen or the combined immunogen and carrier is preferably added and mixed with an adjuvant for immunization injection.
As the adjuvant, known ones such as Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide adjuvant or pertussis adjuvant can be used.
Immunization may be performed at a site such as subcutaneous, intravenous, intraperitoneal or back.
After the initial immunization, booster injections of the immunogen or a conjugate of the immunogen and the carrier are given at sites such as subcutaneous, intravenous, intraperitoneal or back at intervals of 2 to 3 weeks. Also in this case, the immunogen or the conjugate of the immunogen and the carrier is preferably boosted by adding an adjuvant and mixing.
After the initial immunization, the antibody titer in the sera of the immunized animal is repeatedly measured by ELISA or the like. When the antibody titer reaches a plateau, the whole blood is collected, and the serum is separated to contain the anti-HMGB1 degradation product antibody. Get.
The antiserum is subjected to antibody purification by a salting-out method using ammonium sulfate, sodium sulfate or the like, ion exchange chromatography, gel filtration method or affinity chromatography, or a combination of these methods to obtain a polyclonal antibody.
(Ii) The polyclonal antibody obtained here comprises an antibody (this anti-HMGB1 degradation product antibody) having an affinity for the HMGB1 degradation product of at least 1.5 times that of HMGB1, and the HMGB1 degradation product. Since both of the antibodies (conventional antibodies) having an affinity for HMGB1 of less than 1.5 times compared to the affinity for HMGB1 are included, this is further used for affinity chromatography immobilized on a solid phase using HMGB1 as a ligand. Pass through the column.
The conventional antibody binds to the solid phase via the ligand (HMGB1) of this column and is collected.
In contrast, the anti-HMGB1 degradation product antibody is difficult to bind to the ligand (HMGB1) of this column, and many of these columns pass through this column. By obtaining a passed fraction, the anti-HMGB1 degradation product antibody is obtained. Can be obtained.
This is passed through an affinity chromatography column immobilized on a solid phase with the HMGB1 degradation product as a ligand, and the anti-HMGB1 degradation product antibody is passed through the solid phase via the ligand (the HMGB1 degradation product) of this column. Other antibodies are separated by passing through this column, and then the anti-HMGB1 degradation product antibody bound to the solid phase is separated from the ligand (the HMGB1 degradation product) and separated from this column. By obtaining a fraction that flows out, the anti-HMGB1 degradation product antibody with higher purity can be obtained.
(Iii) When an animal or the like is immunized using a conjugate of an immunogen and a carrier, an antibody against this carrier exists in the obtained antiserum or polyclonal antibody. It is preferable to perform the removal process.
As this removal treatment method, a carrier is added to the obtained polyclonal antibody or antiserum solution to remove aggregates generated, or the carrier is immobilized on an insolubilized solid phase and removed by affinity chromatography, etc. Can be used.
(7) Method for obtaining this anti-HMGB1 degradation product antibody (monoclonal antibody)
In this anti-HMGB1 degradation product antibody, a monoclonal antibody can be obtained by the following operation.
Monoclonal antibodies are antibody-producing cells such as hybridomas by the cell fusion method of Keller et al. (G. Koehler et al., Nature, 256, 495-497, published in 1975), or tumorigenic cells by viruses such as Epstan-Barr virus. Can be obtained.
Preparation of a monoclonal antibody by the cell fusion method can be performed by the following operation.
First, a mammal (mouse, nude mouse, rat, etc., for example, BALB / c of an inbred mouse) or a bird (chicken, etc.) is immunized with the immunogen or a conjugate of the immunogen and a carrier. .
The immunization amount of the immunogen or the conjugate of the immunogen and the carrier can be appropriately determined depending on the type of immunized animal, the site of immunization, and the like. Preferably, 0.1 μg to 5 mg of the immunogen or a combination of the immunogen and a carrier is immunized at a time.
The immunogen or the conjugate of the immunogen and the carrier is preferably immunized by adding an adjuvant and mixing.
Known adjuvants such as Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide adjuvant, or pertussis adjuvant can be used as the adjuvant.
Immunization may be performed at a site such as subcutaneous, intravenous, intraperitoneal or back.
After the first immunization, booster injections of the immunogen or a conjugate of the immunogen and the carrier are given at sites of subcutaneous, intravenous, intraperitoneal, or back at 1-2 week intervals.
The number of booster injections is generally 2 to 6 times.
Also in this case, the immunogen or the combined immunogen and carrier is preferably boosted by adding an adjuvant and mixing.
After the first immunization, the antibody titer in the sera of the immunized animal is repeatedly measured by ELISA or the like. When the antibody titer reaches a plateau, the immunogen or the combined immunogen and carrier is added to physiological saline. A solution dissolved in (0.9% sodium chloride aqueous solution) is injected intravenously or intraperitoneally to obtain final immunization.
Three to five days after the final immunization, cells having antibody-producing ability such as spleen cells, lymph node cells or peripheral lymphocytes of immunized animals are obtained.
The cell having antibody-producing ability obtained from this immunized animal is fused with myeloma cells (myeloma cells) of mammals (mouse, nude mouse, rat, etc.). A cell line deficient in an enzyme such as guanine phosphoribosyl transferase (HGPRT) or thymidine kinase (TK) is preferred. For example, a P3-X63-Ag8 strain (ATCC) which is a HGPRT-deficient cell line derived from BALB / c mice. TIB9), P3-X63-Ag8-U1 strain (Cancer Research Research Source Bank (JCRB) 9085), P3-NS1-1-Ag4-1 strain (JCRB 0009), P3-X63-Ag8.653 strain (JCRB 0028) Alternatively, SP2 / O-Ag-14 strain (JCRB 0029) or the like is used. be able to.
Cell fusion can be performed using a fusion promoter such as polyethylene glycol (PEG) of various molecular weights, liposomes or Sendai virus (HVJ), or by electrofusion.
When myeloma cells are of HGPRT-deficient strain or TK-deficient strain, fusion of cells capable of producing antibodies and myeloma cells by using a selection medium (HAT medium) containing hypoxanthine / aminopterin / thymidine Only cells (hybridomas) can be selectively cultured and propagated.
The hybridoma culture supernatant thus obtained is subjected to immunological measurement such as ELISA or Western blot using the immunogen, the conjugate of the immunogen and the carrier, or the HMGB1 degradation product. By measuring by this method, a hybridoma that produces this anti-HMGB1 degradation product antibody can be selected.
In addition, a hybridoma that produces an antibody that does not bind to HMGB1 or the like is selected by measuring the culture supernatant of the hybridoma by immunoassay such as ELISA or Western blot using HMGB1 or the like. Can do.
The anti-HMGB1 degradation product antibody (monoclonal antibody), that is, “degradation product of HMGB1 by thrombin or thrombin / thrombomodulin complex” is performed by combining these two kinds of hybridoma selection methods and known cloning methods such as limiting dilution. An antibody that binds to a degradation product of HMGB1 by thrombin or thrombin-thrombomodulin complex (monoclonal antibody) having an affinity for HMGB1 that is at least 1.5 times that of HMGB1 Can be obtained.
This monoclonal antibody-producing cell line can be cultured in an appropriate medium, and the anti-HMGB1 degradation product antibody (monoclonal antibody) can be obtained from the culture supernatant. As the medium, a serum-free medium or low-concentration serum medium can be used. In this case, it is preferable from the viewpoint of easy purification of the antibody, and a medium such as DMEM medium, RPMI 1640 medium, or ASF medium 103 can be used.
In addition, a monoclonal antibody-producing cell line is injected into the abdominal cavity of a mammal that is compatible with this and prestimulated with pristane or the like, and after a certain period of time, the anti-HMGB1 degradation product antibody (monoclonal antibody) from ascites collected in the abdominal cavity. ) Can also be obtained.
The monoclonal antibody thus obtained was purified by a salting-out method using ammonium sulfate, sodium sulfate or the like, a method such as ion exchange chromatography, gel filtration or affinity chromatography, or a combination of these methods. This anti-HMGB1 degradation product antibody (monoclonal antibody) can be obtained.
[II] Method for immunological measurement of HMGB1 degradation products contained in a sample
(1) General
The immunological measurement method of HMGB1 degradation product contained in the sample of the present invention (hereinafter sometimes referred to as “the immunological measurement method of the present invention” or “the measurement method of the present invention”) includes the following (a): The measurement method using the antibody of (b) and the antibody of (b).
(A) This anti-HMGB1 degradation product antibody.
(B) An antibody that binds to the HMGB1 degradation product and has high affinity for the HMGB1 degradation product.
The antibody (b) includes HMGB1 thrombin or thrombin at a concentration of the antibody in the range of 0.625 to 2.5 ng / mL (preferably all concentrations in the range). In ELISA method in which degradation product by thrombomodulin complex is immobilized,
(A) an antibody having a value of 0.5 or more when the absorbance value obtained when measuring the amount of the antibody bound to the degradation product is divided by the concentration value of the antibody, or
(B) The absorbance value obtained when measuring the amount of the antibody bound to the degradation product is the absorbance of the antibody produced from the hybridoma MD78 (FERM P-18405), which is the reference antibody-producing cell. Antibodies that are more than 6 times the value
Is preferred.
As the antibody (a) (the anti-HMGB1 degradation product antibody), the affinity of HMGB1 for the degradation product by thrombin or thrombin / thrombomodulin complex is the same as the affinity for HMGB2 and the affinity for HMGB2 by thrombin or thrombin / thrombomodulin complex. Those that are each at least 10 times greater than the affinity for the degradation products are preferred. (Antibody concentration: at least one concentration in the range of 0.625 to 5 ng / mL (preferably all concentrations in the range))
The antibody (a) (anti-HMGB1 degradation product antibody) and / or the antibody (b) are preferably monoclonal antibodies.
The antibody (a) (anti-HMGB1 degradation product antibody) and / or the antibody (b) are polyclonal antibodies, antisera containing polyclonal antibodies, monoclonal antibodies, or fragments of these antibodies ( Fab, F (ab ′) 2 Or Fab ′ or the like.
The measurement method of the present invention uses a solid-phased antibody and a labeled antibody, and any one of the antibody (a) and the antibody (b) is solid-phased. It may be used as an antibody and the other antibody may be used as a labeled antibody.
The measurement method of the present invention uses the antibody (a) and the antibody (b), and has high specificity for the HMGB1 degradation product and accurately measures only the HMGB1 degradation product. Can do.
The antibody (a) can be used without particular limitation as long as it is an antibody as described above.
The antibody (b) can be used without any particular limitation as long as it is an antibody as described above.
The antibody (a) and the antibody (b) are not limited to one type, and a plurality of types may be used simultaneously.
(2) Antibody of (a) above
Details of the antibody (a), that is, the present anti-HMGB1 degradation product antibody, are as described in the above section “[I] Anti-HMGB1 degradation product antibody”.
(3) Antibody of (b) above
(I) The antibody of (b) is “an antibody that binds to the HMGB1 degradation product and has high affinity for the HMGB1 degradation product”.
As the antibody (b), HMGB1 thrombin or at least one of the concentrations of the antibody in the range of 0.625 to 2.5 ng / mL (preferably all concentrations in the range) is used. In the ELISA method in which the degradation product by the thrombin / thrombomodulin complex is immobilized,
(A) an antibody having a value of 0.5 or more when the absorbance value obtained when measuring the amount of the antibody bound to the degradation product is divided by the concentration value of the antibody, or
(B) The absorbance value obtained when measuring the amount of the antibody bound to the degradation product is the absorbance of the antibody produced from the hybridoma MD78 (FERM P-18405), which is the reference antibody-producing cell. Antibodies that are more than 6 times the value
Is preferred.
(Ii) Affinity measurement method
As described above, the antibody (b) is an antibody that binds to the HMGB1 degradation product and has high affinity for the HMGB1 degradation product.
As the antibody (b), HMGB1 thrombin or at least one of the concentrations of the antibody in the range of 0.625 to 2.5 ng / mL (preferably all concentrations in the range) is used. In the ELISA method in which the degradation product by the thrombin / thrombomodulin complex is immobilized,
(A) an antibody having a value of 0.5 or more when the absorbance value obtained when measuring the amount of the antibody bound to the degradation product is divided by the concentration value of the antibody, or
(B) The absorbance value obtained when measuring the amount of the antibody bound to the degradation product is the absorbance of the antibody produced from the hybridoma MD78 (FERM P-18405), which is the reference antibody-producing cell. Antibodies that are more than 6 times the value
Is preferred.
(Ii-1) Method for measuring absorbance of the antibody (a)
The method for measuring the absorbance of the antibody (A) is not particularly limited, and can be performed, for example, as follows. [Same as “Method for Measuring Antibody Affinity” in the present invention. ]
[A] Preparation of measurement reagent
[I] HMGB1 degradation product solid-phased microplate
The HMGB1 degradation products prepared with phosphate buffered saline to a concentration of 1 μg / mL were each prepared in 96-well microtiter plates [Thermo Fisher Scientific Inc. Incorporated (Illinois, USA)] was injected at 100 μL and allowed to stand at 25 ° C. for 18 hours, and the HMGB1 degradation product was immobilized on the wells of the microtiter plate.
Next, after removing the liquid in the well of the microtiter plate, 250 μL each of Tris buffered saline (TBS) [pH 8.0] containing 0.5% casein and 0.1% sodium azide was added to each microtiter plate. The well was dispensed and subjected to blocking treatment. Thereafter, each well was sealed with a plate seal and kept refrigerated until use so as not to evaporate. This was used as the HMGB1 degradation product-immobilized microplate.
[Ii] POD-labeled anti-mouse IgG antibody solution
POD-labeled anti-mouse IgG antibody [DakoCytomation (Denmark)] was diluted 1000-fold with 50 mM Tris-HCl buffer (pH 8.0) containing 0.5% sodium caseinate and 100 mM sodium chloride. This was used as a POD-labeled anti-mouse IgG antibody solution.
[Iii] Cleaning solution
Phosphate buffered saline containing 0.05% Tween 20 was used as a washing solution.
[Iv] Diluent
A 50 mM Tris-HCl buffer solution (pH 8.0) containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide was used as a diluent.
[V] Color developing solution
A 0.045% 3,3 ′, 5,5′-tetramethylbenzidine hydrochloride aqueous solution (pH 2.0) containing 0.2 mM EDTA · disodium was used as a color developing solution.
[Vi] Substrate solution
A 60 mM disodium phosphate aqueous solution (pH 4.3) containing 5 mM hydrogen peroxide, 41 mM citric acid, and 0.2 mM EDTA · disodium was used as a substrate solution.
[Vii] Chromogenic substrate
The chromogenic solution and the substrate solution were returned to room temperature before use, and mixed in equal amounts at the time of use to obtain a chromogenic substrate.
[Viii] reaction stop solution
0.7N sulfuric acid was used as a reaction stop solution.
[B] Sample
Each of the antibodies (b) is diluted with the diluted solution [iv] of [A] so that the concentrations thereof are 0.625 ng / mL, 1.25 ng / mL, and / or 2.5 ng / mL, respectively. The antibody solutions having the above three concentrations were prepared by dilution.
[C] Measurement
[I] Each well of the [i] HMGB1 degradation product-immobilized microplate of [A] was washed 3 times with 400 μL of the washing solution of [A] [iii].
[Ii] Next, 100 μL of each of the three concentrations of the antibody solution of [B] was dispensed to each well as a sample, and then allowed to stand at 25 ° C. for 2 hours, and contained in the antibody solution of each concentration. The antibody (b) was allowed to undergo an antigen-antibody reaction with the HMGB1 degradation product immobilized on the wells of the HMGB1 degradation product-immobilized microplate.
[Iii] Next, each well was washed 3 times with 400 μL of the washing liquid of [iii] in [A].
[Iv] Next, 100 μL of the POD-labeled anti-mouse IgG antibody solution of [ii] in [A] above was dispensed into each well and allowed to stand at 25 ° C. for 1 hour to cause antigen-antibody reaction.
[V] Next, each well was washed three times with 400 μL of the washing solution of [A] [iii].
[Vi] Next, 100 μL of the chromogenic substrate of [vi] of [A] is dispensed into each well and left at room temperature for 20 minutes to cause a chromogenic reaction with peroxidase (POD) as a labeling enzyme. It was.
[Vii] Next, 100 μL of the reaction stop solution of [viii] in [A] was dispensed into each well to stop the color reaction.
[Viii] Next, the absorbance of each well was measured (primary wavelength: 450 nm, subwavelength: 550 nm), and the absorbance when each of the antibody solutions of the antibody (b) at the three concentrations was measured as a sample. Got.
The absorbance value was set to a value indicating the affinity of the antibody (b) with the HMGB1 degradation product.
For example, when the measurement is performed as described above using a solution of an antibody whose affinity is to be measured (for example, a concentration of 2.5 ng / mL) as a sample, it is obtained in a well in which the HMGB1 degradation product is immobilized. When the absorbance difference was 1.25, 1.25 ÷ 2.5 ng / mL = 0.5 (ng / mL) -1 This antibody is expressed as follows: “By the ELISA method in which the degradation product of HMGB1 thrombin or thrombin-thrombomodulin complex is immobilized in the range of the antibody concentration of 0.625 to 2.5 ng / mL, It is an antibody whose value is 0.5 or more when the absorbance value obtained when measuring the amount of the bound antibody is divided by the concentration value of the antibody.
That is, in this case, this antibody is the antibody (b) described above.
(Ii-2) Method for measuring absorbance in the antibody (b)
The method for measuring the absorbance of the antibody (b) is not particularly limited. For example, [A] to [C] in “(ii-1) Method of measuring the absorbance of the antibody (b)” The same can be done. [Same as “Method for Measuring Antibody Affinity” in the present invention. ]
For example, when the measurement was performed as described above in the range where the concentration of the antibody whose affinity was to be measured was 0.625 to 2.5 ng / mL, it was obtained in a well in which the HMGB1 degradation product was immobilized. The value of the absorbance difference obtained when measuring the amount of the antibody bound to the degradation product is 1.2, and is produced from the hybridoma MD78 (FERM P-18405), which is a standard antibody-producing cell. When the absorbance value of the antibody is 0.2, 1.2 ÷ 0.2 = 6, and this antibody is expressed as “in the range of the antibody concentration of 0.625 to 2.5 ng / mL, The absorbance obtained when measuring the amount of the antibody bound to the degradation product by ELISA using a solid phase degradation product of thrombin or thrombin-thrombomodulin complex of HMGB1. There is an antibody to be 6 times or more of the absorbance values in the antibody produced from the antibody-producing cells to the reference ", and said.
That is, in this case, this antibody is the antibody (b) described above.
(Iii) Immunogen
As the immunogen for obtaining the antibody (b), for example, the immunogen described in “(4) Immunogen” of the “[I] anti-HMGB1 degradation product antibody” can be used. .
(Iv) Method for obtaining immunogen etc. of antibody of (b) above
Regarding the method for obtaining an immunogen or the like for immunizing an animal or the like in order to obtain the antibody of (b) above, for example, “(5) Anti-HMGB1 degradation product of“ [I] Anti-HMGB1 degradation product antibody ”above. The acquisition method described in “Method for acquiring antibody immunogen and the like” can be used.
(V) Method for obtaining antibody (polyclonal antibody) of (b) above
In the antibody (b), a polyclonal antibody or antiserum can be obtained by the following operation.
(A) First, a mammal (a mouse, a rabbit, a rat, a sheep, a goat, a horse, etc.) or a bird (a chicken, etc.) is immunized with the immunogen or a conjugate of the immunogen and a carrier.
The immunity of the immunogen or the conjugate of the immunogen and the carrier is determined by the type of animal to be immunized, the site of immunization, and the like. Each time, 10 μg to several tens of mg of the immunogen or a conjugate of the immunogen and a carrier is immunized.
The immunogen or the combined immunogen and carrier is preferably added and mixed with an adjuvant for immunization injection.
As the adjuvant, known ones such as Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide adjuvant or pertussis adjuvant can be used.
Immunization may be performed at a site such as subcutaneous, intravenous, intraperitoneal or back.
After the initial immunization, booster injections of the immunogen or a conjugate of the immunogen and the carrier are given at sites such as subcutaneous, intravenous, intraperitoneal or back at intervals of 2 to 3 weeks. Also in this case, the immunogen or the conjugate of the immunogen and the carrier is preferably boosted by adding an adjuvant and mixing.
After the initial immunization, the antibody titer in the sera of the immunized animal is repeatedly measured by ELISA or the like. When the antibody titer reaches a plateau, the whole blood is collected, and the serum is separated to contain the antibody (b). Obtain serum.
The antiserum is subjected to antibody purification by a salting-out method using ammonium sulfate, sodium sulfate or the like, ion exchange chromatography, gel filtration method or affinity chromatography, or a combination of these methods to obtain a polyclonal antibody.
(B) For the polyclonal antibody obtained here, the affinity of the antibody is measured as described in (ii) above, and an antibody having a high affinity for the HMGB1 degradation product is selected. Antibodies can be obtained.
For example, when the affinity of this antibody is measured as described in (ii) above, the concentration of the antibody whose affinity is to be measured is at least in the range of 0.625 to 2.5 ng / mL. An ELISA method in which a degradation product of HMGB1 by thrombin or thrombin-thrombomodulin complex is immobilized at any concentration (preferably all concentrations in the range),
(A) an antibody having a value of 0.5 or more when the absorbance value obtained when measuring the amount of the antibody bound to the degradation product is divided by the concentration value of the antibody, or
(B) The absorbance value obtained when measuring the amount of the antibody bound to the degradation product is the absorbance of the antibody produced from the hybridoma MD78 (FERM P-18405), which is the reference antibody-producing cell. Antibodies that are more than 6 times the value
Can be said to have a high affinity for the HMGB1 degradation product.
The HMGB1 degradation product is contacted through an affinity chromatography column immobilized on a solid phase with the HMGB1 degradation product as a ligand, and the antibody (b) is immobilized via the ligand (the HMGB1 degradation product) of this column. In addition to binding to the phase, other antibodies are separated by passing through this column, and then the antibody (b) bound to the solid phase is separated from the ligand (the HMGB1 degradation product) By obtaining the fraction flowing out from the column, the antibody (b) having a higher purity can be obtained.
(C) When an animal or the like is immunized using a conjugate of an immunogen and a carrier, an antibody against this carrier exists in the obtained antiserum or polyclonal antibody. It is preferable to perform the removal process.
As this removal treatment method, a carrier is added to the obtained polyclonal antibody or antiserum solution to remove aggregates generated, or the carrier is immobilized on an insolubilized solid phase and removed by affinity chromatography, etc. Can be used.
(Vi) Method for obtaining antibody (monoclonal antibody) of (b) above
In the antibody (b), the monoclonal antibody can be obtained by the following operation.
Monoclonal antibodies are antibody-producing cells such as hybridomas by the cell fusion method of Keller et al. (G. Koehler et al., Nature, 256, 495-497, published in 1975), or tumorigenic cells by viruses such as Epstan-Barr virus. Can be obtained.
Preparation of a monoclonal antibody by the cell fusion method can be performed by the following operation.
First, a mammal (mouse, nude mouse, rat, etc., for example, BALB / c of an inbred mouse) or a bird (chicken, etc.) is immunized with the immunogen or a conjugate of the immunogen and a carrier. .
The immunization amount of the immunogen or the conjugate of the immunogen and the carrier can be appropriately determined depending on the type of immunized animal, the site of immunization, and the like. Preferably, 0.1 μg to 5 mg of the immunogen or a combination of the immunogen and a carrier is immunized at a time.
The immunogen or the conjugate of the immunogen and the carrier is preferably immunized by adding an adjuvant and mixing.
Known adjuvants such as Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide adjuvant, or pertussis adjuvant can be used as the adjuvant.
Immunization may be performed at a site such as subcutaneous, intravenous, intraperitoneal or back.
After the first immunization, booster injections of the immunogen or a conjugate of the immunogen and the carrier are given at sites of subcutaneous, intravenous, intraperitoneal, or back at 1-2 week intervals.
The number of booster injections is generally 2 to 6 times.
Also in this case, the immunogen or the combined immunogen and carrier is preferably boosted by adding an adjuvant and mixing.
After the first immunization, the antibody titer in the sera of the immunized animal is repeatedly measured by ELISA or the like. When the antibody titer reaches a plateau, the immunogen or the combined immunogen and carrier is added to physiological saline. A solution dissolved in (0.9% sodium chloride aqueous solution) is injected intravenously or intraperitoneally to obtain final immunization.
Three to five days after the final immunization, cells having antibody-producing ability such as spleen cells, lymph node cells or peripheral lymphocytes of immunized animals are obtained.
The cell having antibody-producing ability obtained from this immunized animal is fused with myeloma cells (myeloma cells) of mammals (mouse, nude mouse, rat, etc.). A cell line deficient in an enzyme such as guanine phosphoribosyl transferase (HGPRT) or thymidine kinase (TK) is preferred. For example, a P3-X63-Ag8 strain (ATCC) which is a HGPRT-deficient cell line derived from BALB / c mice. TIB9), P3-X63-Ag8-U1 strain (Cancer Research Research Source Bank (JCRB) 9085), P3-NS1-1-Ag4-1 strain (JCRB 0009), P3-X63-Ag8.653 strain (JCRB 0028) Alternatively, SP2 / O-Ag-14 strain (JCRB 0029) or the like is used. be able to.
Cell fusion can be performed using a fusion promoter such as polyethylene glycol (PEG) of various molecular weights, liposomes or Sendai virus (HVJ), or by electrofusion.
When myeloma cells are of HGPRT-deficient strain or TK-deficient strain, fusion of cells capable of producing antibodies and myeloma cells by using a selection medium (HAT medium) containing hypoxanthine / aminopterin / thymidine Only cells (hybridomas) can be selectively cultured and propagated.
The hybridoma culture supernatant thus obtained is subjected to immunological measurement such as ELISA or Western blot using the immunogen, the conjugate of the immunogen and the carrier, or the HMGB1 degradation product. By measuring by this method, a hybridoma producing an antibody having a high affinity for the HMGB1 degradation product can be selected.
Then, with respect to the culture supernatant of the hybridoma thus selected, the affinity of the antibody is measured as described in (ii) above, and an antibody having a high affinity for the HMGB1 degradation product is selected. A hybridoma producing the antibody of (b) can be selected.
By combining these hybridoma selection methods and known cloning methods such as limiting dilution, the above-mentioned antibody (b) (monoclonal antibody), that is, “an antibody that binds to the HMGB1 degradation product, A production cell line of an “antibody with high affinity for HMGB1 degradation product” (monoclonal antibody) can be obtained.
The monoclonal antibody-producing cell line can be cultured in an appropriate medium, and the antibody (b) (monoclonal antibody) can be obtained from the culture supernatant. Examples of the medium include serum-free medium and low-concentration serum medium. In this case, it is preferable in that the antibody can be easily purified, and a medium such as DMEM medium, RPMI 1640 medium, or ASF medium 103 can be used.
In addition, a monoclonal antibody-producing cell line is injected into the abdominal cavity of a mammal that is compatible with this and previously stimulated with pristane or the like, and after a certain period of time, the antibody (monoclonal) (b) from the ascites collected in the abdominal cavity. Antibody).
The monoclonal antibody thus obtained was purified by a salting-out method using ammonium sulfate, sodium sulfate or the like, a method such as ion exchange chromatography, gel filtration or affinity chromatography, or a combination of these methods. The above-mentioned antibody (b) (monoclonal antibody) can be obtained.
(4) Immunological measurement method
The immunological measurement method of the present invention is an immunological measurement method of the HMGB1 degradation product contained in a sample, the antibody (a) [anti-HMGB1 degradation product antibody] and the (b) above. If an antibody is used, the measurement principle is not particularly limited, and the desired effect is achieved.
Examples of the immunological measurement method include enzyme immunoassay (ELISA, EIA), fluorescence immunoassay (FIA), radioimmunoassay (RIA), luminescence immunoassay (LIA), enzyme antibody method, fluorescence Antibody method, immunochromatography method, immunoturbidimetric method, latex turbidimetric method, latex agglutination measurement method, erythrocyte agglutination method, particle agglutination method, JP-A-9-229936 and JP-A-10-132919 A specific binding substance for the described measurement target substance (test substance) is immobilized, and a carrier having a surface coated with this, and particles on which the specific binding substance for the measurement target substance (test substance) is fixed. Measurement method to be used, or ELSA method (Enzyme-linked Ligandsorbent Assay) shown by Dahlbeack et al. . B.Haemost, 79, pp. 767-772 pages, published in 1998; mention may be made of WO98 / 23963) and the like.
In the immunological measurement method described above, the immunological measurement method of the present invention can be applied to any method such as a sandwich method, a competitive method, or a homogeneous method (homogeneous method).
In addition, the measurement in the immunological measurement method of the present invention may be performed by a method or using an apparatus such as an analyzer.
(5) Sample
Samples in the immunological measurement method of the present invention include human blood, serum, plasma, urine, semen, spinal fluid, saliva, sweat, tears, ascites or amniotic fluid; stool; organs such as blood vessels or liver; Samples such as tissues; cells; or biological samples such as extracts of stool, organs, tissues, cells, or the like that may contain the HMGB1 degradation products are targeted.
(6) Immunological measurement method using labeled antibody
The immunological measurement method of the present invention comprises a labeled antibody (or labeled antigen) in which a labeling substance is bound to an antibody (or antigen) and an immobilized antibody (or in which the antibody (or antigen) is immobilized on a solid phase carrier (or When using immunoassay methods such as enzyme immunoassay, fluorescence immunoassay, radioimmunoassay, or luminescent immunoassay using a solid phase antigen), use the sandwich method or competition method. However, it is preferable to carry out by the sandwich method.
When the immunological measurement method of the present invention is carried out by the sandwich method, either one of the antibody (a) and the antibody (b) is used as the immobilized antibody. The other antibody may be used as a labeled antibody.
Examples of the solid phase carrier used for the solid phase antibody (or solid phase antigen) used in the immunological measurement method include polystyrene, polycarbonate, polyvinyl toluene, polypropylene, polyethylene, polyvinyl chloride, nylon, polymethacrylate, poly Microcapsules, beads, microplates (microtiter plates), test tubes, sticks, test pieces, etc. made of materials such as acrylamide, latex, liposomes, gelatin, agarose, cellulose, sepharose, glass, ceramics, metals or magnetic materials A solid support in the shape can be used.
The solid-phased antibody (or solid-phased antigen) is an antibody such as the antibody (a) or the antibody (b) or an antigen and a solid phase carrier, a physical adsorption method, a chemical binding method or the like. It can be prepared by adsorbing and binding by a known method such as combination use.
In the case of the physical adsorption method, the antibody (or antigen) and the solid phase carrier are mixed and brought into contact with a solution such as a buffer solution according to a known method, or the antibody (or antigen) dissolved in the buffer solution or the like is fixed. It can be performed by bringing a phase carrier into contact.
When the chemical binding method is used, the Japanese Society of Clinical Pathology, “Special Issue on Extraordinary Clinical Pathology No. 53, Immunoassay for Clinical Examination—Technology and Applications”, Clinical Pathology Publications, 1983; Japan Biochemical Society In accordance with a known method described in ed. "New Generation Chemistry Experiment Course 1 Protein IV", published by Tokyo Kagaku Dojin, published in 1991, etc., the antibody (or antigen) and the solid support are divalent, such as glutaraldehyde, carbodiimide, imide ester or maleimide. It is possible to carry out the reaction by mixing and bringing into contact with a functional crosslinking reagent and reacting with the amino group, carboxyl group, thiol group, aldehyde group or hydroxyl group of the antibody (or antigen) and the solid phase carrier.
Further, if it is necessary to carry out treatment in order to suppress non-specific reaction or spontaneous aggregation of the solid phase carrier, the surface or inner wall surface of the solid phase carrier on which the antibody (or antigen) is immobilized, for example, Treated with a known method such as bovine serum albumin (BSA), human serum albumin (HSA), ovalbumin, casein, gelatin or a salt thereof, a surfactant, or skim milk powder, etc. The solid phase carrier may be subjected to blocking treatment (masking treatment).
In the case of enzyme immunoassay, for example, peroxidase (POD), alkaline phosphatase (ALP), β-galactosidase, urease, catalase, glucose oxidase, lactate dehydrogenase or amylase can be used as the labeling substance. it can.
In the case of a fluorescence immunoassay, for example, fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, substituted rhodamine isothiocyanate or dichlorotriazine isothiocyanate can be used.
In the case of radioimmunoassay, for example, tritium, iodine 125, iodine 131, or the like can be used.
In the luminescence immunoassay, for example, NADH-FMNH 2 A substance related to a reaction system such as a luciferase reaction system, a luminol-hydrogen peroxide-POD reaction system, an acridinium ester reaction system, or a dioxetane compound reaction system can be used.
The method for binding an antibody (or antigen) such as the antibody (a) or the antibody (b) and a labeling substance such as an enzyme is described in the “Special Issue on Clinical Pathology Special Issue No. 53 Clinical Examination” published by the Japanese Society of Clinical Pathology. Immunoassay for -Technology and Application- ", Clinical Pathology Publication, published in 1983; Japanese Biochemical Society edited by" Neochemistry Experiment Course 1 Protein IV ", Tokyo Kagaku Dojin, published in 1991, etc., The antibody (or antigen) and the labeling substance are mixed with and contacted with a bivalent cross-linking reagent such as glutaraldehyde, carbodiimide, imide ester or maleimide, and the amino group, carboxyl group or thiol of each of the antibody (or antigen) and the labeling substance is contacted. Bonding can be performed by reacting with a group, an aldehyde group or a hydroxyl group.
The measurement operation method in the immunoassay method such as the enzyme immunoassay method, the fluorescence immunoassay method, the radioimmunoassay method or the luminescence immunoassay method described above is a known method (“Special Issue on Clinical Pathology” 53, Immunoassay for Clinical Testing -Technology and Application- ", Clinicopathology Publishing Society, published in 1983; edited by Yuji Ishikawa et al.," Enzyme immunoassay ", 3rd edition, Medical School, published in 1987; Ed. “Protein Nucleic Acid Enzyme Separate Volume No. 31 Enzyme Immunoassay”, Kyoritsu Shuppan, published in 1987).
For example, by reacting a sample with a solid-phased antibody (“solid-phase carrier-antibody”) and simultaneously reacting with a labeled antibody (“antibody-labeled substance”), or by reacting the labeled antibody after washing, “ A complex of “solid phase carrier-antibody” = “degradation product of HMGB1” = “antibody-labeled substance” is formed.
Then, the unbound labeled antibody is washed and separated, and the amount of the labeled antibody indirectly bound to the solid phase carrier by the binding of “solid phase carrier-antibody” = “said HMGB1 degradation product” = “antibody-labeled substance” Alternatively, the amount (concentration) of the HMGB1 degradation product contained in the sample can be measured by measuring the amount of unbound labeled antibody.
Specifically, in the case of an enzyme immunoassay, a substrate is reacted with an enzyme labeled with an antibody under the optimum conditions, and the amount of the enzyme reaction product is measured by an optical method or the like.
In the case of the fluorescence immunoassay, the fluorescence intensity by the fluorescent substance label is measured.
In the case of a radioimmunoassay, the radiation dose due to the radioactive substance label is measured.
Furthermore, in the case of a luminescence immunoassay, the amount of luminescence by the luminescence reaction system is measured.
(7) Immunological measurement method by agglutination method
According to the immunological measurement method of the present invention, the formation of immune complex aggregates is measured by optically measuring the transmitted light or scattered light, or by visually measuring the HMGB1 degradation contained in the sample. It can also be carried out by immunoassay methods such as immunoturbidimetry, latex turbidimetry, latex agglutination, erythrocyte agglutination, or particle agglutination, which measure the amount (concentration) of the product.
When the antibody (a) and / or the antibody (b) is used after being immobilized on a solid phase carrier, examples of the solid phase carrier include polystyrene, styrene-styrenesulfonate copolymer Polymer, acrylonitrile-butadiene-styrene copolymer, vinyl chloride-acrylic acid ester copolymer, vinyl acetate-acrylic acid copolymer, polyacrolein, styrene-methacrylic acid copolymer, styrene-glycidyl (meth) acrylic acid copolymer Materials such as polymers, styrene-butadiene copolymers, methacrylic acid polymers, acrylic acid polymers, latex, gelatin, liposomes, microcapsules, erythrocytes, silica, alumina, carbon black, metal compounds, ceramics, metals or magnetic materials The particles can be used.
The method of immobilizing the antibody (a) and / or the antibody (b) on a solid support is performed by a known method such as physical adsorption, chemical binding, or a combination thereof. be able to.
In the case of physical adsorption, according to a known method, the antibody and the solid phase carrier are mixed and brought into contact in a solution such as a buffer solution, or the antibody dissolved in the buffer solution or the like is brought into contact with the solid phase carrier, etc. It can be carried out.
When the chemical binding method is used, the Japanese Society of Clinical Pathology, “Special Issue on Extraordinary Clinical Pathology No. 53, Immunoassay for Clinical Examination—Technology and Applications”, Clinical Pathology Publications, 1983; Japan Biochemical Society In accordance with known methods described in ed. “Shinsei Kagaku Kenkyu Ken 1 Protein IV”, published by Tokyo Kagaku Dojin, published in 1991, etc., the antibody and solid phase carrier are divalent cross-linking reagents such as glutaraldehyde, carbodiimide, imide ester or maleimide. It is possible to carry out the reaction by reacting with the amino group, carboxyl group, thiol group, aldehyde group or hydroxyl group of the antibody and the solid phase carrier.
Further, if it is necessary to carry out treatment to further suppress non-specific reaction or spontaneous aggregation of the solid phase carrier, the antibody (a) and / or the antibody (b) may be immobilized. A bovine serum albumin (BSA), human serum albumin (HSA), ovalbumin, casein, gelatin or a salt thereof, a surfactant, skim milk powder or the like is coated on the surface or inner wall surface of the solid support. The solid phase carrier may be subjected to a blocking process (masking process) by a known method.
When the latex turbidimetry is used as a measurement principle, the particle size of the latex particles used as the solid phase carrier is not particularly limited, but the latex particles are the antibody (a) or the antibody (b). The particle size of the latex particles is determined based on the average particle size for reasons such as the degree of formation of an aggregate by binding through the substance to be measured (the HMGB1 degradation product) and the ease of measurement of the generated aggregate. Is preferably 0.04 to 1 μm.
When the latex turbidimetry is used as the measurement principle, the concentration of the latex particles obtained by solidifying the antibody (a) and / or the antibody (b) is included in the sample. Various concentrations such as the concentration of the HMGB1 degradation product, the antibody (a) and / or the distribution density of the antibody (b) on the latex particle surface, the particle size of the latex particle, and the mixing ratio of the sample and the measuring reagent Since the optimum concentration differs depending on the conditions, it cannot be generally stated.
However, normally, the above-mentioned antibody (a) and / or the above-mentioned antibody (b) mixed with a sample and a measurement reagent and immobilized on latex particles and the above-mentioned HMGB1 degradation In the measurement reaction in which the antigen-antibody reaction with the “product” is performed, the concentration of the latex particles obtained by immobilizing the antibody (a) and / or the antibody (b) is 0. In general, the concentration of the antibody and / or the above-mentioned (a) is such that the concentration in the reaction mixture is such that the concentration is 005 to 1% (w / v). The latex reagent obtained by immobilizing the antibody (b) is included in the measurement reagent.
In addition, when the indirect agglutination reaction method such as latex agglutination reaction method, erythrocyte agglutination reaction method or particle agglutination reaction method is used as the measurement principle, the particle size of the particles used as the solid phase carrier is not particularly limited, but the average particle The diameter is preferably in the range of 0.01 to 100 μm, more preferably in the range of 0.5 to 10 μm.
The specific gravity of these particles is preferably in the range of 1 to 10, and more preferably in the range of 1 to 2.
In addition, as a container used for the measurement when an indirect agglutination reaction method such as a latex agglutination reaction method, an erythrocyte agglutination reaction method or a particle agglutination reaction method is used as a measurement principle, for example, glass, polystyrene, polyvinyl chloride, polymethacrylate, etc. And a test tube, a microplate (microtiter plate), a tray, and the like.
The bottom surface of the solution storage portion (such as a well of a microplate) of these containers preferably has a shape having an inclination from the center to the periphery of the bottom, such as U-type, V-type, or UV-type.
When the immunoassay method of the present invention is performed by an immunoassay method such as an immunoturbidimetric method, latex turbidimetric method, latex agglutination method, hemagglutination method or particle agglutination method, In addition, a phosphate buffer, a glycine buffer, a Tris buffer, a Good buffer, or the like may be used, and a reaction accelerator such as polyethylene glycol or a nonspecific reaction inhibitor may be further included.
The measuring operation method in the immunological measurement method such as the immunoturbidimetric method, latex turbidimetric method, latex agglutination method, erythrocyte agglutination method or particle agglutination method can be performed by a known method or the like. For example, in the case of measuring by an optical method, the sample and the above-mentioned “the antibody of (a) and / or the antibody of (b)” or “the sample and the solid phase carrier immobilized on the solid phase” are used. The antibody (a) and / or the antibody (b) is reacted, and transmitted light or scattered light is measured by an endpoint method or a rate method.
In the case of visual measurement, the above-mentioned antibody (a) and / or (b) above, which is solid-phased on a sample and a solid support in a container such as a plate or a microplate. The “antibody” is reacted and the state of aggregation is visually determined.
In addition, you may measure using apparatuses, such as a microplate reader, instead of measuring visually.
[III] Reagent for immunoassay of HMGB1 degradation product contained in sample
(1) General
The reagent for immunological measurement of the degradation product of HMGB1 contained in the sample of the present invention (hereinafter sometimes referred to as “immunological measurement reagent of the present invention” or “measurement reagent of the present invention”) is the following (a): An assay reagent comprising an antibody and the antibody (b).
(A) This anti-HMGB1 degradation product antibody.
(B) An antibody that binds to the HMGB1 degradation product and has high affinity for the HMGB1 degradation product.
The antibody (b) includes HMGB1 thrombin or thrombin at a concentration of the antibody in the range of 0.625 to 2.5 ng / mL (preferably all concentrations in the range). In ELISA method in which degradation product by thrombomodulin complex is immobilized,
(A) an antibody having a value of 0.5 or more when the absorbance value obtained when measuring the amount of the antibody bound to the degradation product is divided by the concentration value of the antibody, or
(B) The absorbance value obtained when measuring the amount of the antibody bound to the degradation product is the absorbance of the antibody produced from the hybridoma MD78 (FERM P-18405), which is the reference antibody-producing cell. Antibodies that are more than 6 times the value
Is preferred.
As the antibody (a) (the anti-HMGB1 degradation product antibody), the affinity of HMGB1 for the degradation product by thrombin or thrombin / thrombomodulin complex is the same as the affinity for HMGB2 and the affinity for HMGB2 by thrombin or thrombin / thrombomodulin complex. Those that are each at least 10 times greater than the affinity for the degradation products are preferred. (Antibody concentration: at least one concentration in the range of 0.625 to 5 ng / mL (preferably all concentrations in the range))
The antibody (a) (anti-HMGB1 degradation product antibody) and / or the antibody (b) are preferably monoclonal antibodies.
The antibody (a) (anti-HMGB1 degradation product antibody) and / or the antibody (b) are polyclonal antibodies, antisera containing polyclonal antibodies, monoclonal antibodies, or fragments of these antibodies ( Fab, F (ab ′) 2 Or Fab ′ or the like.
The measurement reagent of the present invention uses a solid-phased antibody and a labeled antibody, and any one of the antibody (a) and the antibody (b) is solid-phased. It may be used as an antibody and the other antibody may be used as a labeled antibody.
The measurement reagent of the present invention uses the antibody (a) and the antibody (b), and has high specificity for the HMGB1 degradation product and accurately measures only the HMGB1 degradation product. Can do.
The antibody (a) can be used without particular limitation as long as it is an antibody as described above.
The antibody (b) can be used without any particular limitation as long as it is an antibody as described above.
The antibody (a) and the antibody (b) are not limited to one type, and a plurality of types may be used simultaneously.
(2) Antibody of (a) above
Details of the antibody (a), that is, the present anti-HMGB1 degradation product antibody, are as described in the above section “[I] Anti-HMGB1 degradation product antibody”.
(3) Antibody of (b) above
The details of the antibody (b) are described in the section “(3) Antibody (b)” in “[II] Immunological measurement method of degradation product of HMGB1 contained in the sample” above. That's right.
(4) Immunological measurement reagent
In the immunological measurement reagent of the present invention, the immunological measurement method (sandwich method or the like) using a labeling substance such as enzyme immunoassay, fluorescent immunoassay, radioimmunoassay or luminescence immunoassay is used as the measurement principle. Competing methods, etc.), or immunological measurement methods that measure the formation of immune complex aggregates such as immunoturbidimetry, latex turbidimetry, latex agglutination, erythrocyte agglutination, or particle agglutination It can be applied without particular limitation.
For example, in the immunoassay reagent based on the sandwich method in enzyme immunoassay (ELISA), fluorescent immunoassay or luminescence immunoassay, the antibody (a) and the (b) Of these antibodies, any one of the antibodies may be used as a solid-phase antibody, and the other antibody may be used as a labeled antibody.
Further, for example, in an immunological measurement reagent having a measurement principle such as latex turbidimetry, latex agglutination, erythrocyte agglutination, or particle agglutination, an antibody to be immobilized on a solid phase carrier such as latex particles May be the antibody (a) and / or the antibody (b), and in the measurement reagent based on the immunoturbidimetric method, the antibody (a) and the antibody The antibody of (b) may be used.
The immunoassay reagent of the present invention is characterized by containing the antibody (a) and the antibody (b), and therefore contained in the immunoassay reagent of the present invention. The details of “the antibody of (a)” described above are as described in the above section “[I] Anti-HMGB1 degradation product antibody” and the like, and are contained in the immunoassay reagent of the present invention. The details of “the antibody of (b)” are as described in the section “(3) Antibody of (b)” above in “[II] Immunological measurement method of HMGB1 degradation product contained in the sample” above. The details of the measurement principle and the like of the immunological measurement reagent of the present invention are as described in the above-mentioned section “[II] Immunological measurement method of HMGB1 degradation product contained in sample”.
(5) Other reagent components
In the immunological measurement reagent of the present invention, various aqueous solvents can be used as the solvent.
Examples of the aqueous solvent include purified water, physiological saline, and various buffer solutions such as Tris buffer, phosphate buffer, and phosphate buffered saline.
The pH of the buffer solution may be appropriately selected and used as appropriate. Although there is no particular limitation, it is general to select and use a pH within the range of pH 3 to 12.
In addition, the immunoassay reagent of the present invention includes a “solid phase antibody” obtained by immobilizing an antibody such as the antibody (a) or the antibody (b) on a solid phase carrier, and / or In addition to the reagent component such as the “labeled antibody” obtained by binding the antibody such as the antibody (b) or the antibody (a) and a labeling substance such as an enzyme, bovine serum albumin (BSA), human serum albumin (HSA), proteins such as ovalbumin, casein, gelatin or salts thereof; various salts; various sugars; skim milk powder; various animal sera such as normal rabbit serum; various preservatives such as sodium azide or antibiotics; A reaction promoting substance, a sensitivity increasing substance such as polyethylene glycol, a nonspecific reaction inhibiting substance, or various nonionic surfactants, amphoteric surfactants or anionic surfactants. One or more may suitably be incorporated, such as surface active agents.
The concentration of these in the measurement reagent is not particularly limited, but is preferably 0.001 to 10% (w / v), and particularly preferably 0.01 to 5% (w / v). .
Examples of the surfactant include sorbitan fatty acid ester, glycerin fatty acid ester, decaglycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene glycerin fatty acid ester, polyethylene glycol fatty acid ester, polyoxyethylene alkyl ether, Nonionic surfactants such as polyoxyethylene phytosterol, phytostanol, polyoxyethylene polyoxypropylene alkyl ether, polyoxyethylene alkylphenyl ether, polyoxyethylene castor oil, hydrogenated castor oil or polyoxyethylene lanolin; betaine acetate, etc. Amphoteric surfactants; cationic surfactants such as amine salts or quaternary ammonium salts; or polyoxyethylene alcohols Anionic surfactants such as ether sulfate or a polyoxyethylene alkyl ether acetate and the like.
(6) Configuration of measurement reagent
The immunoassay reagent of the present invention can be used alone for measuring the HMGB1 degradation product contained in the sample. And it can be sold by itself.
The immunoassay reagent of the present invention can also be used for measurement of the HMGB1 degradation product contained in a sample in combination with other reagents. And it can also be sold in combination with other reagents.
Examples of the other reagents include buffers, sample diluents, reagent diluents, reagents containing labeling substances, reagents containing substances that generate signals such as color development, and signals related to color development. A reagent containing a substance, a reagent containing a substance for performing calibration (calibration), a reagent containing a substance for performing accuracy control, and the like can be given.
The other reagent is the first reagent and the immunological measurement reagent of the present invention is the second reagent, or the immunological measurement reagent of the present invention is the first reagent, and the other reagent is the first reagent. Two reagents can be used and sold in various combinations as appropriate.
Further, the immunological measurement reagent of the present invention may be a measurement reagent kit comprising a plurality of constituent reagents such as the first reagent and the second reagent, or the other reagents described above.
　以下、実施例により本発明をより具体的に詳述するが、本発明はこれらの実施例によって限定されるものではない。
〔参考例１〕（ＨＭＧＢ１、ＨＭＧＢ１分解産物、ＨＭＧＢ２、及びＨＭＧＢ２分解産物の調製）
　ＨＭＧＢ１、ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物、ＨＭＧＢ２、及びＨＭＧＢ２のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物それぞれの調製を下記のようにして行った。
〔１〕　ＨＭＧＢ１及びＨＭＧＢ２の調製
（１）　まず、ウシの胸腺５００ｇを、１４０ｍＭの塩化ナトリウム及び０．５ｍＭのＰＭＳＦを含む６００ｍＬの緩衝液中で破砕を行った。
（２）　次に、この破砕物を遠心分離機で遠心分離を行い、その上澄み液を除去した。
（３）　これに、１４０ｍＭの塩化ナトリウム及び０．５ｍＭのＰＭＳＦを含む緩衝液を加えて撹拌した後、遠心分離機で遠心分離を行い、その上澄み液を除去した。この洗浄操作を２回繰り返して行った。
（４）　次に、得られた沈殿物に、０．７５Ｍの過塩素酸の３００ｍＬを加えた。
　そして、遠心分離機で遠心分離した後、上澄み液を分取した。残った沈殿物に０．７５Ｍの過塩素酸の４００ｍＬを加えた。これについても、遠心分離機で遠心分離した後、上澄み液を分取した。この上澄み液と先に分取した上澄み液とを合わせた。なお、沈殿物は廃棄した。
（５）　前記の合わせた上澄み液に０．７５Ｍの過塩素酸を加えて、全体の容量を１，０００ｍＬとした。次に、遠心分離機で遠心分離した後、上澄み液をグラスフィルター（グレード４）で濾過した。
（６）　前記の濾過の濾液に、３，５００ｍＬのアセトンと２１ｍＬの濃塩酸の混合液を加えた。濁りが生じてくるので、遠心分離機で遠心分離して、上澄み液を分取した。この上澄み液に、アセトン２，５００ｍＬを加えた。そして、再度、濁りが生じてくるので、これを遠心分離機で遠心分離して、上澄み液を分離し、残った沈殿物を集めた。
（７）　この集めた沈殿物を室温で自然乾燥させた。以上の操作により、ＨＭＧＢ１及びＨＭＧＢ２を含むタンパク質画分が、およそ２００ｍｇ得られた。
（８）　前記のＨＭＧＢ１及びＨＭＧＢ２を含むタンパク質画分を、２００ｍＭ塩化ナトリウムを含む７．５ｍＭホウ酸ナトリウム緩衝液（ｐＨ９．０）の１０ｍＬに溶解した後、この２００ｍＭ塩化ナトリウムを含む７．５ｍＭホウ酸ナトリウム緩衝液（ｐＨ９．０）で充分に透析を行った。
（９）　この透析の後、７．５ｍＭホウ酸ナトリウム緩衝液（ｐＨ９．０）で平衡化しておいたＣＭ−セファデックスＣ２５のカラムに添加した。そしてその後、２００ｍＭ塩化ナトリウムを含む７．５ｍＭホウ酸ナトリウム緩衝液（ｐＨ９．０）により溶出させて、陽イオン交換クロマトグラフィーを行った。
（１０）　ここで溶出した各画分を、ＳＤＳ−ポリアクリルアミド電気泳動にかけ、その易動度よりＨＭＧＢ１を含む画分、及びＨＭＧＢ２を含む画分を各々特定した。
　以上の操作により、ＨＭＧＢ１及びＨＭＧＢ２をそれぞれ調製した。
〔２〕　ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物の調製
　ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物の調製を次のようにして行った。
（１）　前記〔１〕において調製したＨＭＧＢ１（３００μｇ／ｍＬ）に、トロンビン〔ＧＥ　Ｈｅａｌｔｈｃａｒｅ　Ｂｉｏ−Ｓｃｉｅｎｃｅｓ社（スウェーデン国）〕を１００Ｕ／ｍＬとなるように添加し〔場合により更に、遺伝子組み換え可溶性トロンボモジュリン（旭化成ファーマ社〔日本国〕）を含んでもよい〕、３７℃で３時間インキュベートした。
（２）　前記（１）の反応により、ＨＭＧＢ１の１０番目のアルギニン（Ｒ１０）と１１番目のグリシン（Ｇ１１）の間を切断し、新たに露呈したＮ末端「ＧＫＭＳＳ・・・・・」を有するＨＭＧＢ１の分解産物を生成させ、すなわち前記ＨＭＧＢ１分解産物を調製した。
〔３〕　ＨＭＧＢ２のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物の調製
　前記〔１〕において調製したＨＭＧＢ２について、前記〔２〕の記載の通りに操作を行い、前記ＨＭＧＢ２分解産物の調製を行った。
〔参考例２〕（調製したＨＭＧＢ１分解産物等の確認）
　前記参考例１で調製したＨＭＧＢ１、前記ＨＭＧＢ１分解産物、ＨＭＧＢ２、及び前記ＨＭＧＢ２分解産物を、ＳＤＳ−ポリアクリルアミドゲル電気泳動法で確認した。
１．試薬
　次の（１）~（４）の試薬をそれぞれ調製又は用意した。
（１）ＳＤＳ−ポリアクリルアミドゲル
　Ｆｕｎａｋｏｓｈｉ　Ｅａｓｙ−Ｇｅｌ（ＩＩＩ）　プリキャスト・ゲル（１５％）（フナコシ社〔日本国〕）を使用した。
（２）泳動槽用緩衝液
　トリス（ヒドロキシメチル）アミノメタン〔Ｔｒｉｓ〕１．５ｇ、ドデシル硫酸ナトリウム〔ＳＤＳ〕０．５ｇ、及びグリシン７．２ｇを純水に添加、混合した後５００ｍＬとして、泳動槽用緩衝液〔０．１％ＳＤＳ−１９２ｍＭグリシン−２５ｍＭトリス緩衝液〕を調製した。
（３）クマシーブリリアントブルー染色液
　Ｑｕｉｃｋ−ＣＢＢ（和光純薬工業社〔日本国〕）を使用した。
（４）試料処理液
　ドデシル硫酸ナトリウム〔ＳＤＳ〕０．４ｇ、２−メルカプトエタノール１．２ｍＬ、１Ｍのトリス（ヒドロキシメチル）アミノメタン〔Ｔｒｉｓ〕−塩酸緩衝液（ｐＨ６．８）の１ｍＬ、及びグリセリン２ｍＬを純水に添加、混合した後１０ｍＬとして、試料処理液〔４％ＳＤＳ−１２％２−メルカプトエタノール−２０％グリセリン−１００ｍＭトリス緩衝液〕を調製した。
２．試料
　次の前記参考例１の〔１〕~〔３〕で調製したＨＭＧＢ１、前記ＨＭＧＢ１分解産物、ＨＭＧＢ２、及び前記ＨＭＧＢ２分解産物をそれぞれ試料とした。また、次の分子量マーカー及びトロンビンも試料とした。
（ａ）分子量マーカー〔Ｐｒｅｃｉｓｉｏｎ　Ｐｌｕｓ　Ｐｒｏｔｅｉｎ　Ａｌｌ　Ｂｌｕｅ　Ｓｔａｎｄａｒｄｓマーカー；マーカー分子量　１０ＫＤａ、１５ＫＤａ、２０ＫＤａ、２５ＫＤａ、３７ＫＤａ、５０ＫＤａ、７５ＫＤａ、１００ＫＤａ、１５０ＫＤａ及び２５０ＫＤａ；ＢＩＯ−ＲＡＤ　Ｌａｂｏｒａｔｏｒｉｅｓ社（米国）〕
（ｂ）ＨＭＧＢ１
（ｃ）前記ＨＭＧＢ１分解産物
（ｄ）ＨＭＧＢ２
（ｅ）前記ＨＭＧＢ２分解産物
（ｆ）トロンビン〔ＧＥ　Ｈｅａｌｔｈｃａｒｅ　Ｂｉｏ−Ｓｃｉｅｎｃｅｓ社（スウェーデン国）〕
３．電気泳動
　前記１で調製した試薬を使用し、前記２の試料のそれぞれについて、次の操作によりＳＤＳ−ポリアクリルアミドゲル電気泳動法での電気泳動を行った。
（１）　前記２の（ｂ）~（ｆ）の試料のそれぞれについて、前記１の（４）の試料処理液と１：１の量比で混合し、１００℃で５分間処理した。
（２）　下部泳動槽に前記１の（２）の泳動槽用緩衝液を入れた。次に、前記１の（１）のゲルを泳動槽にセットした。そして次に、上部泳動槽に前記１の（２）の泳動槽用緩衝液を入れた。
（３）　前記（１）の処理を行った各試料の１０μＬ及び前記２の（ａ）の分子量マーカーの試料２μＬのそれぞれについて、前記（２）のゲルのコウム穴に注入した。
　なお、このゲルに注入した前記（１）の処理を行った試料及び前記２の（ａ）の分子量マーカーの試料であるが、レーン１に前記の「分子量マーカー」を、その右側のレーン２に前記の「ＨＭＧＢ１」を、その右側のレーン３に前記の「前記ＨＭＧＢ１分解産物」を、その右側のレーン４に前記の「ＨＭＧＢ２」を、その右側のレーン５に前記の「前記ＨＭＧＢ２分解産物」を、そしてその右側のレーン６に前記の「トロンビン」を注入した。
（４）　次に、２０ｍＡの電流で９０分間泳動を行った。
（５）　前記（４）の泳動を終了した後、ゲルを前記１の（３）のＱｕｉｃｋ−ＣＢＢを用いて染色した。
４．結果
　前記３の（５）で染色したゲルを図１に示した。
　このゲルの電気泳動像において、「前記ＨＭＧＢ１分解産物」（「３」と表記したレーン）のバンドは「ＨＭＧＢ１」（「２」と表記したレーン）のバンドよりも低分子量側に存在し、そして、「前記ＨＭＧＢ２分解産物」（「５」と表記したレーン）のバンドは「ＨＭＧＢ２」（「４」と表記したレーン）のバンドよりも低分子量側に存在することが分かる。
　すなわち、「前記ＨＭＧＢ１分解産物」及び「前記ＨＭＧＢ２分解産物」をそれぞれ調製できていることが確認できた。
〔実施例１〕（ＨＭＧＢ１分解産物に結合する抗体の調製−１）
　前記ＨＭＧＢ１分解産物に結合する抗体の調製を下記のようにして行った。
（１）　前記参考例１の〔１〕で調製したＨＭＧＢ１（全体長のもの）を免疫原として用いた。
　この免疫原としての前記参考例１の〔１〕で調製したＨＭＧＢ１（全体長のもの）の溶液の１容量に対して、化学合成アジュバントとしてのＦＲＥＵＮＤ完全アジュバント（ＤＩＦＣＯ　ＬＡＢＯＲＡＴＯＲＩＥＳ社）を１容量の割合で混合して、ＨＭＧＢ１溶液とＦＲＥＵＮＤ完全アジュバントとの混合物を調製した。
　また、この免疫原としての前記参考例１の〔１〕で調製したＨＭＧＢ１（全体長のもの）の溶液の１容量に対して、化学合成アジュバントとしてのＦＲＥＵＮＤ不完全アジュバント（ＤＩＦＣＯ　ＬＡＢＯＲＡＴＯＲＩＥＳ社）を１容量の割合で混合して、ＨＭＧＢ１溶液とＦＲＥＵＮＤ不完全アジュバントとの混合物を調製した。
（２）　次に、マウス（ＢＡＬＢ／ｃ）の腹腔内に免疫原として、３００~５００μｇ／匹／回の前記のＨＭＧＢ１溶液とＦＲＥＵＮＤ完全アジュバントとの混合物を注射し、２週間後及び４週間後に、マウスの腹腔内に前記のＨＭＧＢ１溶液とＦＲＥＵＮＤ不完全アジュバントとの混合物を注射した。
（３）　最終免疫より４週間後に、前記参考例１の〔１〕で調製したＨＭＧＢ１（全体長のもの）の原液を３００μｇ／匹によりブースターを行い、その翌日に、免疫したマウスの脾臓の細胞と、ミエローマ細胞（Ｐ３Ｕ１）を１対１から１０対１の割合で混合し、一般的な方法でポリエチレングリコール〔ＰＥＧ１５００；Ｒｏｃｈｅ社（スイス国）〕を加えて細胞融合させ、生育したハイブリドーマコロニーを選別した。
　具体的には、細胞融合は次のように行った。
　混合した前記脾臓細胞とミエローマ細胞（Ｐ３Ｕ１）を遠心して上清を除き、室温でポリエチレングリコール〔ＰＥＧ１５００；Ｒｏｃｈｅ社（スイス国）〕１ｍＬに１分間かけて懸濁した後、３７℃で１分間撹拌した。
　血清不含培地１ｍＬを１分間かけて加える操作を２回行い、その後、血清不含培地７ｍＬを２分間かけて加えた。
　細胞を数回洗浄した後、ヒポキサンチン、アミノプテリン、チミジン含有培地に懸濁して９６穴マイクロタイタープレートに分注して、３７℃において５％ＣＯ２存在下で培養した。
　生育したモノクローナル抗体産生細胞株（融合細胞株）の選別の方法としては、細胞融合から７~１４日後、前記の参考例１で調製したＨＭＧＢ１（全体長のもの）を固相化し、融合細胞培養上清を一次抗体としたＥＬＩＳＡ法の系にて行った。
　このＥＬＩＳＡ法は具体的には、次のように行った。
（ｉ）　前記の参考例１で調製したＨＭＧＢ１、及びＨＭＧＢ２のそれぞれをリン酸緩衝生理食塩水（０．９％塩化ナトリウム水溶液）により１μｇ／ｍＬの濃度となるように調製したもの、又は対照としてのリン酸緩衝生理食塩水（０．９％塩化ナトリウム水溶液）を、各々９６穴マイクロタイタープレート〔Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ　Ｉｎｃ．社（米国・イリノイ州）〕のウェルに１００μＬ注入し、５℃、１６~２４時間（又は３７℃、２時間）静置し、前記のＨＭＧＢ１、及びＨＭＧＢ２のそれぞれを、前記マイクロタイタープレートのウェルに固相化し、更に１％ＢＳＡを含むリン酸緩衝生理食塩水にて５℃で１６~２４時間（又は３７℃、２時間）静置し、ブロッキングした。
（ｉｉ）　次に、前記（ｉ）のマイクロタイタープレートの各ウェルを洗浄液〔０．０５％のＴｗｅｅｎ２０を含有するリン酸緩衝生理食塩水〕で３回洗浄した。
（ｉｉｉ）　次に、前記（ｉｉ）で洗浄を行ったマイクロタイタープレートのウェルに、これらの融合細胞培養上清溶液のそれぞれを試料として１００μＬ注入し、３７℃で２時間静置し、前記マイクロタイタープレートのウェルに固相化された前記のＨＭＧＢ１、及びＨＭＧＢ２のそれぞれと、前記の各融合細胞培養上清溶液に含まれるモノクローナル抗体とを各々反応させた。
（ｉｖ）　次に、前記（ｉｉｉ）のマイクロタイタープレートの各ウェルを前記の洗浄液で３回洗浄した。
（ｖ）　次に、前記（ｉｖ）で洗浄を行ったマイクロタイタープレートの各ウェルに、１％ＢＳＡを含むリン酸緩衝生理食塩水によって１０００倍希釈したＰＯＤ標識抗マウスＩｇＧ抗体〔ＤａｋｏＣｙｔｏｍａｔｉｏｎ社（デンマーク国）〕を１００μＬ注入し、３７℃で２時間静置し、反応を行わせた。
（ｖｉ）　次に、前記（ｖ）のマイクロタイタープレートの各ウェルを前記の洗浄液で３回洗浄した。
（ｖｉｉ）　次に、０．２ｍＭのＥＤＴＡ・２ナトリウムを含む０．０４５％の３，３’，５，５’−テトラメチルベンジジン塩酸塩水溶液（ｐＨ２．０）よりなる発色液と、５ｍＭ過酸化水素、４１ｍＭクエン酸、０．２ｍＭのＥＤＴＡ・２ナトリウムを含む６０ｍＭリン酸二ナトリウム水溶液（ｐＨ４．３）よりなる基質液とを、１：１で混合して調製した発色基質液の１００μＬを、前記（ｖｉ）で洗浄を行ったマイクロタイタープレートの各ウェルに注入し、室温にて５~３０分間静置し、反応を行わせ、発色させた。その後、０．７Ｎ硫酸よりなる反応停止液を各ウェルに１００μＬ分注し、発色反応を停止させた。
（ｖｉｉｉ）　次に、前記（ｖｉｉ）のマイクロタイタープレートの各ウェルについて、分光光度計を用いて、主波長４５０ｎｍ及び副波長５５０ｎｍにおける吸光度を測定した。前記の測定により、ＨＭＧＢ１に結合する抗体を産生する融合細胞株を選別し、そして、生育した融合細胞株の中から１つのクローンを確立し、２Ｈ６株と命名した。
（４）　この選別したモノクローナル抗体産生細胞株からＩｇＧ（免疫グロブリンＧ）を次のように精製した。このモノクローナル抗体産生細胞株を、ＰＦＨＭ−ＩＩ（ＧＩＢＣＯ社）を用いてＣＯ２インキュベータ内３７℃で培養した。培養後、上清中のＩｇＧをプロテインＡカラム〔ＧＥ　Ｈｅａｌｔｈｃａｒｅ　Ｂｉｏ−Ｓｃｉｅｎｃｅｓ社（スウェーデン国）〕に結合させた。結合させたＩｇＧを、１００ｍＭクエン酸水溶液（ｐＨ３．０）で溶出した。溶出液１容量に対して、０．５Ｍリン酸緩衝液（ｐＨ７．５）緩衝液１容量を添加し、精製ＩｇＧとして、前記のＨＭＧＢ１に結合する抗体を前記のモノクローナル抗体産生細胞株より取得した。
　この抗体は、前記ＨＭＧＢ１分解産物に結合する抗体であった。すなわち、２Ｈ６株のモノクローナル抗体産生細胞株から前記ＨＭＧＢ１分解産物に結合する抗体（モノクローナル抗体）〔以下、「本抗ＨＭＧＢ１分解産物抗体（２Ｈ６）」という）を得ることができた。
　なお、この本抗ＨＭＧＢ１分解産物抗体（２Ｈ６）は、後述するように、前記ＨＭＧＢ１分解産物に対する親和性が、ＨＭＧＢ１に対する親和性と比較して、１．５倍以上であった。
　また、この本抗ＨＭＧＢ１分解産物抗体（２Ｈ６）は、これも後述するように、前記ＨＭＧＢ１分解産物に対する親和性が、ＨＭＧＢ２に対する親和性及び前記ＨＭＧＢ２分解産物に対する親和性とそれぞれ比較して、各々１０倍以上であった。
　すなわち、この本抗ＨＭＧＢ１分解産物抗体（２Ｈ６）は、本発明の前記ＨＭＧＢ１分解産物の測定方法及び測定試薬における（ａ）の抗体である。
　なお、この「本抗ＨＭＧＢ１分解産物抗体（２Ｈ６）」のモノクローナル抗体産生細胞株である２Ｈ６株は、独立行政法人製品評価技術基盤機構の特許微生物寄託センター（日本国千葉県木更津市かずさ鎌足二丁目５番８号）に、「受領番号：ＮＩＴＥ　ＡＰ−１５７０」として２０１３年３月１５日付けにて受領されている。
〔実施例２〕（ＨＭＧＢ１分解産物に結合する抗体の調製−２）
　前記ＨＭＧＢ１分解産物に結合する抗体の調製に当たり、前記参考例１の〔１〕で調製したＨＭＧＢ１（全体長のもの）を免疫原として用いた。
　以下、前記実施例１の（１）~（４）の記載の通りに操作を行い、前記ＨＭＧＢ１分解産物に結合する抗体の調製を行った。
　その結果、生育した融合細胞株の中から１つのクローンを確立し、５Ｄ１株と命名した。そして、５Ｄ１株のモノクローナル抗体産生細胞株から前記ＨＭＧＢ１分解産物に結合する抗体（モノクローナル抗体）〔以下、「抗ＨＭＧＢ１分解産物等抗体（５Ｄ１）」という）を得ることができた。なお、この抗ＨＭＧＢ１分解産物等抗体（５Ｄ１）は、後述するように、前記ＨＭＧＢ１分解産物に対する親和性が高い抗体であった。すなわち、この抗ＨＭＧＢ１分解産物等抗体（５Ｄ１）は、本発明の前記ＨＭＧＢ１分解産物の測定方法及び測定試薬における（ｂ）の抗体である。
　なお、この「抗ＨＭＧＢ１分解産物等抗体（５Ｄ１）」のモノクローナル抗体産生細胞株である５Ｄ１株は、独立行政法人製品評価技術基盤機構の特許微生物寄託センター（日本国千葉県木更津市かずさ鎌足二丁目５番８号）に、「受領番号：ＮＩＴＥ　ＡＰ−１５７１」として２０１３年３月１５日付けにて受領されている。
〔実施例３〕（ＨＭＧＢ１分解産物に結合する抗体の調製−３）
　前記ＨＭＧＢ１分解産物に結合する抗体の調製に当たり、前記参考例１の〔１〕で調製したＨＭＧＢ１（全体長のもの）を免疫原として用いた。
　以下、前記実施例１の（１）~（４）の記載の通りに操作を行い、前記ＨＭＧＢ１分解産物に結合する抗体の調製を行った。
　その結果、生育した融合細胞株の中から１つのクローンを確立し、２Ａ１０株と命名した。そして、２Ａ１０株のモノクローナル抗体産生細胞株から前記ＨＭＧＢ１分解産物に結合する抗体（モノクローナル抗体）〔以下、「抗ＨＭＧＢ１分解産物等抗体（２Ａ１０）」という）を得ることができた。なお、この抗ＨＭＧＢ１分解産物等抗体（２Ａ１０）は、後述するように、前記ＨＭＧＢ１分解産物に対する親和性が高い抗体であった。すなわち、この抗ＨＭＧＢ１分解産物等抗体（２Ａ１０）は、本発明の前記ＨＭＧＢ１分解産物の測定方法及び測定試薬における（ｂ）の抗体である。
　なお、この「抗ＨＭＧＢ１分解産物等抗体（２Ａ１０）」のモノクローナル抗体産生細胞株である２Ａ１０株は、独立行政法人製品評価技術基盤機構の特許微生物寄託センター（日本国千葉県木更津市かずさ鎌足二丁目５番８号）に、「受領番号：ＮＩＴＥ　ＡＰ−１５７２」として２０１３年３月１５日付けにて受領されている。
〔実施例４〕（ＨＭＧＢ１分解産物に結合する抗体の調製−４）
　前記ＨＭＧＢ１分解産物に結合する抗体の調製に当たり、前記参考例１の〔１〕で調製したＨＭＧＢ１（全体長のもの）を免疫原として用いた。
　以下、前記実施例１の（１）~（４）の記載の通りに操作を行い、前記ＨＭＧＢ１分解産物に結合する抗体の調製を行った。
　その結果、生育した融合細胞株の中から１つのクローンを確立し、６Ｈ３株と命名した。そして、６Ｈ３株のモノクローナル抗体産生細胞株から前記ＨＭＧＢ１分解産物に結合する抗体（モノクローナル抗体）〔以下、「抗ＨＭＧＢ１分解産物等抗体（６Ｈ３）」という）を得ることができた。なお、この抗ＨＭＧＢ１分解産物等抗体（６Ｈ３）は、後述するように、前記ＨＭＧＢ１分解産物に対する親和性が高い抗体であった。すなわち、この抗ＨＭＧＢ１分解産物等抗体（６Ｈ３）は、本発明の前記ＨＭＧＢ１分解産物の測定方法及び測定試薬における（ｂ）の抗体である。
　なお、この「抗ＨＭＧＢ１分解産物等抗体（６Ｈ３）」のモノクローナル抗体産生細胞株である６Ｈ３株は、独立行政法人製品評価技術基盤機構の特許微生物寄託センター（日本国千葉県木更津市かずさ鎌足二丁目５番８号）に、「受領番号：ＮＩＴＥ　ＡＰ−１５７３」として２０１３年３月１５日付けにて受領されている。
〔実施例５〕（ＨＭＧＢ１分解産物に結合する抗体の反応性の確認）
　前記実施例１、及び実施例２において取得した、ＨＭＧＢ１分解産物に結合する抗体の各々について、ＨＭＧＢ１、前記ＨＭＧＢ１分解産物、ＨＭＧＢ２、及び前記ＨＭＧＢ２分解産物それぞれとの反応性を確認した。
〔１〕抗ＨＭＧＢ１分解産物等抗体（５Ｄ１）
　前記実施例２において取得した「抗ＨＭＧＢ１分解産物等抗体（５Ｄ１）」について、ＨＭＧＢ１、前記ＨＭＧＢ１分解産物、ＨＭＧＢ２、及び前記ＨＭＧＢ２分解産物それぞれとの反応性を以下の通り確かめた。
１．ＳＤＳ−ポリアクリルアミドゲル電気泳動法
（１）試薬
　次の（ａ）~（ｃ）の試薬をそれぞれ調製した。
（ａ）ＳＤＳ−ポリアクリルアミドゲル
　Ｆｕｎａｋｏｓｈｉ　Ｅａｓｙ−Ｇｅｌ（ＩＩＩ）　プリキャスト・ゲル（１５％）（フナコシ社〔日本国〕）を使用した。
（ｂ）泳動槽用緩衝液
　前記参考例２の１の（２）の記載の通りに調製を行い、泳動槽用緩衝液〔０．１％ＳＤＳ−１９２ｍＭグリシン−２５ｍＭトリス緩衝液〕を調製した。
（ｃ）試料処理液
　ドデシル硫酸ナトリウム〔ＳＤＳ〕０．１ｇ、２−メルカプトエタノール０．１ｍＬ、５００ｍＭのトリス（ヒドロキシメチル）アミノメタン〔Ｔｒｉｓ〕−塩酸緩衝液（ｐＨ６．８）の１ｍＬ、及びグリセリン２ｍＬを純水に添加、混合した後１０ｍＬとして、試料処理液〔１％ＳＤＳ−１％２−メルカプトエタノール−２０％グリセリン−５０ｍＭトリス緩衝液〕を調製した。
（２）試料
　前記参考例１の〔１〕~〔３〕で調製した、ＨＭＧＢ１、前記ＨＭＧＢ１分解産物、ＨＭＧＢ２、及び前記ＨＭＧＢ２分解産物のそれぞれを試料（標準試料）として用いた。
　また、分子量マーカー〔Ｐｒｅｃｉｓｉｏｎ　Ｐｌｕｓ　Ｐｒｏｔｅｉｎ　Ａｌｌ　Ｂｌｕｅ　Ｓｔａｎｄａｒｄｓマーカー；マーカー分子量　１０ＫＤａ、１５ＫＤａ、２０ＫＤａ、２５ＫＤａ、３７ＫＤａ、５０ＫＤａ、７５ＫＤａ、１００ＫＤａ、１５０ＫＤａ及び２５０ＫＤａ；ＢＩＯ−ＲＡＤ　Ｌａｂｏｒａｔｏｒｉｅｓ社（米国）〕も試料として用いた。
（３）電気泳動
　前記（１）で調製した試薬を使用し、前記（２）の試料のそれぞれについて、次の操作によりＳＤＳ−ポリアクリルアミドゲル電気泳動法での電気泳動を行った。
（ａ）　前記（２）の試料のそれぞれについて、前記（１）の（ｃ）の試料処理液により試料濃度が１μｇ／ｍＬとなるように調製し、１００℃で５分間、煮沸処理を行った。
（ｂ）　下部泳動槽に前記（１）の（ｂ）の泳動槽用緩衝液を入れた。次に、前記（１）の（ａ）のＳＤＳ−ポリアクリルアミドゲルを泳動槽にセットした。そして次に、上部泳動槽に前記（１）の（ｂ）の泳動槽用緩衝液を入れた。
（ｃ）　前記（ａ）の処理を行った試料の１０μＬ（１０ｎｇ）を前記（ｂ）のゲルのコウム穴に注入した。また、前記（２）の分子量マーカーの試料の２μＬを前記（ｂ）のゲルのコウム穴に注入した。
　なお、このゲルに注入した前記の試料及び分子量マーカーであるが、向かって左側のレーンより、次の「１」の「分子量マーカー」、「２」の「ＨＭＧＢ１」、「３」の「前記ＨＭＧＢ１分解産物」、「４」の「ＨＭＧＢ２」、及び「５」の「前記ＨＭＧＢ２分解産物」を順に注入した。
　「１」：「分子量マーカー」
　「２」：「ＨＭＧＢ１」
　「３」：「前記ＨＭＧＢ１分解産物」
　「４」：「ＨＭＧＢ２」
　「５」：「前記ＨＭＧＢ２分解産物」
（ｄ）　次に、２０ｍＡの電流で９０分間泳動を行った。
（ｅ）　前記（ｄ）の泳動を終了した後、ゲルをガラスプレートより取り出した。
　以上の操作により、ＨＭＧＢ１、前記ＨＭＧＢ１分解産物、ＨＭＧＢ２、及び前記ＨＭＧＢ２分解産物のそれぞれを、その分子量に従ってゲル中に位置させ、このゲルを取得した。
２．ウエスタンブロッティング法
（１）　抗体希釈液〔０．５％のカゼイン、１００ｍＭの塩化ナトリウム、及び０．１％のアジ化ナトリウムを含有する５０ｍＭのトリス（ヒドロキシメチル）アミノメタン緩衝液［Ｔｒｉｓ緩衝液］（ｐＨ８．０）〕により２μｇ／ｍＬとなるように、実施例２で取得した「抗ＨＭＧＢ１分解産物等抗体（５Ｄ１）」を希釈し、この抗体の溶液を調製した。
（２）　前記１の（３）の（ｅ）で取得したゲルの転写を、トランスブロットＳＤセル（ＢＩＯ−ＲＡＤ　Ｌａｂｏｒａｔｏｒｉｅｓ社〔米国〕）を用いて、その使用説明書に従い、セミドライ方式で行った。
　まず、前記１の（３）の（ｅ）で取得したゲルを転写用装置上に置いた。
　次に、このゲルの上に、９ｃｍ×９ｃｍのポリビニルジフルオライド膜（ＢＩＯ−ＲＡＤ　Ｌａｂｏｒａｔｏｒｉｅｓ社〔米国〕）を重ね、４８ｍＭトリス（ヒドロキシメチル）アミノメタン〔Ｔｒｉｓ〕、３９ｍＭグリシン、及び２０％（Ｖ／Ｖ）メタノールよりなる転写用緩衝液を用いて、電流１００ｍＡで１時間転写を行い、その分子量に従ってゲル中に位置している前記のＨＭＧＢ１、前記ＨＭＧＢ１分解産物、ＨＭＧＢ２、及び前記ＨＭＧＢ２分解産物等のタンパク質を前記ゲルより前記ポリビニルジフルオライド膜に転写した。
（３）　この前記の各々のＨＭＧＢ１、前記ＨＭＧＢ１分解産物、ＨＭＧＢ２、及び前記ＨＭＧＢ２分解産物等のタンパク質の転写を行ったポリビニルジフルオライド膜を、ブロッキング液〔０．５％のカゼイン、１００ｍＭの塩化ナトリウム、及び０．１％のアジ化ナトリウムを含有する５０ｍＭのトリス（ヒドロキシメチル）アミノメタン緩衝液［Ｔｒｉｓ緩衝液］（ｐＨ８．０）〕の２０ｍＬに室温で一晩浸漬して、ブロッキングを行った。
（４）　次に、前記（１）で調製した「抗ＨＭＧＢ１分解産物等抗体（５Ｄ１）」の溶液に、前記（３）のポリビニルジフルオライド膜を室温で２時間浸漬して反応させた。
　これにより、ポリビニルジフルオライド膜に転写された、前記の各々のＨＭＧＢ１、前記ＨＭＧＢ１分解産物、ＨＭＧＢ２、及び前記ＨＭＧＢ２分解産物と、前記の「抗ＨＭＧＢ１分解産物等抗体（５Ｄ１）」とを反応させた。
（５）　前記（４）の操作を行ったポリビニルジフルオライド膜を、２０ｍＬの洗浄液〔０．０５％のＴｗｅｅｎ２０を含むリン酸緩衝生理食塩水〕の中で５分間振とう洗浄を行った。これを３回行った。
（６）　次に、ＰＯＤ標識抗マウスＩｇＧ抗体〔ＤａｋｏＣｙｔｏｍａｔｉｏｎ社（デンマーク国）〕を、標識抗体希釈液〔０．５％のカゼイン、及び１００ｍＭの塩化ナトリウムを含有する５０ｍＭのトリス（ヒドロキシメチル）アミノメタン緩衝液［Ｔｒｉｓ緩衝液］（ｐＨ８．０）〕により１０００倍に希釈して調製した溶液に、前記（５）のポリビニルジフルオライド膜を室温で９０分間浸漬して反応させた。
（７）　前記（６）の操作を行ったポリビニルジフルオライド膜を、２０ｍＬの前記洗浄液中で５分間振とう洗浄を行った。この操作を３回行った。
（８）　０．０２５％の３，３’−ジアミノベンジジン四塩酸塩及び０．０１％過酸化水素を含むリン酸緩衝生理食塩水の２０ｍＬに、前記（７）のポリビニルジフルオライド膜を室温で１５分間浸漬して発色させた。
　これにより、このポリビニルジフルオライド膜において、前記の「抗ＨＭＧＢ１分解産物等抗体（５Ｄ１）」が認識し結合した、前記の各々のＨＭＧＢ１、前記ＨＭＧＢ１分解産物、ＨＭＧＢ２、又は前記ＨＭＧＢ２分解産物の分子量に応じた位置に、発色を生じさせた。
　このポリビニルジフルオライド膜上の発色の有無及びその発色した位置（分子量）より、前記の「抗ＨＭＧＢ１分解産物等抗体（５Ｄ１）」について、ＨＭＧＢ１、前記ＨＭＧＢ１分解産物、ＨＭＧＢ２、及び前記ＨＭＧＢ２分解産物のそれぞれとの反応性を確認した。
３．結果
（１）　前記２の（８）で発色させたポリビニルジフルオライド膜を図２に示した。
　この図において、左側のレーンより順に、「分子量マーカー」のレーン（「１」と表記）、「ＨＭＧＢ１」のレーン（「２」と表記）、「前記ＨＭＧＢ１分解産物」のレーン（「３」と表記）、「ＨＭＧＢ２」のレーン（「４」と表記）、及び「前記ＨＭＧＢ２分解産物」のレーン（「５」と表記）を表す。
（２）　この図より、「抗ＨＭＧＢ１分解産物等抗体（５Ｄ１）」においては、ＨＭＧＢ１、前記ＨＭＧＢ１分解産物、ＨＭＧＢ２、及び前記ＨＭＧＢ２分解産物をそれぞれ示す位置において発色が認められることが分かる。
　このことより、「抗ＨＭＧＢ１分解産物等抗体（５Ｄ１）」は、ＨＭＧＢ１、前記ＨＭＧＢ１分解産物、ＨＭＧＢ２、及び前記ＨＭＧＢ２分解産物のいずれをも認識し結合することが確かめられた。
〔２〕本抗ＨＭＧＢ１分解産物抗体（２Ｈ６）
　前記実施例１において取得した「本抗ＨＭＧＢ１分解産物抗体（２Ｈ６）」について、ＨＭＧＢ１、前記ＨＭＧＢ１分解産物、ＨＭＧＢ２、及び前記ＨＭＧＢ２分解産物それぞれとの反応性を確かめた。
　すなわち、「抗ＨＭＧＢ１分解産物等抗体（５Ｄ１）」に代えて、前記実施例１において取得した「本抗ＨＭＧＢ１分解産物抗体（２Ｈ６）」を用いること以外は、前記〔１〕の記載の通りに操作を行い、「本抗ＨＭＧＢ１分解産物抗体（２Ｈ６）」のＨＭＧＢ１、前記ＨＭＧＢ１分解産物、ＨＭＧＢ２、及び前記ＨＭＧＢ２分解産物それぞれとの反応性を確かめた。
　この反応性を確かめた結果である、発色させたポリビニルジフルオライド膜を図３に示した。
　この図において、左側のレーンより順に、「分子量マーカー」のレーン（「１」と表記）、「ＨＭＧＢ１」のレーン（「２」と表記）、「前記ＨＭＧＢ１分解産物」のレーン（「３」と表記）、「ＨＭＧＢ２」のレーン（「４」と表記）、及び「前記ＨＭＧＢ２分解産物」のレーン（「５」と表記）を表す。
　この図より、「本抗ＨＭＧＢ１分解産物抗体（２Ｈ６）」においては、前記ＨＭＧＢ１分解産物を示す位置において濃い発色が認められるものの、ＨＭＧＢ１を示す位置においては極めて薄い発色らしきものが見られるにすぎない。そして、ＨＭＧＢ２、及び前記ＨＭＧＢ２分解産物をそれぞれ示す位置においては、発色は認められない。
　このことより、「本抗ＨＭＧＢ１分解産物抗体（２Ｈ６）」は、前記ＨＭＧＢ１分解産物を特異性高く認識し結合することが確かめられた。そして、ＨＭＧＢ１とはわずかに結合し、ＨＭＧＢ２及び前記ＨＭＧＢ２分解産物とは結合しないことが確かめられた。
〔実施例６〕（ＨＭＧＢ１分解産物に結合する抗体の親和性の確認）
　前記実施例１~実施例４において取得したＨＭＧＢ１分解産物に結合する抗体及び他のＨＭＧＢ１に結合する抗体等の各々について、ＨＭＧＢ１、前記ＨＭＧＢ１分解産物、ＨＭＧＢ２、及び前記ＨＭＧＢ２分解産物それぞれとの親和性を、酵素免疫測定法（ＥＬＩＳＡ法）により確認した。
１．測定試薬
（１）ＨＭＧＢ１分解産物等固相化マイクロプレート
　前記参考例１の〔１〕~〔３〕で調製した、ＨＭＧＢ１、前記ＨＭＧＢ１分解産物、ＨＭＧＢ２、及び前記ＨＭＧＢ２分解産物のそれぞれをリン酸緩衝生理食塩水により１μｇ／ｍＬの濃度となるように調製したものを、各々９６穴マイクロタイタープレート〔Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ　Ｉｎｃ．社（米国・イリノイ州）〕のウェルに１００μＬ注入し、２５℃で１８時間静置し、前記のＨＭＧＢ１、前記ＨＭＧＢ１分解産物、ＨＭＧＢ２、及び前記ＨＭＧＢ２分解産物のそれぞれを、前記マイクロタイタープレートのウェルに固相化した。
　次に、このマイクロタイタープレートのウェル中の液を除去した後、０．５％カゼイン及び０．１％アジ化ナトリウムを含むトリス緩衝生理食塩水（ＴＢＳ）〔ｐＨ８．０〕の２５０μＬずつを各ウェルに分注し、ブロッキング処理を行った。この後、各ウェルの上をプレートシールで封をし、蒸発しないようにして、使用時まで冷蔵保存した。これを、ＨＭＧＢ１分解産物等固相化マイクロプレートとした。
（２）ＰＯＤ標識抗マウスＩｇＧ抗体液
　ＰＯＤ標識抗マウスＩｇＧ抗体〔ＤａｋｏＣｙｔｏｍａｔｉｏｎ社（デンマーク国）〕を、０．５％カゼインナトリウム、及び１００ｍＭ塩化ナトリウムを含む５０ｍＭトリス−塩酸緩衝液（ｐＨ８．０）によって１０００倍希釈した。これをＰＯＤ標識抗マウスＩｇＧ抗体液とした。
（３）洗浄液
　０．０５％のＴｗｅｅｎ２０を含むリン酸緩衝生理食塩水を、洗浄液とした。
（４）希釈液
　０．５％カゼインナトリウム、１００ｍＭ塩化ナトリウム及び０．１％アジ化ナトリウムを含む５０ｍＭトリス−塩酸緩衝液（ｐＨ８．０）を、希釈液とした。
（５）発色液
　０．２ｍＭのＥＤＴＡ・２ナトリウムを含む０．０４５％の３，３’，５，５’−テトラメチルベンジジン塩酸塩水溶液（ｐＨ２．０）を、発色液とした。
（６）基質液
　５ｍＭ過酸化水素、４１ｍＭクエン酸、０．２ｍＭのＥＤＴＡ・２ナトリウムを含む６０ｍＭリン酸二ナトリウム水溶液（ｐＨ４．３）を、基質液とした。
（７）発色基質
　前記の発色液と基質液を使用前に室温に戻した上で、使用時に等量混合し、発色基質とした。
（８）反応停止液
　０．７Ｎ硫酸を、反応停止液とした。
２．試料
　ＨＭＧＢ１、前記ＨＭＧＢ１分解産物、ＨＭＧＢ２、及び前記ＨＭＧＢ２分解産物それぞれとの親和性を確認する抗体の溶液を、次の通り調製し、試料として用いた。
（１）抗ＨＭＧＢ１等抗体（２Ｄ４）
　モノクローナル抗体産生細胞株の２Ｄ４株から産生されるＨＭＧＢ１等に結合する抗体（モノクローナル抗体）〔以下、「抗ＨＭＧＢ１等抗体（２Ｄ４）」という〕を、その濃度が各々、０．６２５ｎｇ／ｍＬ、１．２５ｎｇ／ｍＬ、２．５ｎｇ／ｍＬ、及び５ｎｇ／ｍＬとなるように前記１の（４）の希釈液により希釈して、各濃度の抗ＨＭＧＢ１等抗体（２Ｄ４）の抗体溶液を調製した。
（２）抗ＨＭＧＢ１等抗体（４Ｆ１２）
　モノクローナル抗体産生細胞株の４Ｆ１２株から産生されるＨＭＧＢ１等に結合する抗体（モノクローナル抗体）〔以下、「抗ＨＭＧＢ１等抗体（４Ｆ１２）」という〕を、その濃度が各々、０．６２５ｎｇ／ｍＬ、１．２５ｎｇ／ｍＬ、２．５ｎｇ／ｍＬ、及び５ｎｇ／ｍＬとなるように前記１の（４）の希釈液により希釈して、各濃度の抗ＨＭＧＢ１等抗体（４Ｆ１２）の抗体溶液を調製した。
（３）抗ＨＭＧＢ１等抗体（８Ｈ４）
　モノクローナル抗体産生細胞株の８Ｈ４株から産生されるＨＭＧＢ１等に結合する抗体（モノクローナル抗体）〔以下、「抗ＨＭＧＢ１等抗体（８Ｈ４）」という〕を、その濃度が各々、０．６２５ｎｇ／ｍＬ、１．２５ｎｇ／ｍＬ、２．５ｎｇ／ｍＬ、及び５ｎｇ／ｍＬとなるように前記１の（４）の希釈液により希釈して、各濃度の抗ＨＭＧＢ１等抗体（８Ｈ４）の抗体溶液を調製した。
（４）本抗ＨＭＧＢ１分解産物抗体（２Ｈ６）
　本抗ＨＭＧＢ１分解産物抗体（２Ｈ６）を、その濃度が各々、０．６２５ｎｇ／ｍＬ、１．２５ｎｇ／ｍＬ、２．５ｎｇ／ｍＬ、及び５ｎｇ／ｍＬとなるように前記１の（４）の希釈液により希釈して、各濃度の本抗ＨＭＧＢ１分解産物抗体（２Ｈ６）の抗体溶液を調製した。
（５）抗ＨＭＧＢ１分解産物等抗体（５Ｄ１）
　抗ＨＭＧＢ１分解産物等抗体（５Ｄ１）を、その濃度が各々、０．６２５ｎｇ／ｍＬ、１．２５ｎｇ／ｍＬ、２．５ｎｇ／ｍＬ、及び５ｎｇ／ｍＬとなるように前記１の（４）の希釈液により希釈して、各濃度の抗ＨＭＧＢ１分解産物等抗体（５Ｄ１）の抗体溶液を調製した。
（６）抗ＨＭＧＢ１分解産物等抗体（２Ａ１０）
　抗ＨＭＧＢ１分解産物等抗体（２Ａ１０）を、その濃度が各々、０．６２５ｎｇ／ｍＬ、１．２５ｎｇ／ｍＬ、２．５ｎｇ／ｍＬ、及び５ｎｇ／ｍＬとなるように前記１の（４）の希釈液により希釈して、各濃度の抗ＨＭＧＢ１分解産物等抗体（２Ａ１０）の抗体溶液を調製した。
（７）抗ＨＭＧＢ１分解産物等抗体（６Ｈ３）
　抗ＨＭＧＢ１分解産物等抗体（６Ｈ３）を、その濃度が各々、０．６２５ｎｇ／ｍＬ、１．２５ｎｇ／ｍＬ、２．５ｎｇ／ｍＬ、及び５ｎｇ／ｍＬとなるように前記１の（４）の希釈液により希釈して、各濃度の抗ＨＭＧＢ１分解産物等抗体（６Ｈ３）の抗体溶液を調製した。
（８）抗ＨＭＧＢ１等抗体（ＭＤ７８）
　モノクローナル抗体産生細胞株のＭＤ７８株（独立行政法人産業技術総合研究所特許生物寄託センター（日本国茨城県つくば市東１丁目１番地１中央第６）にＦＥＲＭ　Ｐ−１８４０５として平成１３年７月４日付けにて寄託されている。）から産生されるＨＭＧＢ１等に結合する抗体（モノクローナル抗体）〔以下、「抗ＨＭＧＢ１等抗体（ＭＤ７８）」という〕を、その濃度が各々、０．６２５ｎｇ／ｍＬ、１．２５ｎｇ／ｍＬ、２．５ｎｇ／ｍＬ、及び５ｎｇ／ｍＬとなるように前記１の（４）の希釈液により希釈して、各濃度の抗ＨＭＧＢ１等抗体（ＭＤ７８）の抗体溶液を調製した。
（９）抗ＨＭＧＢ１等抗体（ＭＤ７７）
　モノクローナル抗体産生細胞株のＭＤ７７株（独立行政法人産業技術総合研究所特許生物寄託センター（日本国茨城県つくば市東１丁目１番地１中央第６）にＦＥＲＭ　Ｐ−１８４０４として平成１３年７月４日付けにて寄託されている。）から産生されるＨＭＧＢ１等に結合する抗体（モノクローナル抗体）〔以下、「抗ＨＭＧＢ１等抗体（ＭＤ７７）」という〕を、その濃度が各々、０．６２５ｎｇ／ｍＬ、１．２５ｎｇ／ｍＬ、２．５ｎｇ／ｍＬ、及び５ｎｇ／ｍＬとなるように前記１の（４）の希釈液により希釈して、各濃度の抗ＨＭＧＢ１等抗体（ＭＤ７７）の抗体溶液を調製した。
（１０）抗ＨＭＧＢ１等抗体（４Ｃ３）
　モノクローナル抗体産生細胞株の４Ｃ３株から産生されるＨＭＧＢ１等に結合する抗体（モノクローナル抗体；商品名：Ａｎｔｉ−ＨＭＧＢ１　ａｎｔｉｂｏｄｙ　［４Ｃ３］（Ａｂｃａｍ社））〔以下、「抗ＨＭＧＢ１等抗体（４Ｃ３）」という〕を、その濃度が各々、０．６２５ｎｇ／ｍＬ、１．２５ｎｇ／ｍＬ、２．５ｎｇ／ｍＬ、及び５ｎｇ／ｍＬとなるように前記１の（４）の希釈液により希釈して、各濃度の抗ＨＭＧＢ１等抗体（４Ｃ３）の抗体溶液を調製した。
（１１）抗ＨＭＧＢ１等抗体（Ｊ２Ｅ１）
　モノクローナル抗体産生細胞株のＪ２Ｅ１株から産生されるＨＭＧＢ１等に結合する抗体（モノクローナル抗体；商品名：ＨＭＧ−１　Ａｎｔｉｂｏｄｙ（Ｊ２Ｅ１）：ｓｃ−１３５８０９（Ｓａｎｔａ　ｃｒｕｚ　ｂｉｏｔｅｃｈｎｏｌｏｇｙ，Ｉｎｃ．社））〔以下、「抗ＨＭＧＢ１等抗体（Ｊ２Ｅ１）」という〕を、その濃度が各々、０．６２５ｎｇ／ｍＬ、１．２５ｎｇ／ｍＬ、２．５ｎｇ／ｍＬ、及び５ｎｇ／ｍＬとなるように前記１の（４）の希釈液により希釈して、各濃度の抗ＨＭＧＢ１等抗体（Ｊ２Ｅ１）の抗体溶液を調製した。
（１２）抗ＨＭＧＢ１等抗体（ＨＡＰ４６．５）
　モノクローナル抗体産生細胞株のＨＡＰ４６．５株から産生されるＨＭＧＢ１等に結合する抗体（モノクローナル抗体；商品名：Ｍｏｕｓｅ　ｍｏｎｏｃｌｏｎａｌ［ＨＡＰ４６．５］　ｔｏ　ＨＭＧＢ１（Ａｂｃａｍ社））〔以下、「抗ＨＭＧＢ１等抗体（ＨＡＰ４６．５）」という〕を、その濃度が各々、０．６２５ｎｇ／ｍＬ、１．２５ｎｇ／ｍＬ、２．５ｎｇ／ｍＬ、及び５ｎｇ／ｍＬとなるように前記１の（４）の希釈液により希釈して、各濃度の抗ＨＭＧＢ１等抗体（ＨＡＰ４６．５）の抗体溶液を調製した。
３．測定
（１）　前記１の（１）のＨＭＧＢ１分解産物等固相化マイクロプレートの各ウェルを、前記１の（３）の洗浄液の４００μＬで３回洗浄した。
（２）　次に、前記２の（１）の４濃度の抗ＨＭＧＢ１等抗体（２Ｄ４）の溶液それぞれの１００μＬを試料として各ウェルに分注した後、２５℃で２時間静置し、これらの各濃度の抗ＨＭＧＢ１等抗体（２Ｄ４）を、このＨＭＧＢ１分解産物等固相化マイクロプレートのウェルに固相化された前記のＨＭＧＢ１、前記ＨＭＧＢ１分解産物、ＨＭＧＢ２、及び前記ＨＭＧＢ２分解産物の各々と、それぞれ抗原抗体反応を行わせた。
（３）　次に、各ウェルを、前記１の（３）の洗浄液の４００μＬで３回洗浄した。
（４）　次に、前記１の（２）のＰＯＤ標識抗マウスＩｇＧ抗体液の１００μＬを各ウェルに分注し、２５℃で１時間静置し、それぞれ抗原抗体反応を行わせた。
（５）　次に、各ウェルを、前記１の（３）の洗浄液の４００μＬで３回洗浄した。
（６）　次に、各ウェルに、前記１の（７）の発色基質を１００μＬ分注し、室温で２０分間静置し、標識酵素であるパーオキシダーゼ（ＰＯＤ）による発色反応を行わせた。
（７）　次に、各ウェルに、前記１の（８）の反応停止液を１００μＬ分注し、発色反応を停止させた。
（８）　次に、各ウェルの液の吸光度（主波長：４５０ｎｍ、副波長：５５０ｎｍ）を測定し、前記の４濃度の抗ＨＭＧＢ１等抗体（２Ｄ４）の溶液それぞれを試料として測定した場合の吸光度を得た。
（９）　前記（２）における４濃度の抗ＨＭＧＢ１等抗体（２Ｄ４）の溶液の代わりに、前記２の（２）~（１２）の各抗体の抗体溶液（各々４濃度ずつ）をそれぞれ試料として用いる以外は、前記（１）~（８）の記載の通りに行い、これらの各抗体の抗体溶液それぞれを試料として測定した場合の吸光度を得た。
４．測定結果
（１）　前記３における測定結果、すなわち、前記の各抗体について、ＨＭＧＢ１、前記ＨＭＧＢ１分解産物、ＨＭＧＢ２、及び前記ＨＭＧＢ２分解産物それぞれとの親和性を確認した結果を、図４Ａ~Ｌに示した。
　これらの図において、図の上方に当該親和性を確認した抗体の産生細胞株を表す文字を示した。
　また、これらの図において、横軸は試料として用いた抗体溶液中の抗体濃度（ｎｇ／ｍＬ）を示し、縦軸は測定により得られた吸光度〔主波長（４５０ｎｍ）における吸光度から副波長（５５０ｎｍ）における吸光度を差し引いたもの〕を示す。
　また、これらの図において、「◆」はＨＭＧＢ１を固相化したウェルにおける測定値（前記の吸光度）を示し、「■」は前記ＨＭＧＢ１分解産物を固相化したウェルにおける測定値（前記の吸光度）を示し、「▲」はＨＭＧＢ２を固相化したウェルにおける測定値（前記の吸光度）を示し、そして「●」は前記ＨＭＧＢ２分解産物を固相化したウェルにおける測定値（前記の吸光度）を示す。
（２）　また、前記３における測定結果の測定値を、表１に示した。
Figure JPOXMLDOC01-appb-T000001
 　これらの表において、表の上方に当該親和性を確認した抗体の産生細胞株を表す文字を示した。
　また、これらの表において、左側の欄には試料として用いた抗体溶液中の抗体濃度（ｎｇ／ｍＬ）を示し、そしてウェルに固相化した物ごとに測定により得られた吸光度〔主波長（４５０ｎｍ）における吸光度から副波長（５５０ｎｍ）における吸光度を差し引いたもの〕を示した。
　なお、これらの表においては、前記ＨＭＧＢ１分解産物を固相化したウェルにおける測定値（前記の吸光度）を試料として用いた抗体溶液中の抗体濃度（ｎｇ／ｍＬ）値で除したときの値を示した欄（ウェルに固相化した物ごとに左から３番目の欄）も設けた。
５．まとめ
（１）　前記の酵素免疫測定法の結果である図４及び表１より、前記測定により当該親和性を確認した各抗体は、次の（ｉ）~（ｉｉｉ）の３種類のものに分けることができることが分かる。
（ｉ）　前記ＨＭＧＢ１分解産物に結合する抗体であって、前記ＨＭＧＢ１分解産物に対する親和性が、ＨＭＧＢ１に対する親和性と比較して、少なくとも１．５倍である抗体（抗体濃度：０．６２５ｎｇ／ｍＬ~５ｎｇ／ｍＬ）：　「本抗ＨＭＧＢ１分解産物抗体（２Ｈ６）」
〔　※　なお、前記ＨＭＧＢ１分解産物に対する親和性において、ＨＭＧＢ２に対する親和性及び前記ＨＭＧＢ２分解産物に対する親和性とそれぞれ比較して、各々少なくとも１０倍である。（抗体濃度：０．６２５ｎｇ／ｍＬ~５ｎｇ／ｍＬ）〕
〔　※　本発明の試料に含まれる前記ＨＭＧＢ１分解産物の免疫学的測定方法及び測定試薬における、（ａ）の抗体〕
（ｉｉ）　前記ＨＭＧＢ１分解産物に結合する抗体であって、前記ＨＭＧＢ１分解産物に対する親和性が高い抗体：　「抗ＨＭＧＢ１分解産物等抗体（５Ｄ１）」、「抗ＨＭＧＢ１分解産物等抗体（２Ａ１０）」、及び「抗ＨＭＧＢ１分解産物等抗体（６Ｈ３）」
〔　※　なお、前記ＨＭＧＢ１分解産物を固相化したウェルにおける測定値（前記の吸光度）を試料として用いた抗体溶液中の抗体濃度（ｎｇ／ｍＬ）値で除したときの値が０．５以上である。（抗体濃度：０．６２５ｎｇ／ｍＬ~５ｎｇ／ｍＬ）〕
〔　※　また、前記ＨＭＧＢ１分解産物を固相化したウェルにおける測定値（前記の吸光度）が、基準とする抗体産生細胞（「ＭＤ７８」）より産生される抗体における当該吸光度の値の６倍以上である。（抗体濃度：０．６２５ｎｇ／ｍＬ~５ｎｇ／ｍＬ）〕
〔　※　本発明の試料に含まれる前記ＨＭＧＢ１分解産物の免疫学的測定方法及び測定試薬における、（ｂ）の抗体〕
（ｉｉｉ）　前記（ｉ）及び（ｉｉ）以外の抗体：　「抗ＨＭＧＢ１等抗体（２Ｄ４）」、「抗ＨＭＧＢ１等抗体（４Ｆ１２）」、「抗ＨＭＧＢ１等抗体（８Ｈ４）」、「抗ＨＭＧＢ１等抗体（ＭＤ７８）」、「抗ＨＭＧＢ１等抗体（ＭＤ７７）」、「抗ＨＭＧＢ１等抗体（４Ｃ３）」、「抗ＨＭＧＢ１等抗体（Ｊ２Ｅ１）」、及び「抗ＨＭＧＢ１等抗体（ＨＡＰ４６．５）」
（２）　これらのことより、「本抗ＨＭＧＢ１分解産物抗体（２Ｈ６）」は、前記ＨＭＧＢ１分解産物に結合する抗体であって、前記ＨＭＧＢ１分解産物に対する親和性が、ＨＭＧＢ１に対する親和性と比較して、少なくとも１．５倍である抗体であることが確かめられた。
　また、「抗ＨＭＧＢ１分解産物等抗体（５Ｄ１）」、「抗ＨＭＧＢ１分解産物等抗体（２Ａ１０）」、及び「抗ＨＭＧＢ１分解産物等抗体（６Ｈ３）」はそれぞれ、前記ＨＭＧＢ１分解産物に結合する抗体であって、前記ＨＭＧＢ１分解産物に対する親和性が高い抗体であることが確かめられた。
〔実施例７〕（ＨＭＧＢ１分解産物の測定の確認）
　前記実施例１~実施例４において取得したＨＭＧＢ１分解産物に結合する抗体及び他のＨＭＧＢ１に結合する抗体等を用い、本発明によるＨＭＧＢ１分解産物の測定が可能であることを確認した。
１．測定試薬
（１）抗体固相化マイクロプレート
　下記の（ｉ）~（ｖ）の５種類の抗体の各々について、プロテインＡを用いて精製し、これらの抗体をリン酸緩衝生理食塩水（ＰＢＳ）でその濃度が２．５μｇ／ｍＬとなるようにそれぞれ希釈した。
（ｉ）本抗ＨＭＧＢ１分解産物抗体（２Ｈ６）
（ｉｉ）抗ＨＭＧＢ１分解産物等抗体（５Ｄ１）
（ｉｉｉ）抗ＨＭＧＢ１分解産物等抗体（２Ａ１０）
（ｉｖ）抗ＨＭＧＢ１分解産物等抗体（６Ｈ３）
（ｖ）抗ＨＭＧＢ１等抗体（ＭＤ７７）
　次に、これらの抗体溶液の各々を、マイクロタイタープレート（マイクロプレート）〔Ｎｕｎｃ社、商品名：Ｍａｘｉｓｏｒｐ〕の各ウェルに１００μＬずつ分注し、２５℃で一晩静置し、これらの抗体のそれぞれを個別にマイクロプレートのウェルに固相化した。
　次に、マイクロプレートのウェル中の液を除去し、０．５％カゼインナトリウム及び０．１％アジ化ナトリウムを含むトリス緩衝生理食塩水（ＴＢＳ）〔ｐＨ８．０〕の２５０μＬずつを各ウェルに分注し、各ウェルの上をプレートシールで封をし、蒸発しないようにして、使用時まで冷蔵保存した。
　これを、前記の（ｉ）~（ｖ）の５種類の抗体の各々についての抗体固相化マイクロプレートとした。
（２）ＰＯＤ標識抗体液
　下記の（Ａ）~（Ｆ）の６種類の抗体の各々について、それぞれ「パーオキシダーゼ　ラベリングキット−ＮＨ２」（同仁化学社）により所定の方法にて、６種類のパーオキシダーゼ標識抗体を調製し、これらをＰＯＤ標識抗体液とした。
（Ａ）本抗ＨＭＧＢ１分解産物抗体（２Ｈ６）
（Ｂ）抗ＨＭＧＢ１分解産物等抗体（５Ｄ１）
（Ｃ）抗ＨＭＧＢ１分解産物等抗体（２Ａ１０）
（Ｄ）抗ＨＭＧＢ１分解産物等抗体（６Ｈ３）
（Ｅ）抗ＨＭＧＢ１等抗体（ＭＤ７７）
（Ｆ）抗ＨＭＧＢ１等抗体（０４）　〔モノクローナル抗体産生細胞株の０４株から産生されるＨＭＧＢ１等に結合するマウス抗体（モノクローナル抗体）［以下、「抗ＨＭＧＢ１等抗体（０４）」という］。〕
（３）洗浄液
　０．０５％のＴｗｅｅｎ２０を含むリン酸緩衝生理食塩水を、洗浄液とした。
（４）希釈液
　０．５％カゼインナトリウム、１００ｍＭ塩化ナトリウム及び０．１％アジ化ナトリウムを含む５０ｍＭトリス−塩酸緩衝液（ｐＨ８．０）を、希釈液とした。
（５）発色液
　０．２ｍＭのＥＤＴＡ・２ナトリウムを含む０．０４５％の３，３’，５，５’−テトラメチルベンジジン塩酸塩水溶液（ｐＨ２．０）を、発色液とした。
（６）基質液
　５ｍＭ過酸化水素、４１ｍＭクエン酸、０．２ｍＭのＥＤＴＡ・２ナトリウムを含む６０ｍＭリン酸二ナトリウム水溶液（ｐＨ４．３）を、基質液とした。
（７）発色基質
　前記の発色液と基質液を使用前に室温に戻した上で、使用時に等量混合し、発色基質とした。
（８）反応停止液
　０．７Ｎ硫酸を、反応停止液とした。
２．試料
（１）試料（前記ＨＭＧＢ１分解産物）
　前記参考例１の〔２〕で調製した前記ＨＭＧＢ１分解産物を、０．５％カゼインナトリウム、１００ｍＭ塩化ナトリウム及び０．１％アジ化ナトリウムを含む５０ｍＭトリス−塩酸緩衝液（ｐＨ８．０）により、１６ｎｇ／ｍＬの濃度となるように調製したものを試料（前記ＨＭＧＢ１分解産物）とした。
（２）試料（ＨＭＧＢ１）
　前記参考例１の〔１〕で調製したＨＭＧＢ１を、０．５％カゼインナトリウム、１００ｍＭ塩化ナトリウム及び０．１％アジ化ナトリウムを含む５０ｍＭトリス−塩酸緩衝液（ｐＨ８．０）により、１６ｎｇ／ｍＬの濃度となるように調製したものを試料（ＨＭＧＢ１）とした。
（３）試料（試薬盲検）
　対照としての試薬盲検（試薬ブランク）測定のための、０．５％カゼインナトリウム、１００ｍＭ塩化ナトリウム及び０．１％アジ化ナトリウムを含む５０ｍＭトリス−塩酸緩衝液（ｐＨ８．０）〔前記ＨＭＧＢ１分解産物及びＨＭＧＢ１の濃度が０ｎｇ／ｍＬ〕を、試料（試薬盲検）とした。
３．測定
（１）　前記１の（１）の（ｉ）~（ｖ）の５種類の抗体の各々についての抗体固相化マイクロプレートそれぞれの各ウェルを、前記１の（３）の洗浄液の４００μＬで３回洗浄した。
（２）　次に、前記２の（１）の試料（前記ＨＭＧＢ１分解産物）の１００μＬを各ウェルに分注した後、３７℃で一晩静置し、この試料（前記ＨＭＧＢ１分解産物）に含まれる前記ＨＭＧＢ１分解産物を、それぞれの抗体固相化マイクロプレートのウェルに固相化された前記の５種類の抗体の各々と、それぞれ抗原抗体反応を行わせた。
（３）　次に、各ウェルを、前記１の（３）の洗浄液の４００μＬで５回洗浄した。
（４）　次に、前記１の（２）の（Ａ）の抗体をパーオキシダーゼ標識したＰＯＤ標識抗体液の１００μＬを各ウェルに分注し、３７℃で９０分間静置し、それぞれ抗原抗体反応を行わせた。
（５）　次に、各ウェルを、前記１の（３）の洗浄液の４００μＬで５回洗浄した。
（６）　次に、各ウェルに、前記１の（７）の発色基質を１００μＬ分注し、室温で３０分間静置し、標識酵素であるパーオキシダーゼ（ＰＯＤ）による発色反応を行わせた。
（７）　次に、各ウェルに、前記１の（８）の反応停止液を１００μＬ分注し、発色反応を停止させた。
（８）　次に、各ウェルの液の吸光度（４５０ｎｍ）を測定し、前記の試料（前記ＨＭＧＢ１分解産物）を測定した場合の吸光度を得た。
（９）　前記（２）における試料（前記ＨＭＧＢ１分解産物）の代わりに、前記２の（２）の試料（ＨＭＧＢ１）、及び前記２の（３）の試料（試薬盲検）をそれぞれ試料として用いる以外は、前記（１）~（８）の記載の通りに行い、これらの各試料を測定した場合の吸光度を得た。
（１０）　前記（４）における「前記１の（２）の（Ａ）の抗体をパーオキシダーゼ標識したＰＯＤ標識抗体液」の代わりに、「前記１の（２）の（Ｂ）の抗体をパーオキシダーゼ標識したＰＯＤ標識抗体液」、「前記１の（２）の（Ｃ）の抗体をパーオキシダーゼ標識したＰＯＤ標識抗体液」、「前記１の（２）の（Ｄ）の抗体をパーオキシダーゼ標識したＰＯＤ標識抗体液」、「前記１の（２）の（Ｅ）の抗体をパーオキシダーゼ標識したＰＯＤ標識抗体液」、及び「前記１の（２）の（Ｆ）の抗体をパーオキシダーゼ標識したＰＯＤ標識抗体液」をそれぞれＰＯＤ標識抗体液として用いる以外は、前記（１）~（９）の記載の通りに行い、各試料を測定した場合の吸光度を得た。〔なお、測定に使用した抗体固相化マイクロプレートとＰＯＤ標識抗体液の組み合わせは、図５及び表２に示した通りである。〕
４．測定結果
（１）　前記３における測定結果、すなわち、前記実施例１~実施例４において取得した前記ＨＭＧＢ１分解産物に結合する抗体及び他のＨＭＧＢ１に結合する抗体等を用い、酵素免疫測定法（ＥＬＩＳＡ法）により、前記ＨＭＧＢ１分解産物及びＨＭＧＢ１の測定を行った結果を、図５に示した。
　この図において、横軸の上段は当該測定に使用したＰＯＤ標識抗体液の抗体の産生細胞株を表す文字を示し、横軸の下段は当該測定に使用した抗体固相化マイクロプレートの抗体の産生細胞株を表す文字を示し、縦軸は測定により得られた吸光度（４５０ｎｍ）を示す。
　また、この図（棒グラフ）において、各々の棒は、当該測定に使用したＰＯＤ標識抗体液及び抗体固相化マイクロプレート毎に、左側より順に、試料（ＨＭＧＢ１）について測定したときの測定値（前記の吸光度）、試料（前記ＨＭＧＢ１分解産物）について測定したときの測定値（前記の吸光度）、及び試料（試薬盲検）について測定したときの測定値（前記の吸光度）をそれぞれ示す。
（２）　また、前記３における測定結果の測定値を、表２に示した。
Figure JPOXMLDOC01-appb-T000002
 　この表においては、左側より順に、当該測定に使用した抗体固相化マイクロプレートの抗体の産生細胞株を表す文字、当該測定に使用したＰＯＤ標識抗体液の抗体の産生細胞株を表す文字、試料（ＨＭＧＢ１）について測定したときの測定値（前記の吸光度）、試料（前記ＨＭＧＢ１分解産物）について測定したときの測定値（前記の吸光度）、及び試料（試薬盲検）について測定したときの測定値（前記の吸光度）をそれぞれ示した。
５．まとめ
（１）　前記の酵素免疫測定法による測定の結果である図５及び表２より、次のことが分かる。
　すなわち、「本抗ＨＭＧＢ１分解産物抗体（２Ｈ６）」と、「抗ＨＭＧＢ１分解産物等抗体（５Ｄ１）」、「抗ＨＭＧＢ１分解産物等抗体（２Ａ１０）」又は「抗ＨＭＧＢ１分解産物等抗体（６Ｈ３）」を組み合わせて測定に使用した場合には、ＨＭＧＢ１の測定値（吸光度）は低いものであるのに対し、前記ＨＭＧＢ１分解産物の測定値（吸光度）は高いことが分かる。また、この場合、試薬盲検（試薬ブランク）はいずれも低いものであることも分かる。
　つまり、「前記実施例６の５の（１）の（ｉ）の抗体」（本発明の前記ＨＭＧＢ１分解産物の免疫学的測定方法及び免疫学的測定試薬における（ａ）の抗体）と、「前記実施例６の５の（１）の（ｉｉ）の抗体」（本発明の前記ＨＭＧＢ１分解産物の免疫学的測定方法及び免疫学的測定試薬における（ｂ）の抗体）を組み合わせて測定に使用した場合には、試料に含まれるＨＭＧＢ１の測り込みを抑制することができ、そして試料に含まれる前記ＨＭＧＢ１分解産物を感度高く測定できることが分かる。
（２）　これに対して、「抗ＨＭＧＢ１等抗体（ＭＤ７７）」又は「抗ＨＭＧＢ１等抗体（０４）」を測定に使用した場合には、前記ＨＭＧＢ１分解産物の測定値（吸光度）が極めて低いものであったり、又は前記ＨＭＧＢ１分解産物の測定値（吸光度）とＨＭＧＢ１の測定値（吸光度）が同程度のものであって殆ど差がないものであることが分かる。
　つまり、「前記実施例６の５の（１）の（ｉｉｉ）の抗体」（前記実施例６の５の（１）の（ｉ）及び（ｉｉ）以外の抗体）を測定に使用した場合には、試料に含まれる前記ＨＭＧＢ１分解産物を感度高くかつ特異性高く測定することができないことが分かる。
（３）　従って、本実施例における検討結果より、本発明の「試料に含まれる前記ＨＭＧＢ１分解産物の免疫学的測定方法及び免疫学的測定試薬」における「（ａ）の抗体」と「（ｂ）の抗体」を組み合わせて測定に使用することにより、試料に含まれるＨＭＧＢ１の測り込みを抑制することができ、そして試料に含まれる前記ＨＭＧＢ１分解産物を感度高く測定できることが分かった。
〔実施例８〕（ＨＭＧＢ１分解産物の免疫学的測定方法及び測定試薬−１）
　本発明の前記ＨＭＧＢ１分解産物の免疫学的測定方法及び免疫学的測定試薬、並びに従来の免疫学的測定方法及び免疫学的測定試薬により、試料に含まれる前記ＨＭＧＢ１分解産物及び試料に含まれるＨＭＧＢ１の測定を行った。
〔１〕従来の免疫学的測定方法及び免疫学的測定試薬による測定
１．測定試薬
　固相化抗ＨＭＧＢ１ポリクローナル抗体、及びパーオキシダーゼ（ＰＯＤ）標識抗ＨＭＧＢ１，２モノクローナル抗体によるＥＬＩＳＡ・サンドイッチ法を測定原理とする、ＨＭＧＢ１の測定試薬（研究用試薬）である「ＨＭＧＢ１　ＥＬＩＳＡ　Ｋｉｔ　ＩＩ」（シノテスト社〔日本国〕）を、従来の免疫学的測定試薬として用いた。
２．試料
（１）試料（前記ＨＭＧＢ１分解産物）
　前記参考例１の〔２〕で調製した前記ＨＭＧＢ１分解産物を、０．５％カゼインナトリウム、１００ｍＭ塩化ナトリウム及び０．１％アジ化ナトリウムを含むリン酸緩衝生理食塩水により、各々２．５ｎｇ／ｍＬ、５ｎｇ／ｍＬ、１０ｎｇ／ｍＬ、２０ｎｇ／ｍＬ、４０ｎｇ／ｍＬ、及び８０ｎｇ／ｍＬの濃度となるように調製したものをそれぞれ試料（前記ＨＭＧＢ１分解産物）とした。
　また、０．５％カゼインナトリウム、１００ｍＭ塩化ナトリウム及び０．１％アジ化ナトリウムを含むリン酸緩衝生理食塩水を、前記ＨＭＧＢ１分解産物の濃度が０ｎｇ／ｍＬの試料（前記ＨＭＧＢ１分解産物）とした。
（２）試料（ＨＭＧＢ１）
　前記参考例１の〔１〕で調製したＨＭＧＢ１を、０．５％カゼインナトリウム、１００ｍＭ塩化ナトリウム及び０．１％アジ化ナトリウムを含むリン酸緩衝生理食塩水により、各々２．５ｎｇ／ｍＬ、５ｎｇ／ｍＬ、１０ｎｇ／ｍＬ、２０ｎｇ／ｍＬ、４０ｎｇ／ｍＬ、及び８０ｎｇ／ｍＬの濃度となるように調製したものをそれぞれ試料（ＨＭＧＢ１）とした。
　また、０．５％カゼインナトリウム、１００ｍＭ塩化ナトリウム及び０．１％アジ化ナトリウムを含むリン酸緩衝生理食塩水を、ＨＭＧＢ１の濃度が０ｎｇ／ｍＬの試料（ＨＭＧＢ１）とした。
３．測定
（１）　前記２の（１）の試料（前記ＨＭＧＢ１分解産物）〔計７濃度〕のそれぞれ、及び前記２の（２）の試料（ＨＭＧＢ１）〔計７濃度〕のそれぞれについて、前記１の従来の免疫学的測定試薬である「ＨＭＧＢ１　ＥＬＩＳＡ　Ｋｉｔ　ＩＩ」（シノテスト社〔日本国〕）を用いて、その添付文書の記載の通りに測定の操作を行った。
（２）　前記（１）の測定により、各試料の測定の結果として、それぞれ吸光度（４５０ｎｍ）を得た。
４．測定結果
（１）　前記３における測定結果、すなわち、従来の免疫学的測定方法及び免疫学的測定試薬により、試料に含まれる前記ＨＭＧＢ１分解産物及び試料に含まれるＨＭＧＢ１の測定を行った結果を、図６に示した。
　なお、この図において、横軸は試料に含まれる前記ＨＭＧＢ１分解産物又は試料に含まれるＨＭＧＢ１の濃度（ｎｇ／ｍＬ）を示し、縦軸は測定により得られた吸光度（４５０ｎｍ）を示す。
　また、この図において、「■」は前記２の（１）の試料（前記ＨＭＧＢ１分解産物）〔計７濃度〕について測定したときの測定値（前記の吸光度）を示し、「◆」は前記２の（２）の試料（ＨＭＧＢ１）〔計７濃度〕について測定したときの測定値（前記の吸光度）を示す。
（２）　また、前記３における測定結果の測定値を、表３に示した。
Figure JPOXMLDOC01-appb-T000003
 　なお、この表においては、左側より順に、「（ｉ）　試料に含まれる前記ＨＭＧＢ１分解産物又は試料に含まれるＨＭＧＢ１の濃度（ｎｇ／ｍＬ）」、「（ｉｉ）　前記２の（２）の試料（ＨＭＧＢ１）〔計７濃度〕について測定したときの測定値（４５０ｎｍにおける吸光度）」、「（ｉｉｉ）　前記（ｉｉ）の測定値から試薬盲検の値〔ＨＭＧＢ１の濃度が０ｎｇ／ｍＬの試料（ＨＭＧＢ１）における当該測定値（４５０ｎｍにおける吸光度）〕を差し引いた値（吸光度差）」、「（ｉｖ）　後記（ｖｉ）の試料（前記ＨＭＧＢ１分解産物）における値（吸光度差）を前記（ｉｉｉ）の試料（ＨＭＧＢ１）における値（吸光度差）で除した値」、「（ｖ）　前記２の（１）の試料（前記ＨＭＧＢ１分解産物）〔計７濃度〕について測定したときの測定値（４５０ｎｍにおける吸光度）」、「（ｖｉ）　前記（ｖ）の測定値から試薬盲検の値〔前記ＨＭＧＢ１分解産物の濃度が０ｎｇ／ｍＬの試料（前記ＨＭＧＢ１分解産物）における当該測定値（４５０ｎｍにおける吸光度）〕を差し引いた値（吸光度差）」をそれぞれ示した。
〔２〕本発明の前記ＨＭＧＢ１分解産物の免疫学的測定方法及び免疫学的測定試薬による測定
１．測定試薬
（１）抗体固相化マイクロプレート（５Ｄ１）
　前記実施例２の抗ＨＭＧＢ１分解産物等抗体（５Ｄ１）をプロテインＡを用いて精製し、これをリン酸緩衝生理食塩水（ＰＢＳ）でその濃度が２．５μｇ／ｍＬとなるように希釈した。
　次に、これを、マイクロタイタープレート（マイクロプレート）〔Ｎｕｎｃ社、商品名：Ｍａｘｉｓｏｒｐ〕の各ウェルに１００μＬずつ分注し、２５℃で一晩静置し、前記の抗ＨＭＧＢ１分解産物等抗体（５Ｄ１）をマイクロプレートのウェルに固相化した。
　次に、マイクロプレートのウェル中の液を除去し、０．５％カゼインナトリウム及び０．１％アジ化ナトリウムを含むトリス緩衝生理食塩水（ＴＢＳ）〔ｐＨ８．０〕の２５０μＬずつを各ウェルに分注し、各ウェルの上をプレートシールで封をし、蒸発しないようにして、使用時まで冷蔵保存した。
　これを、抗体固相化マイクロプレート（５Ｄ１）とした。
（２）ビオチン標識抗体液（２Ｈ６）
　前記実施例１の本抗ＨＭＧＢ１分解産物抗体（２Ｈ６）を、Ｓｕｌｆｏ−ＮＨＳ−ＬＣ−Ｂｉｏｔｉｎ（Ｐｉｅｒｃｅ社、商品コード番号：２１３３５）を用いてビオチン標識を行った。
　このビオチン標識抗体を、１００ｍＭ塩化ナトリウム、０．５％カゼインナトリウム、２ｍＭのＥＤＴＡ・２ナトリウム、０．１％アジ化ナトリウム、及び１０％マウス血清を含む５０ｍＭトリス−塩酸緩衝液（ｐＨ７．８）に、２μｇ／ｍＬの濃度となるように溶解し、これをビオチン標識抗体液（２Ｈ６）とした。
（３）ストレプトアビジン−パーオキシダーゼコンジュゲート液
　Ｓｔｒｅｐｔａｖｉｄｉｎ−ＰｏｌｙＨＲＰ４０（Ｓｔｅｒｅｏｓｐｅｃｉｆｉｃ　Ｄｅｔｅｃｔｉｏｎ　Ｔｅｃｈｎｏｌｏｇｉｅｓ社〔ドイツ国〕；商品コード番号：ＳＰ４０Ｃ）を、１００ｍＭ塩化ナトリウム、０．５％カゼイン（ビタミンフリー）及び０．５ｍＭ塩化カルシウムを含む１００ｍＭトリス−塩酸緩衝液（ｐＨ７．８）で１０，０００倍に希釈した。
　これを、ストレプトアビジン−パーオキシダーゼコンジュゲート液とした。
（４）洗浄液
　０．０５％のＴｗｅｅｎ２０を含むリン酸緩衝生理食塩水を、洗浄液とした。
（５）希釈液
　１００ｍＭ塩化ナトリウム、０．５％カゼインナトリウム、２ｍＭのＥＤＴＡ・２ナトリウム、０．１％アジ化ナトリウム、及び１０％マウス血清を含む１００ｍＭのＣＨＥＳ緩衝液（ｐＨ９．５）を、希釈液とした。
（６）発色液
　０．２ｍＭのＥＤＴＡ・２ナトリウムを含む０．０４５％の３，３’，５，５’−テトラメチルベンジジン塩酸塩水溶液（ｐＨ２．０）を、発色液とした。
（７）基質液
　５ｍＭ過酸化水素、４１ｍＭクエン酸、０．２ｍＭのＥＤＴＡ・２ナトリウムを含む６０ｍＭリン酸二ナトリウム水溶液（ｐＨ４．３）を、基質液とした。
（８）発色基質
　前記の発色液と基質液を使用前に室温に戻した上で、使用時に等量混合し、発色基質とした。
（９）反応停止液
　０．７Ｎ硫酸を、反応停止液とした。
２．試料
（１）試料（前記ＨＭＧＢ１分解産物）
　前記〔１〕の２の（１）の記載の通りに、前記ＨＭＧＢ１分解産物の濃度がそれぞれ、０ｎｇ／ｍＬ、２．５ｎｇ／ｍＬ、５ｎｇ／ｍＬ、１０ｎｇ／ｍＬ、２０ｎｇ／ｍＬ、４０ｎｇ／ｍＬ、及び８０ｎｇ／ｍＬの試料（前記ＨＭＧＢ１分解産物）を調製した。
（２）試料（ＨＭＧＢ１）
　前記〔１〕の２の（２）の記載の通りに、ＨＭＧＢ１の濃度がそれぞれ、０ｎｇ／ｍＬ、２．５ｎｇ／ｍＬ、５ｎｇ／ｍＬ、１０ｎｇ／ｍＬ、２０ｎｇ／ｍＬ、４０ｎｇ／ｍＬ、及び８０ｎｇ／ｍＬの試料（ＨＭＧＢ１）を調製した。
３．測定
（１）　前記１の（１）の抗体固相化マイクロプレート（５Ｄ１）の各ウェルを、前記１の（４）の洗浄液の２５０μＬで３回洗浄した。
（２）　次に、各ウェルに、前記１の（５）の希釈液の１００μＬを分注した。
（３）　次に、前記２の（１）の計７濃度の試料（前記ＨＭＧＢ１分解産物）それぞれの１０μＬを各ウェルに分注した後、各ウェルの上をプレートシールで封をし、５℃で一晩静置し、このマイクロプレートのウェルに固相化された抗体と試料に含まれていた前記ＨＭＧＢ１分解産物との抗原抗体反応を行わせた。
（４）　次に、各ウェルを、前記１の（４）の洗浄液の４００μＬで５回洗浄した。
（５）　次に、前記１の（２）のビオチン標識抗体液（２Ｈ６）の１００μＬを各ウェルに分注し、２５℃で２時間静置し、前記の固相化抗体に結合した前記ＨＭＧＢ１分解産物とビオチン標識抗体との抗原抗体反応を行わせた。
（６）　次に、各ウェルを、前記１の（４）の洗浄液の４００μＬで５回洗浄した。
（７）　次に、各ウェルに、前記１の（３）のストレプトアビジン−パーオキシダーゼコンジュゲート液を１００μＬ分注し、２５℃で１時間静置し、「ビオチン−ストレプトアビジン」の結合反応を行わせた。
（８）　次に、各ウェルを、前記１の（４）の洗浄液の４００μＬで５回洗浄した。
（９）　次に、各ウェルに、前記１の（８）の発色基質を１００μＬ分注し、室温で２０分間静置し、標識酵素であるパーオキシダーゼによる発色反応を行わせた。
（１０）　次に、各ウェルに、前記１の（９）の反応停止液を１００μＬ分注し、発色反応を停止させた。
（１１）　次に、各ウェルの液の吸光度（４５０ｎｍ）を測定し、前記の計７濃度の試料（前記ＨＭＧＢ１分解産物）をそれぞれ測定した場合の吸光度を得た。
（１２）　前記（３）における計７濃度の試料（前記ＨＭＧＢ１分解産物）の代わりに、前記２の（２）の計７濃度の試料（ＨＭＧＢ１）を用いる以外は、前記（１）~（１１）の記載の通りに行い、前記の計７濃度の試料（ＨＭＧＢ１）をそれぞれ測定した場合の吸光度を得た。
４．測定結果
（１）　前記３における測定結果、すなわち、本発明の前記ＨＭＧＢ１分解産物の免疫学的測定方法及び免疫学的測定試薬により、試料に含まれる前記ＨＭＧＢ１分解産物及び試料に含まれるＨＭＧＢ１の測定を行った結果を、図７に示した。
　なお、この図において、横軸は試料に含まれる前記ＨＭＧＢ１分解産物又は試料に含まれるＨＭＧＢ１の濃度（ｎｇ／ｍＬ）を示し、縦軸は測定により得られた吸光度（４５０ｎｍ）を示す。
　また、この図において、「■」は前記２の（１）の試料（前記ＨＭＧＢ１分解産物）〔計７濃度〕について測定したときの測定値（前記の吸光度）を示し、「◆」は前記２の（２）の試料（ＨＭＧＢ１）〔計７濃度〕について測定したときの測定値（前記の吸光度）を示す。
（２）　また、前記３における測定結果の測定値を、表４に示した。
Figure JPOXMLDOC01-appb-T000004
 　なお、この表においては、左側より順に、「（ｉ）　試料に含まれる前記ＨＭＧＢ１分解産物又は試料に含まれるＨＭＧＢ１の濃度（ｎｇ／ｍＬ）」、「（ｉｉ）　前記２の（２）の試料（ＨＭＧＢ１）〔計７濃度〕について測定したときの測定値（４５０ｎｍにおける吸光度）」、「（ｉｉｉ）　前記（ｉｉ）の測定値から試薬盲検の値〔ＨＭＧＢ１の濃度が０ｎｇ／ｍＬの試料（ＨＭＧＢ１）における当該測定値（４５０ｎｍにおける吸光度）〕を差し引いた値（吸光度差）」、「（ｉｖ）　後記（ｖｉ）の試料（前記ＨＭＧＢ１分解産物）における値（吸光度差）を前記（ｉｉｉ）の試料（ＨＭＧＢ１）における値（吸光度差）で除した値」、「（ｖ）　前記２の（１）の試料（前記ＨＭＧＢ１分解産物）〔計７濃度〕について測定したときの測定値（４５０ｎｍにおける吸光度）」、「（ｖｉ）　前記（ｖ）の測定値から試薬盲検の値〔前記ＨＭＧＢ１分解産物の濃度が０ｎｇ／ｍＬの試料（前記ＨＭＧＢ１分解産物）における当該測定値（４５０ｎｍにおける吸光度）〕を差し引いた値（吸光度差）」をそれぞれ示した。
〔３〕まとめ
（１）　前記〔１〕における測定結果である図６及び表３、並びに前記〔２〕における測定結果である図７及び表４より、次のことが分かる。
　すなわち、本発明の前記ＨＭＧＢ１分解産物の免疫学的測定方法及び免疫学的測定試薬においては、測定を行った試料（前記ＨＭＧＢ１分解産物）〔２．５~８０ｎｇ／ｍＬ〕のいずれにおいても、試料（前記ＨＭＧＢ１分解産物）における値（吸光度差）を試料（ＨＭＧＢ１）における値（吸光度差）で除した値は、約８．５~１４．９の範囲にあった。
　つまり、本発明の前記ＨＭＧＢ１分解産物の免疫学的測定方法及び免疫学的測定試薬においては、ＨＭＧＢ１の測定値（吸光度差）は大変低いものであり、試料に含まれるＨＭＧＢ１の測り込みが非常に抑制されていることが分かる。
　これに対して、前記ＨＭＧＢ１分解産物の測定値（吸光度差）は高く、試料に含まれる前記ＨＭＧＢ１分解産物を特異性高く測定できていることが分かる。
　また、本発明の前記ＨＭＧＢ１分解産物の免疫学的測定方法及び免疫学的測定試薬においては、当該測定の検量線のグラフである図７より、この検量線が試料に含まれる前記ＨＭＧＢ１分解産物の濃度に比例してほぼ直線的に伸びており、試料に含まれる前記ＨＭＧＢ１分解産物を低濃度域から高濃度域までの広い範囲にわたって定量的に測定できていることも分かる。
（２）　これに対して、従来の免疫学的測定方法及び免疫学的測定試薬においては、測定を行った試料（前記ＨＭＧＢ１分解産物）〔２．５~８０ｎｇ／ｍＬ〕のいずれにおいても、試料（前記ＨＭＧＢ１分解産物）における値（吸光度差）を試料（ＨＭＧＢ１）における値（吸光度差）で除した値は、約０．７２~０．７９の範囲にあった。
　つまり、従来の免疫学的測定方法及び免疫学的測定試薬においては、いずれの濃度においても、同じ濃度における前記ＨＭＧＢ１分解産物の測定値（吸光度差）よりもＨＭＧＢ１の測定値（吸光度差）の方が高いことが分かる。
　すなわち、従来の免疫学的測定方法及び免疫学的測定試薬においては、試料に含まれるＨＭＧＢ１の方をより高感度に測り込んでしまうものであり、このＨＭＧＢ１に由来する正の誤差による影響が非常に大きいものであり、試料に含まれる前記ＨＭＧＢ１分解産物を特異性高く測定することが不可能なものであることが分かる。
（３）　従って、本実施例における検討結果より、本発明の前記ＨＭＧＢ１分解産物の免疫学的測定方法及び免疫学的測定試薬は、前記ＨＭＧＢ１分解産物に対する特異性が高いものであって、ＨＭＧＢ１の測り込みが抑制され、すなわちＨＭＧＢ１に由来する正の誤差が生じることが抑制され、前記ＨＭＧＢ１分解産物のみを特異性高く、正確かつ高感度に定量測定することができるものであることが確かめられた。
〔実施例９〕（ＨＭＧＢ１分解産物の免疫学的測定方法及び測定試薬−２）
　本発明の前記ＨＭＧＢ１分解産物の免疫学的測定方法及び免疫学的測定試薬により、試料に含まれる前記ＨＭＧＢ１分解産物、試料に含まれるＨＭＧＢ１、試料に含まれる前記ＨＭＧＢ２分解産物、及び試料に含まれるＨＭＧＢ２の測定を行った。
１．測定試薬
（１）抗体固相化マイクロプレート（５Ｄ１）
　前記実施例８の〔２〕の１の（１）の記載の通りに調製し、これを、抗体固相化マイクロプレート（５Ｄ１）とした。
（２）ビオチン標識抗体液（２Ｈ６）
　前記実施例８の〔２〕の１の（２）の記載の通りに調製し、これを、ビオチン標識抗体液（２Ｈ６）とした。
（３）ストレプトアビジン−パーオキシダーゼコンジュゲート液
　前記実施例８の〔２〕の１の（３）の記載の通りに調製し、これを、ストレプトアビジン−パーオキシダーゼコンジュゲート液とした。
（４）洗浄液
　前記実施例８の〔２〕の１の（４）の記載の通りに調製し、これを、洗浄液とした。
（５）希釈液
　前記実施例８の〔２〕の１の（５）の記載の通りに調製し、これを、希釈液とした。
（６）発色液
　前記実施例８の〔２〕の１の（６）の記載の通りに調製し、これを、発色液とした。
（７）基質液
　前記実施例８の〔２〕の１の（７）の記載の通りに調製し、これを、基質液とした。
（８）発色基質
　前記実施例８の〔２〕の１の（８）の記載の通りに調製し、これを、発色基質とした。
（９）反応停止液
　前記実施例８の〔２〕の１の（９）の記載の通りに調製し、これを、反応停止液とした。
２．試料
（１）試料（前記ＨＭＧＢ１分解産物）
　前記参考例１の〔２〕で調製した前記ＨＭＧＢ１分解産物を、０．５％カゼインナトリウム、１００ｍＭ塩化ナトリウム及び０．１％アジ化ナトリウムを含むリン酸緩衝生理食塩水により、各々１．５ｎｇ／ｍＬ、３．１ｎｇ／ｍＬ、６．２ｎｇ／ｍＬ、１２．５ｎｇ／ｍＬ、２５ｎｇ／ｍＬ、５０ｎｇ／ｍＬ、及び１００ｎｇ／ｍＬの濃度となるように調製したものをそれぞれ試料（前記ＨＭＧＢ１分解産物）とした。
　また、０．５％カゼインナトリウム、１００ｍＭ塩化ナトリウム及び０．１％アジ化ナトリウムを含むリン酸緩衝生理食塩水を、前記ＨＭＧＢ１分解産物の濃度が０ｎｇ／ｍＬの試料（前記ＨＭＧＢ１分解産物）とした。
（２）試料（ＨＭＧＢ１）
　前記参考例１の〔１〕で調製したＨＭＧＢ１を、０．５％カゼインナトリウム、１００ｍＭ塩化ナトリウム及び０．１％アジ化ナトリウムを含むリン酸緩衝生理食塩水により、各々１．５ｎｇ／ｍＬ、３．１ｎｇ／ｍＬ、６．２ｎｇ／ｍＬ、１２．５ｎｇ／ｍＬ、２５ｎｇ／ｍＬ、５０ｎｇ／ｍＬ、及び１００ｎｇ／ｍＬの濃度となるように調製したものをそれぞれ試料（ＨＭＧＢ１）とした。
　また、０．５％カゼインナトリウム、１００ｍＭ塩化ナトリウム及び０．１％アジ化ナトリウムを含むリン酸緩衝生理食塩水を、ＨＭＧＢ１の濃度が０ｎｇ／ｍＬの試料（ＨＭＧＢ１）とした。
（３）試料（前記ＨＭＧＢ２分解産物）
　前記参考例１の〔３〕で調製した前記ＨＭＧＢ２分解産物を、０．５％カゼインナトリウム、１００ｍＭ塩化ナトリウム及び０．１％アジ化ナトリウムを含むリン酸緩衝生理食塩水により、各々１．５ｎｇ／ｍＬ、３．１ｎｇ／ｍＬ、６．２ｎｇ／ｍＬ、１２．５ｎｇ／ｍＬ、２５ｎｇ／ｍＬ、５０ｎｇ／ｍＬ、及び１００ｎｇ／ｍＬの濃度となるように調製したものをそれぞれ試料（前記ＨＭＧＢ２分解産物）とした。
　また、０．５％カゼインナトリウム、１００ｍＭ塩化ナトリウム及び０．１％アジ化ナトリウムを含むリン酸緩衝生理食塩水を、前記ＨＭＧＢ２分解産物の濃度が０ｎｇ／ｍＬの試料（前記ＨＭＧＢ２分解産物）とした。
（４）試料（ＨＭＧＢ２）
　前記参考例１の〔１〕で調製したＨＭＧＢ２を、０．５％カゼインナトリウム、１００ｍＭ塩化ナトリウム及び０．１％アジ化ナトリウムを含むリン酸緩衝生理食塩水により、各々１．５ｎｇ／ｍＬ、３．１ｎｇ／ｍＬ、６．２ｎｇ／ｍＬ、１２．５ｎｇ／ｍＬ、２５ｎｇ／ｍＬ、５０ｎｇ／ｍＬ、及び１００ｎｇ／ｍＬの濃度となるように調製したものをそれぞれ試料（ＨＭＧＢ２）とした。
　また、０．５％カゼインナトリウム、１００ｍＭ塩化ナトリウム及び０．１％アジ化ナトリウムを含むリン酸緩衝生理食塩水を、ＨＭＧＢ２の濃度が０ｎｇ／ｍＬの試料（ＨＭＧＢ２）とした。
３．測定
（１）　前記１の（１）の抗体固相化マイクロプレート（５Ｄ１）の各ウェルを、前記１の（４）の洗浄液の２５０μＬで３回洗浄した。
（２）　次に、各ウェルに、前記１の（５）の希釈液の１００μＬを分注した。
（３）　次に、前記２の（１）の計８濃度の試料（前記ＨＭＧＢ１分解産物）それぞれの１０μＬを各ウェルに分注した後、各ウェルの上をプレートシールで封をし、５℃で一晩静置し、このマイクロプレートのウェルに固相化された抗体と試料に含まれていた前記ＨＭＧＢ１分解産物との抗原抗体反応を行わせた。
（４）　次に、各ウェルを、前記１の（４）の洗浄液の４００μＬで５回洗浄した。
（５）　次に、前記１の（２）のビオチン標識抗体液（２Ｈ６）の１００μＬを各ウェルに分注し、２５℃で２時間静置し、前記の固相化抗体に結合した前記ＨＭＧＢ１分解産物とビオチン標識抗体との抗原抗体反応を行わせた。
（６）　次に、各ウェルを、前記１の（４）の洗浄液の４００μＬで５回洗浄した。
（７）　次に、各ウェルに、前記１の（３）のストレプトアビジン−パーオキシダーゼコンジュゲート液を１００μＬ分注し、２５℃で１時間静置し、「ビオチン−ストレプトアビジン」の結合反応を行わせた。
（８）　次に、各ウェルを、前記１の（４）の洗浄液の４００μＬで５回洗浄した。
（９）　次に、各ウェルに、前記１の（８）の発色基質を１００μＬ分注し、室温で２０分間静置し、標識酵素であるパーオキシダーゼによる発色反応を行わせた。
（１０）　次に、各ウェルに、前記１の（９）の反応停止液を１００μＬ分注し、発色反応を停止させた。
（１１）　次に、各ウェルの液の吸光度（主波長：４５０ｎｍ、副波長：５５０ｎｍ）を測定し、前記の計８濃度の試料（前記ＨＭＧＢ１分解産物）をそれぞれ測定した場合の吸光度を得た。
（１２）　前記（３）における計８濃度の試料（前記ＨＭＧＢ１分解産物）の代わりに、前記２の（２）の計８濃度の試料（ＨＭＧＢ１）を用いる以外は、前記（１）~（１１）の記載の通りに行い、前記の計８濃度の試料（ＨＭＧＢ１）をそれぞれ測定した場合の吸光度を得た。
（１３）　前記（３）における計８濃度の試料（前記ＨＭＧＢ１分解産物）の代わりに、前記２の（３）の計８濃度の試料（前記ＨＭＧＢ２分解産物）を用いる以外は、前記（１）~（１１）の記載の通りに行い、前記の計８濃度の試料（前記ＨＭＧＢ２分解産物）をそれぞれ測定した場合の吸光度を得た。
（１４）　前記（３）における計８濃度の試料（前記ＨＭＧＢ１分解産物）の代わりに、前記２の（４）の計８濃度の試料（ＨＭＧＢ２）を用いる以外は、前記（１）~（１１）の記載の通りに行い、前記の計８濃度の試料（ＨＭＧＢ２）をそれぞれ測定した場合の吸光度を得た。
４．測定結果
（１）　前記３における測定結果、すなわち、本発明の前記ＨＭＧＢ１分解産物の免疫学的測定方法及び免疫学的測定試薬により、試料に含まれる前記ＨＭＧＢ１分解産物、試料に含まれるＨＭＧＢ１、試料に含まれる前記ＨＭＧＢ２分解産物、及び試料に含まれるＨＭＧＢ２それぞれの測定を行った結果を、図８に示した。
　この図において、図の上方に、測定に使用した固相化抗体及び標識抗体それぞれの産生細胞株を表す文字を示した。
　なお、この図において、横軸は試料に含まれる前記ＨＭＧＢ１分解産物、試料に含まれるＨＭＧＢ１、試料に含まれる前記ＨＭＧＢ２分解産物、又は試料に含まれるＨＭＧＢ２の濃度（ｎｇ／ｍＬ）を示し、縦軸は測定により得られた吸光度〔主波長（４５０ｎｍ）における吸光度から副波長（５５０ｎｍ）における吸光度を差し引いたもの〕を示す。
　また、この図において、「■」は前記２の（１）の試料（前記ＨＭＧＢ１分解産物）〔計８濃度〕について測定したときの測定値（前記の吸光度）を示し、「◆」は前記２の（２）の試料（ＨＭＧＢ１）〔計８濃度〕について測定したときの測定値（前記の吸光度）を示し、「●」は前記２の（３）の試料（前記ＨＭＧＢ２分解産物）〔計８濃度〕について測定したときの測定値（前記の吸光度）を示し、「▲」は前記２の（４）の試料（ＨＭＧＢ２）〔計８濃度〕について測定したときの測定値（前記の吸光度）を示す。
（２）　また、前記３における測定結果の測定値を、表５に示した。
Figure JPOXMLDOC01-appb-T000005
 　この表において、表の左上方に、測定に使用した固相化抗体及び標識抗体それぞれの産生細胞株を表す文字を示した。
　なお、この表においては、左側より順に、「（ｉ）　試料に含まれる前記ＨＭＧＢ１分解産物、試料に含まれるＨＭＧＢ１、試料に含まれる前記ＨＭＧＢ２分解産物、又は試料に含まれるＨＭＧＢ２の濃度（ｎｇ／ｍＬ）」、「（ｉｉ）　前記２の（２）の試料（ＨＭＧＢ１）〔計８濃度〕について測定したときの測定値〔主波長（４５０ｎｍ）における吸光度から副波長（５５０ｎｍ）における吸光度を差し引いたもの〕」、「（ｉｉｉ）　前記（ｉｉ）の測定値から試薬盲検の値〔ＨＭＧＢ１の濃度が０ｎｇ／ｍＬの試料（ＨＭＧＢ１）における当該測定値［主波長（４５０ｎｍ）における吸光度から副波長（５５０ｎｍ）における吸光度を差し引いたもの］〕を差し引いた値（吸光度差）」、「（ｉｖ）　後記（ｖｉ）の試料（前記ＨＭＧＢ１分解産物）における値（吸光度差）を前記（ｉｉｉ）の試料（ＨＭＧＢ１）における値（吸光度差）で除した値」、「（ｖ）　前記２の（１）の試料（前記ＨＭＧＢ１分解産物）〔計８濃度〕について測定したときの測定値〔主波長（４５０ｎｍ）における吸光度から副波長（５５０ｎｍ）における吸光度を差し引いたもの〕」、「（ｖｉ）　前記（ｖ）の測定値から試薬盲検の値〔前記ＨＭＧＢ１分解産物の濃度が０ｎｇ／ｍＬの試料（前記ＨＭＧＢ１分解産物）における当該測定値［主波長（４５０ｎｍ）における吸光度から副波長（５５０ｎｍ）における吸光度を差し引いたもの］〕を差し引いた値（吸光度差）」、「（ｖｉｉ）　前記２の（４）の試料（ＨＭＧＢ２）〔計８濃度〕について測定したときの測定値〔主波長（４５０ｎｍ）における吸光度から副波長（５５０ｎｍ）における吸光度を差し引いたもの〕」、「（ｖｉｉｉ）　前記（ｖｉｉ）の測定値から試薬盲検の値〔ＨＭＧＢ２の濃度が０ｎｇ／ｍＬの試料（ＨＭＧＢ２）における当該測定値［主波長（４５０ｎｍ）における吸光度から副波長（５５０ｎｍ）における吸光度を差し引いたもの］〕を差し引いた値（吸光度差）」、「（ｉｘ）　前記（ｖｉ）の試料（前記ＨＭＧＢ１分解産物）における値（吸光度差）を前記（ｖｉｉｉ）の試料（ＨＭＧＢ２）における値（吸光度差）で除した値」、「（ｘ）　前記２の（３）の試料（前記ＨＭＧＢ２分解産物）〔計８濃度〕について測定したときの測定値〔主波長（４５０ｎｍ）における吸光度から副波長（５５０ｎｍ）における吸光度を差し引いたもの〕」、「（ｘｉ）　前記（ｘ）の測定値から試薬盲検の値〔前記ＨＭＧＢ２分解産物の濃度が０ｎｇ／ｍＬの試料（前記ＨＭＧＢ２分解産物）における当該測定値［主波長（４５０ｎｍ）における吸光度から副波長（５５０ｎｍ）における吸光度を差し引いたもの］〕を差し引いた値（吸光度差）」、「（ｘｉｉ）　前記（ｖｉ）の試料（前記ＨＭＧＢ１分解産物）における値（吸光度差）を前記（ｘｉ）の試料（前記ＨＭＧＢ２分解産物）における値（吸光度差）で除した値」をそれぞれ示した。
５．まとめ
（１）　前記３における測定結果である図８及び表５より、次のことが分かる。
　すなわち、本発明の前記ＨＭＧＢ１分解産物の免疫学的測定方法及び免疫学的測定試薬においては、試料（前記ＨＭＧＢ１分解産物）〔濃度：１２．５~１００ｎｇ／ｍＬ〕において、試料（前記ＨＭＧＢ１分解産物）における値（吸光度差）を試料（ＨＭＧＢ１）における値（吸光度差）で除した値は、約７~３０の範囲にあった。
　また、本発明の前記ＨＭＧＢ１分解産物の免疫学的測定方法及び免疫学的測定試薬においては、試料（前記ＨＭＧＢ１分解産物）〔濃度：１２．５~１００ｎｇ／ｍＬ〕において、試料（前記ＨＭＧＢ１分解産物）における値（吸光度差）を試料（ＨＭＧＢ２）における値（吸光度差）で除した値は、約１７~３９の範囲にあった。
　そして、本発明の前記ＨＭＧＢ１分解産物の免疫学的測定方法及び免疫学的測定試薬においては、試料（前記ＨＭＧＢ１分解産物）〔濃度：１２．５~１００ｎｇ／ｍＬ〕において、試料（前記ＨＭＧＢ１分解産物）における値（吸光度差）を試料（前記ＨＭＧＢ２分解産物）における値（吸光度差）で除した値は、約−３３~７６の範囲にあった。
　つまり、本発明の前記ＨＭＧＢ１分解産物の免疫学的測定方法及び免疫学的測定試薬においては、ＨＭＧＢ１の測定値（吸光度差）、前記ＨＭＧＢ２分解産物の測定値（吸光度差）、及びＨＭＧＢ２の測定値（吸光度差）はいずれも大変低いものであり、試料に含まれるＨＭＧＢ１、前記ＨＭＧＢ２分解産物、及びＨＭＧＢ２それぞれの測り込みが非常に抑制されていることが分かる。
　これに対して、前記ＨＭＧＢ１分解産物の測定値（吸光度差）は高く、試料に含まれる前記ＨＭＧＢ１分解産物を特異性高く測定できていることが分かる。
　また、本発明の前記ＨＭＧＢ１分解産物の免疫学的測定方法及び免疫学的測定試薬においては、当該測定の検量線のグラフである図８より、この検量線が試料に含まれる前記ＨＭＧＢ１分解産物の濃度に比例してほぼ直線的に伸びており、試料に含まれる前記ＨＭＧＢ１分解産物を低濃度域から高濃度域までの広い範囲にわたって定量的に測定できていることも分かる。
（２）　従って、本実施例における検討結果からも、本発明の前記ＨＭＧＢ１分解産物の免疫学的測定方法及び免疫学的測定試薬は、前記ＨＭＧＢ１分解産物に対する特異性が高いものであって、ＨＭＧＢ１等の測り込みが抑制され、すなわちＨＭＧＢ１等に由来する正の誤差が生じることが抑制され、前記ＨＭＧＢ１分解産物のみを特異性高く、正確かつ高感度に定量測定することができるものであることが確かめられた。
〔実施例１０〕（ＨＭＧＢ１分解産物の免疫学的測定方法及び測定試薬−３）
　本発明の前記ＨＭＧＢ１分解産物の免疫学的測定方法及び免疫学的測定試薬により、試料に含まれる前記ＨＭＧＢ１分解産物、試料に含まれるＨＭＧＢ１、試料に含まれる前記ＨＭＧＢ２分解産物、及び試料に含まれるＨＭＧＢ２の測定を行った。
１．測定試薬
（１）抗体固相化マイクロプレート
（ｉ）抗体固相化マイクロプレート（２Ａ１０）
　前記実施例８の〔２〕の１の（１）における抗ＨＭＧＢ１分解産物等抗体（５Ｄ１）の代わりに、前記実施例３の抗ＨＭＧＢ１分解産物等抗体（２Ａ１０）を用いる以外は、前記実施例８の〔２〕の１の（１）の記載の通りに調製を行い、これを、抗体固相化マイクロプレート（２Ａ１０）とした。
（ｉｉ）抗体固相化マイクロプレート（６Ｈ３）
　前記実施例８の〔２〕の１の（１）における抗ＨＭＧＢ１分解産物等抗体（５Ｄ１）の代わりに、前記実施例４の抗ＨＭＧＢ１分解産物等抗体（６Ｈ３）を用いる以外は、前記実施例８の〔２〕の１の（１）の記載の通りに調製を行い、これを、抗体固相化マイクロプレート（６Ｈ３）とした。
（２）ビオチン標識抗体液（２Ｈ６）
　前記実施例８の〔２〕の１の（２）の記載の通りに調製し、これを、ビオチン標識抗体液（２Ｈ６）とした。
（３）ストレプトアビジン−パーオキシダーゼコンジュゲート液
　前記実施例８の〔２〕の１の（３）の記載の通りに調製し、これを、ストレプトアビジン−パーオキシダーゼコンジュゲート液とした。
（４）洗浄液
　前記実施例８の〔２〕の１の（４）の記載の通りに調製し、これを、洗浄液とした。
（５）希釈液
　前記実施例８の〔２〕の１の（５）の記載の通りに調製し、これを、希釈液とした。
（６）発色液
　前記実施例８の〔２〕の１の（６）の記載の通りに調製し、これを、発色液とした。
（７）基質液
　前記実施例８の〔２〕の１の（７）の記載の通りに調製し、これを、基質液とした。
（８）発色基質
　前記実施例８の〔２〕の１の（８）の記載の通りに調製し、これを、発色基質とした。
（９）反応停止液
　前記実施例８の〔２〕の１の（９）の記載の通りに調製し、これを、反応停止液とした。
２．試料
（１）試料（前記ＨＭＧＢ１分解産物）
　前記参考例１の〔２〕で調製した前記ＨＭＧＢ１分解産物を、０．５％カゼインナトリウム、１００ｍＭ塩化ナトリウム及び０．１％アジ化ナトリウムを含むリン酸緩衝生理食塩水により、各々２．５ｎｇ／ｍＬ、５ｎｇ／ｍＬ、１０ｎｇ／ｍＬ、２０ｎｇ／ｍＬ、４０ｎｇ／ｍＬ、及び８０ｎｇ／ｍＬの濃度となるように調製したものをそれぞれ試料（前記ＨＭＧＢ１分解産物）とした。
　また、０．５％カゼインナトリウム、１００ｍＭ塩化ナトリウム及び０．１％アジ化ナトリウムを含むリン酸緩衝生理食塩水を、前記ＨＭＧＢ１分解産物の濃度が０ｎｇ／ｍＬの試料（前記ＨＭＧＢ１分解産物）とした。
（２）試料（ＨＭＧＢ１）
　前記参考例１の〔１〕で調製したＨＭＧＢ１を、０．５％カゼインナトリウム、１００ｍＭ塩化ナトリウム及び０．１％アジ化ナトリウムを含むリン酸緩衝生理食塩水により、各々２．５ｎｇ／ｍＬ、５ｎｇ／ｍＬ、１０ｎｇ／ｍＬ、２０ｎｇ／ｍＬ、４０ｎｇ／ｍＬ、及び８０ｎｇ／ｍＬの濃度となるように調製したものをそれぞれ試料（ＨＭＧＢ１）とした。
　また、０．５％カゼインナトリウム、１００ｍＭ塩化ナトリウム及び０．１％アジ化ナトリウムを含むリン酸緩衝生理食塩水を、ＨＭＧＢ１の濃度が０ｎｇ／ｍＬの試料（ＨＭＧＢ１）とした。
（３）試料（前記ＨＭＧＢ２分解産物）
　前記参考例１の〔３〕で調製した前記ＨＭＧＢ２分解産物を、０．５％カゼインナトリウム、１００ｍＭ塩化ナトリウム及び０．１％アジ化ナトリウムを含むリン酸緩衝生理食塩水により、各々２．５ｎｇ／ｍＬ、５ｎｇ／ｍＬ、１０ｎｇ／ｍＬ、２０ｎｇ／ｍＬ、４０ｎｇ／ｍＬ、及び８０ｎｇ／ｍＬの濃度となるように調製したものをそれぞれ試料（前記ＨＭＧＢ２分解産物）とした。
　また、０．５％カゼインナトリウム、１００ｍＭ塩化ナトリウム及び０．１％アジ化ナトリウムを含むリン酸緩衝生理食塩水を、前記ＨＭＧＢ２分解産物の濃度が０ｎｇ／ｍＬの試料（前記ＨＭＧＢ２分解産物）とした。
（４）試料（ＨＭＧＢ２）
　前記参考例１の〔１〕で調製したＨＭＧＢ２を、０．５％カゼインナトリウム、１００ｍＭ塩化ナトリウム及び０．１％アジ化ナトリウムを含むリン酸緩衝生理食塩水により、各々２．５ｎｇ／ｍＬ、５ｎｇ／ｍＬ、１０ｎｇ／ｍＬ、２０ｎｇ／ｍＬ、４０ｎｇ／ｍＬ、及び８０ｎｇ／ｍＬの濃度となるように調製したものをそれぞれ試料（ＨＭＧＢ２）とした。
　また、０．５％カゼインナトリウム、１００ｍＭ塩化ナトリウム及び０．１％アジ化ナトリウムを含むリン酸緩衝生理食塩水を、ＨＭＧＢ２の濃度が０ｎｇ／ｍＬの試料（ＨＭＧＢ２）とした。
３．測定
（１）　前記１の（１）の（ｉ）の抗体固相化マイクロプレート（２Ａ１０）の各ウェルを、前記１の（４）の洗浄液の４００μＬで３回洗浄した。
（２）　次に、各ウェルに、前記１の（５）の希釈液の１００μＬを分注した。
（３）　次に、前記２の（１）の計７濃度の試料（前記ＨＭＧＢ１分解産物）それぞれの１０μＬを各ウェルに分注した後、各ウェルの上をプレートシールで封をし、５℃で一晩静置し、このマイクロプレートのウェルに固相化された抗体と試料に含まれていた前記ＨＭＧＢ１分解産物との抗原抗体反応を行わせた。
（４）　次に、各ウェルを、前記１の（４）の洗浄液の４００μＬで５回洗浄した。
（５）　次に、前記１の（２）のビオチン標識抗体液（２Ｈ６）の１００μＬを各ウェルに分注し、２５℃で２時間静置し、前記の固相化抗体に結合した前記ＨＭＧＢ１分解産物とビオチン標識抗体との抗原抗体反応を行わせた。
（６）　次に、各ウェルを、前記１の（４）の洗浄液の４００μＬで５回洗浄した。
（７）　次に、各ウェルに、前記１の（３）のストレプトアビジン−パーオキシダーゼコンジュゲート液を１００μＬ分注し、２５℃で１時間静置し、「ビオチン−ストレプトアビジン」の結合反応を行わせた。
（８）　次に、各ウェルを、前記１の（４）の洗浄液の４００μＬで５回洗浄した。
（９）　次に、各ウェルに、前記１の（８）の発色基質を１００μＬ分注し、室温で２０分間静置し、標識酵素であるパーオキシダーゼによる発色反応を行わせた。
（１０）　次に、各ウェルに、前記１の（９）の反応停止液を１００μＬ分注し、発色反応を停止させた。
（１１）　次に、各ウェルの液の吸光度（主波長：４５０ｎｍ、副波長：５５０ｎｍ）を測定し、前記の計７濃度の試料（前記ＨＭＧＢ１分解産物）をそれぞれ測定した場合の吸光度を得た。
（１２）　前記（３）における計７濃度の試料（前記ＨＭＧＢ１分解産物）の代わりに、前記２の（２）の計７濃度の試料（ＨＭＧＢ１）を用いる以外は、前記（１）~（１１）の記載の通りに行い、前記の計７濃度の試料（ＨＭＧＢ１）をそれぞれ測定した場合の吸光度を得た。
（１３）　前記（３）における計７濃度の試料（前記ＨＭＧＢ１分解産物）の代わりに、前記２の（３）の計７濃度の試料（前記ＨＭＧＢ２分解産物）を用いる以外は、前記（１）~（１１）の記載の通りに行い、前記の計７濃度の試料（前記ＨＭＧＢ２分解産物）をそれぞれ測定した場合の吸光度を得た。
（１４）　前記（３）における計７濃度の試料（前記ＨＭＧＢ１分解産物）の代わりに、前記２の（４）の計７濃度の試料（ＨＭＧＢ２）を用いる以外は、前記（１）~（１１）の記載の通りに行い、前記の計７濃度の試料（ＨＭＧＢ２）をそれぞれ測定した場合の吸光度を得た。
（１５）　前記（１）における抗体固相化マイクロプレート（２Ａ１０）の代わりに、前記１の（１）の（ｉｉ）の抗体固相化マイクロプレート（６Ｈ３）を用いる以外は、前記（１）~（１４）の記載の通りに行い、前記の試料（前記ＨＭＧＢ１分解産物）〔計７濃度〕、試料（ＨＭＧＢ１）〔計７濃度〕、試料（前記ＨＭＧＢ２分解産物）〔計７濃度〕、及び試料（ＨＭＧＢ２）〔計７濃度〕をそれぞれ測定した場合の吸光度を得た。
４．測定結果
（１）　前記３における測定結果、すなわち、本発明の前記ＨＭＧＢ１分解産物の免疫学的測定方法及び免疫学的測定試薬により、試料に含まれる前記ＨＭＧＢ１分解産物、試料に含まれるＨＭＧＢ１、試料に含まれる前記ＨＭＧＢ２分解産物、及び試料に含まれるＨＭＧＢ２それぞれの測定を行った結果を、図９〔抗体固相化マイクロプレートとして抗体固相化マイクロプレート（２Ａ１０）を使用した場合の測定の結果〕及び図１０〔抗体固相化マイクロプレートとして抗体固相化マイクロプレート（６Ｈ３）を使用した場合の測定の結果〕に示した。
　これらの図において、図の上方に、測定に使用した固相化抗体及び標識抗体それぞれの産生細胞株を表す文字を示した。
　なお、これらの図において、横軸は試料に含まれる前記ＨＭＧＢ１分解産物、試料に含まれるＨＭＧＢ１、試料に含まれる前記ＨＭＧＢ２分解産物、又は試料に含まれるＨＭＧＢ２の濃度（ｎｇ／ｍＬ）を示し、縦軸は測定により得られた吸光度〔主波長（４５０ｎｍ）における吸光度から副波長（５５０ｎｍ）における吸光度を差し引いたもの〕を示す。
　また、これらの図において、「■」は前記２の（１）の試料（前記ＨＭＧＢ１分解産物）〔計７濃度〕について測定したときの測定値（前記の吸光度）を示し、「◆」は前記２の（２）の試料（ＨＭＧＢ１）〔計７濃度〕について測定したときの測定値（前記の吸光度）を示し、「●」は前記２の（３）の試料（前記ＨＭＧＢ２分解産物）〔計７濃度〕について測定したときの測定値（前記の吸光度）を示し、「▲」は前記２の（４）の試料（ＨＭＧＢ２）〔計７濃度〕について測定したときの測定値（前記の吸光度）を示す。
（２）　また、前記３における測定結果の測定値を、表６〔抗体固相化マイクロプレートとして抗体固相化マイクロプレート（２Ａ１０）を使用した場合の測定の結果〕及び表７〔抗体固相化マイクロプレートとして抗体固相化マイクロプレート（６Ｈ３）を使用した場合の測定の結果〕に示した。
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000007
 　これらの表において、表の左上方に、測定に使用した固相化抗体及び標識抗体それぞれの産生細胞株を表す文字を示した。
　なお、これらの表においては、左側より順に、「（ｉ）　試料に含まれる前記ＨＭＧＢ１分解産物、試料に含まれるＨＭＧＢ１、試料に含まれる前記ＨＭＧＢ２分解産物、又は試料に含まれるＨＭＧＢ２の濃度（ｎｇ／ｍＬ）」、「（ｉｉ）　前記２の（２）の試料（ＨＭＧＢ１）〔計７濃度〕について測定したときの測定値〔主波長（４５０ｎｍ）における吸光度から副波長（５５０ｎｍ）における吸光度を差し引いたもの〕」、「（ｉｉｉ）　前記（ｉｉ）の測定値から試薬盲検の値〔ＨＭＧＢ１の濃度が０ｎｇ／ｍＬの試料（ＨＭＧＢ１）における当該測定値［主波長（４５０ｎｍ）における吸光度から副波長（５５０ｎｍ）における吸光度を差し引いたもの］〕を差し引いた値（吸光度差）」、「（ｉｖ）　後記（ｖｉ）の試料（前記ＨＭＧＢ１分解産物）における値（吸光度差）を前記（ｉｉｉ）の試料（ＨＭＧＢ１）における値（吸光度差）で除した値」、「（ｖ）　前記２の（１）の試料（前記ＨＭＧＢ１分解産物）〔計７濃度〕について測定したときの測定値〔主波長（４５０ｎｍ）における吸光度から副波長（５５０ｎｍ）における吸光度を差し引いたもの〕」、「（ｖｉ）　前記（ｖ）の測定値から試薬盲検の値〔前記ＨＭＧＢ１分解産物の濃度が０ｎｇ／ｍＬの試料（前記ＨＭＧＢ１分解産物）における当該測定値［主波長（４５０ｎｍ）における吸光度から副波長（５５０ｎｍ）における吸光度を差し引いたもの］〕を差し引いた値（吸光度差）」、「（ｖｉｉ）　前記２の（４）の試料（ＨＭＧＢ２）〔計７濃度〕について測定したときの測定値〔主波長（４５０ｎｍ）における吸光度から副波長（５５０ｎｍ）における吸光度を差し引いたもの〕」、「（ｖｉｉｉ）　前記（ｖｉｉ）の測定値から試薬盲検の値〔ＨＭＧＢ２の濃度が０ｎｇ／ｍＬの試料（ＨＭＧＢ２）における当該測定値［主波長（４５０ｎｍ）における吸光度から副波長（５５０ｎｍ）における吸光度を差し引いたもの］〕を差し引いた値（吸光度差）」、「（ｉｘ）　前記（ｖｉ）の試料（前記ＨＭＧＢ１分解産物）における値（吸光度差）を前記（ｖｉｉｉ）の試料（ＨＭＧＢ２）における値（吸光度差）で除した値」、「（ｘ）　前記２の（３）の試料（前記ＨＭＧＢ２分解産物）〔計７濃度〕について測定したときの測定値〔主波長（４５０ｎｍ）における吸光度から副波長（５５０ｎｍ）における吸光度を差し引いたもの〕」、「（ｘｉ）　前記（ｘ）の測定値から試薬盲検の値〔前記ＨＭＧＢ２分解産物の濃度が０ｎｇ／ｍＬの試料（前記ＨＭＧＢ２分解産物）における当該測定値［主波長（４５０ｎｍ）における吸光度から副波長（５５０ｎｍ）における吸光度を差し引いたもの］〕を差し引いた値（吸光度差）」、「（ｘｉｉ）　前記（ｖｉ）の試料（前記ＨＭＧＢ１分解産物）における値（吸光度差）を前記（ｘｉ）の試料（前記ＨＭＧＢ２分解産物）における値（吸光度差）で除した値」をそれぞれ示した。
５．まとめ
〔ａ〕抗体固相化マイクロプレート（２Ａ１０）を使用した場合
（１）　前記３における測定結果である図９及び表６〔いずれも抗体固相化マイクロプレートとして抗体固相化マイクロプレート（２Ａ１０）を使用した場合の測定の結果〕より、次のことが分かる。
　すなわち、本発明の前記ＨＭＧＢ１分解産物の免疫学的測定方法及び免疫学的測定試薬においては、測定を行った試料（前記ＨＭＧＢ１分解産物）〔２．５~８０ｎｇ／ｍＬ〕のいずれにおいても、試料（前記ＨＭＧＢ１分解産物）における値（吸光度差）を試料（ＨＭＧＢ１）における値（吸光度差）で除した値は、約３．６~１５の範囲にあった。
　また、本発明の前記ＨＭＧＢ１分解産物の免疫学的測定方法及び免疫学的測定試薬においては、測定を行った試料（前記ＨＭＧＢ１分解産物）〔２．５~８０ｎｇ／ｍＬ〕のいずれにおいても、試料（前記ＨＭＧＢ１分解産物）における値（吸光度差）を試料（ＨＭＧＢ２）における値（吸光度差）で除した値は、約−７８~２９の範囲にあった。
　そして、本発明の前記ＨＭＧＢ１分解産物の免疫学的測定方法及び免疫学的測定試薬においては、測定を行った試料（前記ＨＭＧＢ１分解産物）〔２．５~８０ｎｇ／ｍＬ〕のいずれにおいても、試料（前記ＨＭＧＢ１分解産物）における値（吸光度差）を試料（前記ＨＭＧＢ２分解産物）における値（吸光度差）で除した値は、約−５~２９の範囲にあった。
　つまり、本発明の前記ＨＭＧＢ１分解産物の免疫学的測定方法及び免疫学的測定試薬においては、ＨＭＧＢ１の測定値（吸光度差）、前記ＨＭＧＢ２分解産物の測定値（吸光度差）、及びＨＭＧＢ２の測定値（吸光度差）はいずれも大変低いものであり、試料に含まれるＨＭＧＢ１、前記ＨＭＧＢ２分解産物、及びＨＭＧＢ２それぞれの測り込みが非常に抑制されていることが分かる。
　これに対して、前記ＨＭＧＢ１分解産物の測定値（吸光度差）は高く、試料に含まれる前記ＨＭＧＢ１分解産物を特異性高く測定できていることが分かる。
　また、本発明の前記ＨＭＧＢ１分解産物の免疫学的測定方法及び免疫学的測定試薬においては、当該測定の検量線のグラフである図９より、この検量線が試料に含まれる前記ＨＭＧＢ１分解産物の濃度に比例してほぼ直線的に伸びており、試料に含まれる前記ＨＭＧＢ１分解産物を低濃度域から高濃度域までの広い範囲にわたって定量的に測定できていることも分かる。
（２）　従って、本実施例における検討結果〔抗体固相化マイクロプレートとして抗体固相化マイクロプレート（２Ａ１０）を使用した場合の測定の結果〕からも、本発明の前記ＨＭＧＢ１分解産物の免疫学的測定方法及び免疫学的測定試薬は、前記ＨＭＧＢ１分解産物に対する特異性が高いものであって、ＨＭＧＢ１等の測り込みが抑制され、すなわちＨＭＧＢ１等に由来する正の誤差が生じることが抑制され、前記ＨＭＧＢ１分解産物のみを特異性高く、正確かつ高感度に定量測定することができるものであることが確かめられた。
〔ｂ〕抗体固相化マイクロプレート（６Ｈ３）を使用した場合
（１）　前記〔１〕における測定結果である図１０及び表７〔いずれも抗体固相化マイクロプレートとして抗体固相化マイクロプレート（６Ｈ３）を使用した場合の測定の結果〕より、次のことが分かる。
　すなわち、本発明の前記ＨＭＧＢ１分解産物の免疫学的測定方法及び免疫学的測定試薬においては、測定を行った試料（前記ＨＭＧＢ１分解産物）〔２．５~８０ｎｇ／ｍＬ〕のいずれにおいても、試料（前記ＨＭＧＢ１分解産物）における値（吸光度差）を試料（ＨＭＧＢ１）における値（吸光度差）で除した値は、約３．１~５．８の範囲にあった。
　また、本発明の前記ＨＭＧＢ１分解産物の免疫学的測定方法及び免疫学的測定試薬においては、測定を行った試料（前記ＨＭＧＢ１分解産物）〔２．５~８０ｎｇ／ｍＬ〕のいずれにおいても、試料（前記ＨＭＧＢ１分解産物）における値（吸光度差）を試料（ＨＭＧＢ２）における値（吸光度差）で除した値は、約−４７~２２７の範囲にあった。
　そして、本発明の前記ＨＭＧＢ１分解産物の免疫学的測定方法及び免疫学的測定試薬においては、測定を行った試料（前記ＨＭＧＢ１分解産物）〔２．５~８０ｎｇ／ｍＬ〕のいずれにおいても、試料（前記ＨＭＧＢ１分解産物）における値（吸光度差）を試料（前記ＨＭＧＢ２分解産物）における値（吸光度差）で除した値は、約−６８０~７７の範囲にあった。
　つまり、本発明の前記ＨＭＧＢ１分解産物の免疫学的測定方法及び免疫学的測定試薬においては、ＨＭＧＢ１の測定値（吸光度差）、前記ＨＭＧＢ２分解産物の測定値（吸光度差）、及びＨＭＧＢ２の測定値（吸光度差）はいずれも大変低いものであり、試料に含まれるＨＭＧＢ１、前記ＨＭＧＢ２分解産物、及びＨＭＧＢ２それぞれの測り込みが非常に抑制されていることが分かる。
　これに対して、前記ＨＭＧＢ１分解産物の測定値（吸光度差）は高く、試料に含まれる前記ＨＭＧＢ１分解産物を特異性高く測定できていることが分かる。
　また、本発明の前記ＨＭＧＢ１分解産物の免疫学的測定方法及び免疫学的測定試薬においては、当該測定の検量線のグラフである図１０より、この検量線が試料に含まれる前記ＨＭＧＢ１分解産物の濃度に比例してほぼ直線的に伸びており、試料に含まれる前記ＨＭＧＢ１分解産物を低濃度域から高濃度域までの広い範囲にわたって定量的に測定できていることも分かる。
（２）　従って、本実施例における検討結果〔抗体固相化マイクロプレートとして抗体固相化マイクロプレート（６Ｈ３）を使用した場合の測定の結果〕からも、本発明の前記ＨＭＧＢ１分解産物の免疫学的測定方法及び免疫学的測定試薬は、前記ＨＭＧＢ１分解産物に対する特異性が高いものであって、ＨＭＧＢ１等の測り込みが抑制され、すなわちＨＭＧＢ１等に由来する正の誤差が生じることが抑制され、前記ＨＭＧＢ１分解産物のみを特異性高く、正確かつ高感度に定量測定することができるものであることが確かめられた。
EXAMPLES Hereinafter, although an Example demonstrates this invention more specifically in detail, this invention is not limited by these Examples.
[Reference Example 1] (Preparation of HMGB1, HMGB1 degradation products, HMGB2, and HMGB2 degradation products)
Preparation of degradation products of HMGB1, HMGB1 by thrombin or thrombin / thrombomodulin complex, HMGB2, and degradation products of HMGB2 by thrombin or thrombin / thrombomodulin complex were carried out as follows.
[1] Preparation of HMGB1 and HMGB2
(1) First, 500 g of bovine thymus was crushed in 600 mL of a buffer containing 140 mM sodium chloride and 0.5 mM PMSF.
(2) Next, this crushed material was centrifuged with a centrifuge, and the supernatant was removed.
(3) A buffer solution containing 140 mM sodium chloride and 0.5 mM PMSF was added thereto and stirred, and then centrifuged with a centrifuge to remove the supernatant. This washing operation was repeated twice.
(4) Next, 300 mL of 0.75 M perchloric acid was added to the resulting precipitate.
And after centrifuging with a centrifuge, the supernatant liquid was fractionated. 400 mL of 0.75 M perchloric acid was added to the remaining precipitate. Also for this, after centrifuging with a centrifuge, the supernatant was collected. This supernatant was combined with the previously collected supernatant. The precipitate was discarded.
(5) 0.75 M perchloric acid was added to the combined supernatant to a total volume of 1,000 mL. Next, after centrifuging with a centrifuge, the supernatant was filtered through a glass filter (grade 4).
(6) A mixed solution of 3,500 mL of acetone and 21 mL of concentrated hydrochloric acid was added to the filtrate of the filtration. Since turbidity occurred, the mixture was centrifuged with a centrifuge and the supernatant was collected. To this supernatant, 2,500 mL of acetone was added. Then, since turbidity occurs again, this was centrifuged with a centrifuge to separate the supernatant, and the remaining precipitate was collected.
(7) The collected precipitate was naturally dried at room temperature. By the above operation, approximately 200 mg of a protein fraction containing HMGB1 and HMGB2 was obtained.
(8) The protein fraction containing HMGB1 and HMGB2 is dissolved in 10 mL of 7.5 mM sodium borate buffer (pH 9.0) containing 200 mM sodium chloride, and then 7.5 mM boron containing 200 mM sodium chloride is used. Dialysis was sufficiently performed using a sodium acid buffer (pH 9.0).
(9) After this dialysis, it was added to a CM-Sephadex C25 column that had been equilibrated with 7.5 mM sodium borate buffer (pH 9.0). After that, elution was performed with 7.5 mM sodium borate buffer (pH 9.0) containing 200 mM sodium chloride, and cation exchange chromatography was performed.
(10) Each fraction eluted here was subjected to SDS-polyacrylamide electrophoresis, and a fraction containing HMGB1 and a fraction containing HMGB2 were identified from their mobility.
HMGB1 and HMGB2 were prepared by the above operation.
[2] Preparation of degradation product of HMGB1 by thrombin or thrombin-thrombomodulin complex
Preparation of degradation products of HMGB1 with thrombin or thrombin-thrombomodulin complex was performed as follows.
(1) Thrombin [GE Healthcare Bio-Sciences (Sweden)]] was added to HMGB1 (300 μg / mL) prepared in [1] so as to be 100 U / mL [optionally, further recombinant soluble thrombomodulin. (Asahi Kasei Pharma [Japan]) may be included] and incubated at 37 ° C. for 3 hours.
(2) By the reaction of (1) above, cleaves between the 10th arginine (R10) and 11th glycine (G11) of HMGB1 and has a newly exposed N-terminal “GKMSS ...” A degradation product of HMGB1 was generated, that is, the degradation product of HMGB1 was prepared.
[3] Preparation of degradation product of HMGB2 by thrombin or thrombin-thrombomodulin complex
About HMGB2 prepared in said [1], operation was performed as described in said [2], and the said HMGB2 degradation product was prepared.
[Reference Example 2] (Confirmation of prepared HMGB1 degradation products, etc.)
HMGB1, the HMGB1 degradation product, HMGB2, and the HMGB2 degradation product prepared in Reference Example 1 were confirmed by SDS-polyacrylamide gel electrophoresis.
1. reagent
The following reagents (1) to (4) were prepared or prepared.
(1) SDS-polyacrylamide gel
Funakoshi Easy-Gel (III) precast gel (15%) (Funakoshi [Japan]) was used.
(2) Electrophoresis tank buffer
Tris (hydroxymethyl) aminomethane [Tris] 1.5 g, sodium dodecyl sulfate [SDS] 0.5 g, and glycine 7.2 g were added to and mixed with pure water, and the mixture was adjusted to 500 mL, and the electrophoresis buffer solution [0.1 % SDS-192 mM glycine-25 mM Tris buffer].
(3) Coomassie brilliant blue stain
Quick-CBB (Wako Pure Chemical Industries [Japan]) was used.
(4) Sample processing solution
0.4 g of sodium dodecyl sulfate [SDS], 1.2 mL of 2-mercaptoethanol, 1 mL of 1 M tris (hydroxymethyl) aminomethane [Tris] -hydrochloric acid buffer (pH 6.8), and 2 mL of glycerin are added to pure water. After mixing, a sample treatment solution [4% SDS-12% 2-mercaptoethanol-20% glycerin-100 mM Tris buffer] was prepared as 10 mL.
2. sample
HMGB1, the HMGB1 degradation product, HMGB2, and the HMGB2 degradation product prepared in [1] to [3] of the following Reference Example 1 were used as samples. The following molecular weight markers and thrombin were also used as samples.
(A) Molecular weight marker [Precision Plus Protein All Blue Standards marker; Marker molecular weight 10 KDa, 15 KDa, 20 KDa, 25 KDa, 37 KDa, 50 KDa, 75 KDa, 100 KDa, 150 KDa and 250 KDa;
(B) HMGB1
(C) HMGB1 degradation product
(D) HMGB2
(E) HMGB2 degradation product
(F) Thrombin [GE Healthcare Bio-Sciences (Sweden)]
3. Electrophoresis
Using the reagent prepared in 1 above, each of the 2 samples was subjected to electrophoresis by SDS-polyacrylamide gel electrophoresis by the following operation.
(1) Each of the samples (b) to (f) in 2 was mixed with the sample processing solution in 1 (4) at a ratio of 1: 1 and treated at 100 ° C. for 5 minutes.
(2) The buffer solution for electrophoresis tank of (1) above was placed in the lower electrophoresis tank. Next, the gel of (1) above was set in an electrophoresis tank. Then, the buffer solution for electrophoresis tank (1) described above was put in the upper electrophoresis tank.
(3) 10 μL of each sample subjected to the treatment (1) and 2 μL of the molecular weight marker sample (2) were injected into the comb holes of the gel (2).
It should be noted that the sample subjected to the treatment (1) and the sample of the molecular weight marker (a) of 2 (a) injected into this gel, the above-mentioned “molecular weight marker” in lane 1 and the lane 2 on the right side thereof. The above “HMGB1”, the above “HMGB1 degradation product” in the right lane 3, the above “HMGB2” in the right lane 4, and the above “HMGB2 degradation product” in the right lane 5. And the “thrombin” was injected into lane 6 on the right side.
(4) Next, electrophoresis was performed at a current of 20 mA for 90 minutes.
(5) After completion of the electrophoresis in (4), the gel was stained with the Quick-CBB in (1) in (1).
4). result
FIG. 1 shows the gel stained in (3) above.
In the electrophoresis image of this gel, the band of “the HMGB1 degradation product” (lane denoted by “3”) is present on the lower molecular weight side than the band of “HMGB1” (lane denoted by “2”), and It can be seen that the band of “the degradation product of HMGB2” (lane denoted by “5”) is present on the lower molecular weight side than the band of “HMGB2” (lane denoted by “4”).
That is, it was confirmed that “the HMGB1 degradation product” and “the HMGB2 degradation product” could be prepared, respectively.
[Example 1] (Preparation of antibody binding to HMGB1 degradation product-1)
An antibody that binds to the HMGB1 degradation product was prepared as follows.
(1) HMGB1 (total length) prepared in [1] of Reference Example 1 was used as an immunogen.
1 volume of HMGB1 (total length) solution prepared in [1] of Reference Example 1 as the immunogen to 1 volume of FREUND complete adjuvant (DIFCO LABORATORIES) as a chemically synthesized adjuvant To prepare a mixture of HMGB1 solution and FREUND complete adjuvant.
In addition, one volume of the solution of HMGB1 (total length) prepared in [1] of Reference Example 1 as the immunogen is 1 with FREUND incomplete adjuvant (DIFCO LABORATORIES) as a chemical synthesis adjuvant. Mixing by volume, a mixture of HMGB1 solution and FREUND incomplete adjuvant was prepared.
(2) Next, a mixture of the above-mentioned HMGB1 solution and FREUND complete adjuvant was injected into the abdominal cavity of a mouse (BALB / c) as an immunogen at 300 to 500 μg / animal / dose, 2 weeks and 4 weeks later. Mice were injected intraperitoneally with a mixture of the HMGB1 solution and FREUND incomplete adjuvant.
(3) Four weeks after the final immunization, the stock solution of HMGB1 (total length) prepared in [1] of Reference Example 1 was boosted with 300 μg / mouse, and the next day, spleen cells of the immunized mouse were used. Myeloma cells (P3U1) were mixed at a ratio of 1: 1 to 10: 1, and polyethylene glycol [PEG1500; Roche (Switzerland)] was added by a general method to cause cell fusion. Sorted.
Specifically, cell fusion was performed as follows.
The mixed spleen cells and myeloma cells (P3U1) are centrifuged to remove the supernatant, suspended in 1 mL of polyethylene glycol [PEG 1500; Roche (Switzerland)] at room temperature for 1 minute, and stirred at 37 ° C. for 1 minute. did.
The operation of adding 1 mL of serum-free medium over 1 minute was performed twice, and then 7 mL of serum-free medium was added over 2 minutes.
The cells were washed several times, suspended in a medium containing hypoxanthine, aminopterin and thymidine, dispensed into a 96-well microtiter plate, and 5% CO at 37 ° C. 2 Cultured in the presence.
As a method for selecting the grown monoclonal antibody-producing cell line (fused cell line), 7 to 14 days after cell fusion, HMGB1 prepared in Reference Example 1 (whole length) was immobilized and fused cell culture. The supernatant was used as a primary antibody in an ELISA method system.
Specifically, this ELISA method was performed as follows.
(I) Each of HMGB1 and HMGB2 prepared in Reference Example 1 prepared with phosphate buffered saline (0.9% sodium chloride aqueous solution) to a concentration of 1 μg / mL, or as a control Phosphate buffered saline (0.9% aqueous sodium chloride solution) in 96-well microtiter plates [Thermo Fisher Scientific Inc. (Ind., Illinois, USA)] is injected into a well of 100 μL, allowed to stand at 5 ° C. for 16 to 24 hours (or 37 ° C., 2 hours), and each of the above HMGB1 and HMGB2 Then, the mixture was solidified in phosphate buffered saline containing 1% BSA, and allowed to stand at 5 ° C. for 16 to 24 hours (or 37 ° C., 2 hours) for blocking.
(Ii) Next, each well of the microtiter plate of (i) was washed three times with a washing solution [phosphate buffered saline containing 0.05% Tween 20].
(Iii) Next, 100 μL of each of these fusion cell culture supernatant solutions as a sample was injected into the wells of the microtiter plate washed in (ii) above, and allowed to stand at 37 ° C. for 2 hours. Each of the HMGB1 and HMGB2 immobilized on the well of the titer plate was reacted with the monoclonal antibody contained in each of the fused cell culture supernatant solutions.
(Iv) Next, each well of the microtiter plate of (iii) was washed three times with the washing solution.
(V) Next, POD-labeled anti-mouse IgG antibody (DakoCytomation (Denmark), Denmark) diluted 1000-fold with phosphate buffered saline containing 1% BSA in each well of the microtiter plate washed in (iv). 100 μL was injected, and the mixture was allowed to stand at 37 ° C. for 2 hours for reaction.
(Vi) Next, each well of the microtiter plate of (v) was washed three times with the washing solution.
(Vii) Next, a coloring solution consisting of 0.045% 3,3 ′, 5,5′-tetramethylbenzidine hydrochloride aqueous solution (pH 2.0) containing 0.2 mM EDTA · disodium, and 5 mM excess 100 μL of a chromogenic substrate solution prepared by mixing 1: 1 with a substrate solution consisting of 60 mM aqueous disodium phosphate solution (pH 4.3) containing hydrogen oxide, 41 mM citric acid, 0.2 mM EDTA · disodium The solution was injected into each well of the microtiter plate washed in (vi) and allowed to stand at room temperature for 5 to 30 minutes to cause the reaction to develop color. Thereafter, 100 μL of a reaction stop solution composed of 0.7N sulfuric acid was dispensed into each well to stop the color reaction.
(Viii) Next, for each well of the microtiter plate of (vii), the absorbance at a main wavelength of 450 nm and a subwavelength of 550 nm was measured using a spectrophotometer. Based on the above measurement, a fused cell line producing an antibody that binds to HMGB1 was selected, and one clone was established from the grown fused cell lines and was designated as 2H6 strain.
(4) IgG (immunoglobulin G) was purified from the selected monoclonal antibody-producing cell line as follows. This monoclonal antibody-producing cell line was transformed into CO using PFHM-II (GIBCO). 2 The cells were cultured at 37 ° C. in an incubator. After the culture, IgG in the supernatant was bound to a protein A column [GE Healthcare Bio-Sciences (Sweden)]. The bound IgG was eluted with a 100 mM aqueous citric acid solution (pH 3.0). 1 volume of 0.5 M phosphate buffer (pH 7.5) buffer was added to 1 volume of eluate, and the antibody that binds to the HMGB1 was obtained from the monoclonal antibody-producing cell line as purified IgG. .
This antibody was an antibody that binds to the HMGB1 degradation product. That is, an antibody (monoclonal antibody) that binds to the HMGB1 degradation product (hereinafter referred to as “the present anti-HMGB1 degradation product antibody (2H6)”) could be obtained from the 2H6 monoclonal antibody-producing cell line.
The anti-HMGB1 degradation product antibody (2H6) had an affinity for the HMGB1 degradation product of 1.5 times or more compared with the affinity for HMGB1, as described later.
In addition, as will be described later, this anti-HMGB1 degradation product antibody (2H6) has an affinity for the HMGB1 degradation product of 10% each compared with the affinity for HMGB2 and the affinity for the HMGB2 degradation product. It was more than twice.
That is, this anti-HMGB1 degradation product antibody (2H6) is the antibody (a) in the measurement method and measurement reagent of the HMGB1 degradation product of the present invention.
The 2H6 strain, which is a monoclonal antibody-producing cell line of this “anti-HMGB1 degradation product antibody (2H6)”, is the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (Kazusa Kamashiji, Kisarazu City, Chiba Prefecture, Japan). No. 5-8) is received as “Receipt Number: NITE AP-1570” dated March 15, 2013.
[Example 2] (Preparation of antibody binding to HMGB1 degradation product-2)
In preparing the antibody that binds to the HMGB1 degradation product, HMGB1 (total length) prepared in [1] of Reference Example 1 was used as an immunogen.
Thereafter, the procedure described in Example 1 (1) to (4) was followed to prepare an antibody that binds to the HMGB1 degradation product.
As a result, one clone was established from the grown fused cell lines and designated as 5D1 strain. An antibody (monoclonal antibody) that binds to the HMGB1 degradation product (hereinafter referred to as “anti-HMGB1 degradation product antibody (5D1)”) could be obtained from the 5D1 monoclonal antibody-producing cell line. This anti-HMGB1 degradation product antibody (5D1) was an antibody having a high affinity for the HMGB1 degradation product, as will be described later. That is, this anti-HMGB1 degradation product antibody (5D1) is the antibody of (b) in the measurement method and measurement reagent of the HMGB1 degradation product of the present invention.
The 5D1 strain, which is a monoclonal antibody-producing cell line of this “anti-HMGB1 degradation product antibody (5D1)”, is the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (Kazusa Kamashiji, Kisarazu City, Chiba Prefecture, Japan). No. 5-8) has been received as “Reception Number: NITE AP-1571” on March 15, 2013.
[Example 3] (Preparation of antibody binding to HMGB1 degradation product-3)
In preparing the antibody that binds to the HMGB1 degradation product, HMGB1 (total length) prepared in [1] of Reference Example 1 was used as an immunogen.
Thereafter, the procedure described in Example 1 (1) to (4) was followed to prepare an antibody that binds to the HMGB1 degradation product.
As a result, one clone was established from the grown fused cell lines, and named 2A10 strain. An antibody (monoclonal antibody) that binds to the HMGB1 degradation product (hereinafter referred to as “anti-HMGB1 degradation product antibody (2A10)”) could be obtained from the 2A10 monoclonal antibody-producing cell line. This anti-HMGB1 degradation product antibody (2A10) was an antibody having a high affinity for the HMGB1 degradation product, as will be described later. That is, this anti-HMGB1 degradation product antibody (2A10) is the antibody (b) in the measurement method and measurement reagent of the HMGB1 degradation product of the present invention.
The 2A10 strain, which is a monoclonal antibody-producing cell line of this “anti-HMGB1 degradation product antibody (2A10)”, is a patent microorganism deposit center of the National Institute of Technology and Evaluation (Kazusa Kamashiji, Kisarazu City, Chiba Prefecture, Japan). No. 5-8) is received as “Receipt Number: NITE AP-1572” on March 15, 2013.
[Example 4] (Preparation of antibody binding to HMGB1 degradation product-4)
In preparing the antibody that binds to the HMGB1 degradation product, HMGB1 (total length) prepared in [1] of Reference Example 1 was used as an immunogen.
Thereafter, the procedure described in Example 1 (1) to (4) was followed to prepare an antibody that binds to the HMGB1 degradation product.
As a result, one clone was established from the grown fused cell lines and was designated as 6H3 line. An antibody (monoclonal antibody) that binds to the HMGB1 degradation product (hereinafter referred to as “anti-HMGB1 degradation product antibody (6H3)”) could be obtained from the 6H3 monoclonal antibody-producing cell line. This anti-HMGB1 degradation product antibody (6H3) was an antibody having a high affinity for the HMGB1 degradation product, as will be described later. That is, this anti-HMGB1 degradation product antibody (6H3) is the antibody of (b) in the measurement method and measurement reagent of the HMGB1 degradation product of the present invention.
The 6H3 strain, which is a monoclonal antibody-producing cell line of the “anti-HMGB1 degradation product antibody (6H3)”, is a patent microorganism deposit center of the National Institute of Technology and Evaluation (Kazusa Kamashiji, Kisarazu City, Chiba Prefecture, Japan). No. 5-8) has been received as “Receipt Number: NITE AP-1573” on March 15, 2013.
[Example 5] (Confirmation of reactivity of antibody binding to HMGB1 degradation product)
About each of the antibody couple | bonded with the HMGB1 degradation product acquired in the said Example 1 and Example 2, the reactivity with each of HMGB1, the said HMGB1 degradation product, HMGB2, and the said HMGB2 degradation product was confirmed.
[1] Anti-HMGB1 degradation product antibody (5D1)
Regarding the “anti-HMGB1 degradation product antibody (5D1)” obtained in Example 2, the reactivity with each of HMGB1, HMGB1 degradation product, HMGB2, and HMGB2 degradation product was confirmed as follows.
1. SDS-polyacrylamide gel electrophoresis
(1) Reagent
The following reagents (a) to (c) were prepared respectively.
(A) SDS-polyacrylamide gel
Funakoshi Easy-Gel (III) precast gel (15%) (Funakoshi [Japan]) was used.
(B) Electrophoresis tank buffer
Preparation was performed as described in 1 (2) of Reference Example 2 to prepare a buffer solution for electrophoresis tank [0.1% SDS-192 mM glycine-25 mM Tris buffer].
(C) Sample treatment liquid
0.1 g of sodium dodecyl sulfate [SDS], 0.1 mL of 2-mercaptoethanol, 1 mL of 500 mM tris (hydroxymethyl) aminomethane [Tris] -hydrochloric acid buffer (pH 6.8), and 2 mL of glycerin are added to pure water. After mixing, a sample treatment solution [1% SDS-1% 2-mercaptoethanol-20% glycerin-50 mM Tris buffer] was prepared as 10 mL.
(2) Sample
Each of HMGB1, the HMGB1 degradation product, HMGB2, and the HMGB2 degradation product prepared in [1] to [3] of Reference Example 1 was used as a sample (standard sample).
In addition, molecular weight markers [Precision Plus Protein All Blue Standards markers; marker molecular weights 10 KDa, 15 KDa, 20 KDa, 25 KDa, 37 KDa, 50 KDa, 75 KDa, 100 KDa, 150 KDa and 250 KDa;
(3) Electrophoresis
Using the reagent prepared in (1), each sample of (2) was subjected to electrophoresis by SDS-polyacrylamide gel electrophoresis by the following operation.
(A) About each sample of said (2), it prepared so that a sample density | concentration might be 1 microgram / mL with the sample processing liquid of said (1) (c), and the boiling process was performed for 5 minutes at 100 degreeC. .
(B) The buffer solution for the electrophoresis tank of (b) of (1) was placed in the lower electrophoresis tank. Next, the SDS-polyacrylamide gel of (a) of (1) was set in an electrophoresis tank. Then, the buffer solution for electrophoresis tank (1) (b) was placed in the upper electrophoresis tank.
(C) 10 μL (10 ng) of the sample subjected to the treatment (a) was injected into the comb hole of the gel (b). Further, 2 μL of the sample of the molecular weight marker (2) was injected into the comb hole of the gel (b).
The sample and the molecular weight marker injected into this gel are the following “1” “molecular weight marker”, “2” “HMGB1”, “3” “HMGB1” from the left lane. “Degradation product”, “4” “HMGB2”, and “5” “the HMGB2 degradation product” were injected in this order.
“1”: “Molecular weight marker”
“2”: “HMGB1”
“3”: “degradation product of HMGB1”
“4”: “HMGB2”
“5”: “The HMGB2 degradation product”
(D) Next, electrophoresis was performed at a current of 20 mA for 90 minutes.
(E) After finishing the electrophoresis in (d), the gel was taken out of the glass plate.
By the above operation, each of HMGB1, the HMGB1 degradation product, HMGB2, and the HMGB2 degradation product was positioned in the gel according to its molecular weight, and this gel was obtained.
2. Western blotting method
(1) Antibody dilution solution [50 mM Tris (hydroxymethyl) aminomethane buffer [Tris buffer] containing 0.5% casein, 100 mM sodium chloride, and 0.1% sodium azide (pH 8. 0)] was diluted so that the concentration was 2 μg / mL, and an antibody such as anti-HMGB1 degradation product (5D1) obtained in Example 2 was diluted to prepare a solution of this antibody.
(2) The transfer of the gel obtained in (e) of (1) above (3) was performed in a semi-dry manner using a transblot SD cell (BIO-RAD Laboratories [USA]) according to the instructions for use. .
First, the gel obtained in (e) of (1) above was placed on a transfer device.
Next, a 9 cm × 9 cm polyvinyl difluoride membrane (BIO-RAD Laboratories [USA]) was layered on the gel, and 48 mM Tris (hydroxymethyl) aminomethane [Tris], 39 mM glycine, and 20% ( V / V) Using a transfer buffer composed of methanol, transfer was performed at an electric current of 100 mA for 1 hour, and the HMGB1, HMGB1 degradation product, HMGB2 and HMGB2 degradation product located in the gel according to the molecular weight. And the like were transferred from the gel to the polyvinyl difluoride film.
(3) Polyvinyl difluoride membranes on which proteins such as HMGB1, HMGB1 degradation product, HMGB2, and HMGB2 degradation product were transferred to a blocking solution [0.5% casein, 100 mM chloride. Blocking was performed by immersing in 20 mL of 50 mM Tris (hydroxymethyl) aminomethane buffer [Tris buffer] (pH 8.0)] containing sodium and 0.1% sodium azide overnight at room temperature. It was.
(4) Next, the polyvinyl difluoride film of (3) was reacted for 2 hours at room temperature in the solution of “anti-HMGB1 degradation product antibody (5D1)” prepared in (1) above.
Thereby, each said HMGB1, said HMGB1 degradation product, HMGB2, and said HMGB2 degradation product transcribe | transferred to the polyvinyl difluoride film | membrane are made to react with said "anti-HMGB1 degradation product etc. antibodies (5D1)". It was.
(5) The polyvinyl difluoride membrane subjected to the above operation (4) was washed by shaking in 20 mL of a washing solution [phosphate buffered saline containing 0.05% Tween 20] for 5 minutes. This was done three times.
(6) Next, a POD-labeled anti-mouse IgG antibody [DakoCytomation (Denmark)] was added to a labeled antibody diluent [50 mM tris (hydroxymethyl) amino containing 0.5% casein and 100 mM sodium chloride. The polyvinyl difluoride film of (5) was immersed in a solution prepared by diluting 1000 times with a methane buffer [Tris buffer] (pH 8.0)] at room temperature for 90 minutes to react.
(7) The polyvinyl difluoride film subjected to the operation of (6) was washed by shaking in 20 mL of the cleaning solution for 5 minutes. This operation was performed three times.
(8) The polyvinyl difluoride membrane of (7) above is placed at room temperature in 20 mL of phosphate buffered saline containing 0.025% 3,3′-diaminobenzidine tetrahydrochloride and 0.01% hydrogen peroxide. For 15 minutes to develop color.
Thereby, in this polyvinyl difluoride film, the molecular weight of each of the HMGB1, the HMGB1 degradation product, HMGB2, or the HMGB2 degradation product, which is recognized and bound by the “anti-HMGB1 degradation product antibody (5D1)”. Color development was caused at a position corresponding to.
From the presence / absence of color development on the polyvinyl difluoride film and the position (molecular weight) of the color development, with respect to the “anti-HMGB1 degradation product antibody (5D1)”, HMGB1, HMGB1 degradation product, HMGB2, and HMGB2 degradation product The reactivity with each of was confirmed.
3. result
(1) The polyvinyl difluoride film colored in (2) above is shown in FIG.
In this figure, in order from the left lane, the “molecular weight marker” lane (denoted as “1”), the “HMGB1” lane (denoted as “2”), and the “the HMGB1 degradation product” lane (denoted as “3”). Notation), “HMGB2” lane (denoted “4”), and “the HMGB2 degradation product” lane (denoted “5”).
(2) From this figure, it can be seen that in the “anti-HMGB1 degradation product antibody (5D1)”, color development is observed at the positions indicating HMGB1, the HMGB1 degradation product, HMGB2, and the HMGB2 degradation product.
From this, it was confirmed that the “anti-HMGB1 degradation product antibody (5D1)” recognizes and binds to all of HMGB1, the HMGB1 degradation product, HMGB2, and the HMGB2 degradation product.
[2] Anti-HMGB1 degradation product antibody (2H6)
With respect to the “anti-HMGB1 degradation product antibody (2H6)” obtained in Example 1, the reactivity with HMGB1, the HMGB1 degradation product, HMGB2 and the HMGB2 degradation product was confirmed.
That is, in place of the “anti-HMGB1 degradation product antibody (5D1)” instead of using the “anti-HMGB1 degradation product antibody (2H6)” obtained in Example 1, as described in [1] above. The operation was carried out to confirm the reactivity of the “anti-HMGB1 degradation product antibody (2H6)” with HMGB1, the HMGB1 degradation product, HMGB2, and the HMGB2 degradation product.
FIG. 3 shows a developed polyvinyl difluoride film as a result of confirming this reactivity.
In this figure, in order from the left lane, the “molecular weight marker” lane (denoted as “1”), the “HMGB1” lane (denoted as “2”), and the “the HMGB1 degradation product” lane (denoted as “3”). Notation), “HMGB2” lane (denoted “4”), and “the HMGB2 degradation product” lane (denoted “5”).
From this figure, in the “anti-HMGB1 degradation product antibody (2H6)”, a deep color development is observed at the position showing the HMGB1 degradation product, but only an extremely light color development appears at the position showing HMGB1. . Color development is not observed at the positions indicating HMGB2 and the HMGB2 degradation product.
From this, it was confirmed that the “anti-HMGB1 degradation product antibody (2H6)” recognizes and binds to the HMGB1 degradation product with high specificity. And it was confirmed that it binds slightly to HMGB1 and does not bind to HMGB2 and the HMGB2 degradation product.
[Example 6] (Confirmation of affinity of antibody binding to HMGB1 degradation product)
For each of the antibody that binds to the HMGB1 degradation product obtained in Examples 1 to 4 and the antibody that binds to other HMGB1, etc., the affinity with HMGB1, the HMGB1 degradation product, HMGB2, and the HMGB2 degradation product, respectively. Was confirmed by enzyme immunoassay (ELISA method).
1. Reagent
(1) Solid-phase microplate such as HMGB1 degradation products
Each of HMGB1, HMGB1 degradation product, HMGB2 and HMGB2 degradation product prepared in [1] to [3] of Reference Example 1 was prepared with phosphate buffered saline to a concentration of 1 μg / mL. The 96-well microtiter plate [Thermo Fisher Scientific Inc. (Ind., Illinois, USA)] was injected into a well of 100 μL, allowed to stand at 25 ° C. for 18 hours, and the HMGB1, HMGB1 degradation product, HMGB2 and HMGB2 degradation product were respectively added to the wells of the microtiter plate. To solid phase.
Next, after removing the liquid in the well of the microtiter plate, 250 μL each of Tris buffered saline (TBS) [pH 8.0] containing 0.5% casein and 0.1% sodium azide was added to each microtiter plate. The well was dispensed and subjected to blocking treatment. Thereafter, each well was sealed with a plate seal and kept refrigerated until use so as not to evaporate. This was used as a solid-phased microplate such as HMGB1 degradation product.
(2) POD-labeled anti-mouse IgG antibody solution
POD-labeled anti-mouse IgG antibody [DakoCytomation (Denmark)] was diluted 1000-fold with 50 mM Tris-HCl buffer (pH 8.0) containing 0.5% sodium caseinate and 100 mM sodium chloride. This was used as a POD-labeled anti-mouse IgG antibody solution.
(3) Cleaning liquid
Phosphate buffered saline containing 0.05% Tween 20 was used as a washing solution.
(4) Diluent
A 50 mM Tris-HCl buffer solution (pH 8.0) containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide was used as a diluent.
(5) Coloring solution
A 0.045% 3,3 ′, 5,5′-tetramethylbenzidine hydrochloride aqueous solution (pH 2.0) containing 0.2 mM EDTA · disodium was used as a color developing solution.
(6) Substrate solution
A 60 mM disodium phosphate aqueous solution (pH 4.3) containing 5 mM hydrogen peroxide, 41 mM citric acid, and 0.2 mM EDTA · disodium was used as a substrate solution.
(7) Chromogenic substrate
The chromogenic solution and the substrate solution were returned to room temperature before use, and mixed in equal amounts at the time of use to obtain a chromogenic substrate.
(8) Reaction stop solution
0.7N sulfuric acid was used as a reaction stop solution.
2. sample
An antibody solution for confirming the affinity with HMGB1, the HMGB1 degradation product, HMGB2, and the HMGB2 degradation product was prepared as follows and used as a sample.
(1) Anti-HMGB1 antibody (2D4)
An antibody (monoclonal antibody) that binds to HMGB1 produced from the 2D4 monoclonal antibody-producing cell line (hereinafter referred to as “anti-HMGB1 antibody (2D4)”) has a concentration of 0.625 ng / mL, 1 The antibody solution of each concentration of anti-HMGB1 antibody (2D4) was prepared by diluting with the dilution solution of (1) of (1) so as to be .25 ng / mL, 2.5 ng / mL, and 5 ng / mL.
(2) Anti-HMGB1 antibody (4F12)
An antibody (monoclonal antibody) that binds to HMGB1 produced from the 4F12 monoclonal antibody-producing cell line (hereinafter referred to as “anti-HMGB1 antibody (4F12)”) has a concentration of 0.625 ng / mL, 1 The antibody solution of each concentration of anti-HMGB1 antibody (4F12) was prepared by diluting with the diluent (1) of 1 above so as to be .25 ng / mL, 2.5 ng / mL, and 5 ng / mL.
(3) Anti-HMGB1 antibody (8H4)
An antibody (monoclonal antibody) that binds to HMGB1 and the like produced from the 8H4 monoclonal antibody-producing cell line (hereinafter referred to as “anti-HMGB1 antibody (8H4)”) has a concentration of 0.625 ng / mL, 1 The antibody solution of anti-HMGB1 antibody (8H4) of each concentration was prepared by diluting with the diluent (1) of 1 above so as to be .25 ng / mL, 2.5 ng / mL, and 5 ng / mL.
(4) Anti-HMGB1 degradation product antibody (2H6)
The anti-HMGB1 degradation product antibody (2H6) was diluted in the above (4) so that its concentrations were 0.625 ng / mL, 1.25 ng / mL, 2.5 ng / mL, and 5 ng / mL, respectively. The antibody solution of this anti-HMGB1 degradation product antibody (2H6) of each concentration was prepared by diluting with a liquid.
(5) Antibodies such as anti-HMGB1 degradation products (5D1)
The anti-HMGB1 degradation product or the like antibody (5D1) is diluted (1) in (1) so that the concentrations thereof are 0.625 ng / mL, 1.25 ng / mL, 2.5 ng / mL, and 5 ng / mL, respectively. The antibody solution of anti-HMGB1 degradation product antibody (5D1) at each concentration was prepared by diluting with a solution.
(6) Anti-HMGB1 degradation product antibody (2A10)
The anti-HMGB1 degradation product or the like antibody (2A10) is diluted in (4) of 1 above so that the concentrations thereof are 0.625 ng / mL, 1.25 ng / mL, 2.5 ng / mL, and 5 ng / mL, respectively. The antibody solution of antibodies (2A10) such as anti-HMGB1 degradation products at each concentration was prepared by diluting with a solution.
(7) Anti-HMGB1 degradation product antibody (6H3)
The anti-HMGB1 degradation product antibody (6H3) is diluted (4) in the above 1 so that the concentrations thereof are 0.625 ng / mL, 1.25 ng / mL, 2.5 ng / mL, and 5 ng / mL, respectively. The antibody solution of antibody (6H3) such as anti-HMGB1 degradation product at each concentration was prepared by diluting with a solution.
(8) Anti-HMGB1 antibody (MD78)
Monoclonal antibody-producing cell line MD78 strain (incorporated administrative agency National Institute of Advanced Industrial Science and Technology patent biological deposit center (1st, 1st, 1st, 1st, 1st, 1st, Tsukuba, Ibaraki, Japan) as FERM P-18405, July 4, 2001 The antibody (monoclonal antibody) that binds to HMGB1 and the like produced from the above (hereinafter referred to as “anti-HMGB1 antibody (MD78)”) has a concentration of 0.625 ng / mL, The antibody solution of each concentration of anti-HMGB1 antibody (MD78) was prepared by diluting with the diluent (1) of (1) so as to be 1.25 ng / mL, 2.5 ng / mL, and 5 ng / mL. .
(9) Anti-HMGB1 antibody (MD77)
The monoclonal antibody-producing cell line MD77 strain (incorporated administrative agency National Institute of Advanced Industrial Science and Technology patent biological deposit center (1-6, 1st east, Tsukuba city, Ibaraki, Japan) FERM P-18404 July 4, 2001 The antibody (monoclonal antibody) that binds to HMGB1 and the like produced from the above (hereinafter referred to as “anti-HMGB1 antibody (MD77)”) has a concentration of 0.625 ng / mL, The antibody solution of each concentration of anti-HMGB1 antibody (MD77) was prepared by diluting with the diluent of (1) of (1) so as to be 1.25 ng / mL, 2.5 ng / mL, and 5 ng / mL. .
(10) Anti-HMGB1 antibody (4C3)
Antibody that binds to HMGB1 and the like produced from the 4C3 monoclonal antibody-producing cell line (monoclonal antibody; trade name: Anti-HMGB1 antibody [4C3] (Abcam)) (hereinafter referred to as “anti-HMGB1 antibody (4C3)”) Are diluted with the diluent (1) above so that the concentrations thereof are 0.625 ng / mL, 1.25 ng / mL, 2.5 ng / mL, and 5 ng / mL, respectively. An antibody solution of anti-HMGB1 antibody (4C3) was prepared.
(11) Anti-HMGB1 antibody (J2E1)
An antibody that binds to HMGB1 and the like produced from the J2E1 strain of a monoclonal antibody-producing cell line (monoclonal antibody; trade name: HMG-1 Antibody (J2E1): sc-135809 (Santa cruz biotechnology, Inc.)) [hereinafter, “ The anti-HMGB1 antibody (J2E1) ”), so that the concentrations thereof are 0.625 ng / mL, 1.25 ng / mL, 2.5 ng / mL, and 5 ng / mL, respectively. It diluted with the dilution liquid and prepared the antibody solution of anti-HMGB1 antibodies (J2E1) of each concentration.
(12) Anti-HMGB1 antibody (HAP46.5)
An antibody that binds to HMGB1 and the like produced from the monoclonal antibody-producing cell line HAP46.5 (monoclonal antibody; trade name: Mouse monoclonal [HAP46.5] to HMGB1 (Abcam)) [hereinafter referred to as “anti-HMGB1 antibody ( HAP46.5) ”) with the dilution of (1) in (1) above so that the concentrations thereof are 0.625 ng / mL, 1.25 ng / mL, 2.5 ng / mL, and 5 ng / mL, respectively. After dilution, an antibody solution of anti-HMGB1 antibody (HAP46.5) at each concentration was prepared.
3. Measurement
(1) The wells of the solid phase microplate such as the HMGB1 degradation product of 1 (1) were washed 3 times with 400 μL of the washing solution of 1 (3).
(2) Next, 100 μL of each of the 4 concentrations of the anti-HMGB1 antibody (2D4) solution (2D4) in 2 (1) above was dispensed to each well as a sample, and then left at 25 ° C. for 2 hours. Each concentration of anti-HMGB1 antibody (2D4) is immobilized on the well of the HMGB1 degradation product-immobilized microplate, each of the HMGB1, the HMGB1 degradation product, HMGB2, and the HMGB2 degradation product, Each was subjected to an antigen-antibody reaction.
(3) Next, each well was washed three times with 400 μL of the washing liquid of (1) above.
(4) Next, 100 μL of the POD-labeled anti-mouse IgG antibody solution of (1) above was dispensed into each well and allowed to stand at 25 ° C. for 1 hour to cause antigen-antibody reaction.
(5) Next, each well was washed 3 times with 400 μL of the washing liquid of (1) above.
(6) Next, 100 μL of the chromogenic substrate of (7) above was dispensed into each well, and allowed to stand at room temperature for 20 minutes to cause a chromogenic reaction with peroxidase (POD) as a labeling enzyme.
(7) Next, 100 μL of the reaction stop solution of (1) above was dispensed into each well to stop the color reaction.
(8) Next, the absorbance (primary wavelength: 450 nm, subwavelength: 550 nm) of the liquid in each well was measured, and the absorbance when each of the solutions of the above four concentrations of anti-HMGB1 antibody (2D4) was measured as a sample. Got.
(9) Instead of the 4 concentrations of the anti-HMGB1 antibody (2D4) solution (2D4) in the above (2), the antibody solutions (4 concentrations each) of the above (2) to (12) are used as samples. Except for the use, the procedure was carried out as described in (1) to (8) above, and the absorbance was measured when each of these antibody solutions was measured as a sample.
4). Measurement result
(1) The measurement results in 3 above, that is, the results of confirming the affinity of each of the antibodies with HMGB1, HMGB1 degradation product, HMGB2, and HMGB2 degradation product, are shown in FIGS.
In these figures, the letters representing the antibody-producing cell lines whose affinity was confirmed were shown above the figures.
In these figures, the horizontal axis indicates the antibody concentration (ng / mL) in the antibody solution used as the sample, and the vertical axis indicates the absorbance obtained by the measurement [from the absorbance at the main wavelength (450 nm) to the sub-wavelength (550 nm). The value obtained by subtracting the absorbance in FIG.
In these figures, “♦” indicates a measured value (the above-described absorbance) in a well in which HMGB1 is immobilized, and “■” indicates a measured value (in the above-described absorbance in a well on which the HMGB1 degradation product is immobilized). ), “▲” indicates the measured value (the above-mentioned absorbance) in the well in which HMGB2 is immobilized, and “●” indicates the measured value (in the above-described absorbance) in the well on which the HMGB2 degradation product is immobilized. Show.
(2) Table 1 shows the measured values of the measurement results in 3 above.
Figure JPOXMLDOC01-appb-T000001
 In these tables, letters representing antibody producing cell lines whose affinity was confirmed were shown above the tables.
In these tables, the left column indicates the antibody concentration (ng / mL) in the antibody solution used as a sample, and the absorbance [primary wavelength ( 450 nm) minus the absorbance at the sub-wavelength (550 nm)].
In these tables, the value obtained by dividing the measured value in the well in which the HMGB1 degradation product is immobilized (the absorbance described above) by the antibody concentration (ng / mL) value in the antibody solution used as a sample is shown. The indicated column (the third column from the left for each solid-phased well) was also provided.
5. Summary
(1) From FIG. 4 and Table 1 which are the results of the enzyme immunoassay, each antibody whose affinity was confirmed by the measurement is classified into the following three types (i) to (iii). You can see that
(I) An antibody that binds to the HMGB1 degradation product and has an affinity for the HMGB1 degradation product that is at least 1.5 times the affinity for HMGB1 (antibody concentration: 0.625 ng / mL) ~ 5ng / mL): "This anti-HMGB1 degradation product antibody (2H6)"
[* Note that the affinity for the HMGB1 degradation product is at least 10 times greater than the affinity for HMGB2 and the affinity for the HMGB2 degradation product, respectively. (Antibody concentration: 0.625 ng / mL to 5 ng / mL)]
[* Antibody (a) in the immunological measurement method and reagent of the HMGB1 degradation product contained in the sample of the present invention]
(Ii) an antibody that binds to the HMGB1 degradation product and has high affinity for the HMGB1 degradation product: “anti-HMGB1 degradation product antibody (5D1)”, “anti-HMGB1 degradation product antibody (2A10)”, And "Anti-HMGB1 degradation product antibody (6H3)"
[* Note that the value obtained by dividing the measured value (the absorbance described above) in the well in which the HMGB1 degradation product is immobilized by the antibody concentration (ng / mL) value in the antibody solution used as a sample is 0.5 or more. It is. (Antibody concentration: 0.625 ng / mL to 5 ng / mL)]
[* In addition, the measured value (the absorbance described above) in the well in which the HMGB1 degradation product is immobilized is 6 times or more the absorbance value of the antibody produced from the reference antibody-producing cell (“MD78”). is there. (Antibody concentration: 0.625 ng / mL to 5 ng / mL)]
[* The antibody of (b) in the immunological measurement method and reagent for the HMGB1 degradation product contained in the sample of the present invention]
(Iii) Antibodies other than (i) and (ii): “Anti-HMGB1 antibody (2D4)”, “Anti-HMGB1 antibody (4F12)”, “Anti-HMGB1 antibody (8H4)”, “Anti-HMGB1 antibody, etc. (MD78), "anti-HMGB1 antibody (MD77)", "anti-HMGB1 antibody (4C3)", "anti-HMGB1 antibody (J2E1)", and "anti-HMGB1 antibody (HAP46.5)"
(2) From these facts, “the present anti-HMGB1 degradation product antibody (2H6)” is an antibody that binds to the HMGB1 degradation product, and the affinity for the HMGB1 degradation product is higher than the affinity for HMGB1. The antibody was confirmed to be at least 1.5 times.
Further, “anti-HMGB1 degradation product antibody (5D1)”, “anti-HMGB1 degradation product antibody (2A10)” and “anti-HMGB1 degradation product antibody (6H3)” are antibodies that bind to the HMGB1 degradation product, respectively. Thus, it was confirmed that the antibody had a high affinity for the HMGB1 degradation product.
[Example 7] (Confirmation of measurement of HMGB1 degradation product)
It was confirmed that the HMGB1 degradation product according to the present invention can be measured using the antibodies that bind to the HMGB1 degradation products obtained in Examples 1 to 4 and other HMGB1 binding antibodies.
1. Reagent
(1) Antibody-immobilized microplate
Each of the following five types of antibodies (i) to (v) is purified using protein A, and the concentration of these antibodies is 2.5 μg / mL with phosphate buffered saline (PBS). Each was diluted as follows.
(I) Anti-HMGB1 degradation product antibody (2H6)
(Ii) Anti-HMGB1 degradation product antibody (5D1)
(Iii) Antibody such as anti-HMGB1 degradation product (2A10)
(Iv) Antibodies such as anti-HMGB1 degradation products (6H3)
(V) Anti-HMGB1 antibody (MD77)
Next, 100 μL of each of these antibody solutions was dispensed into each well of a microtiter plate (microplate) [Nunc, trade name: Maxisorp] and allowed to stand overnight at 25 ° C. Each was individually immobilized on a well of a microplate.
Next, the liquid in the wells of the microplate was removed, and 250 μL each of Tris-buffered saline (TBS) [pH 8.0] containing 0.5% sodium caseinate and 0.1% sodium azide was added to each well. The aliquots were sealed, and each well was sealed with a plate seal so as not to evaporate and stored refrigerated until use.
This was used as an antibody-immobilized microplate for each of the five types of antibodies (i) to (v).
(2) POD labeled antibody solution
For each of the following 6 types of antibodies (A) to (F), 6 types of peroxidase-labeled antibodies were prepared by “peroxidase labeling kit-NH2” (Dojin Chemical Co., Ltd.) by a predetermined method. These were used as POD-labeled antibody solutions.
(A) Anti-HMGB1 degradation product antibody (2H6)
(B) Anti-HMGB1 degradation product antibody (5D1)
(C) Anti-HMGB1 degradation product antibody (2A10)
(D) Antibody (6H3) such as anti-HMGB1 degradation product
(E) Anti-HMGB1 antibody (MD77)
(F) Anti-HMGB1 antibody (04) [Mouse antibody (monoclonal antibody) that binds to HMGB1 etc. produced from the 04 monoclonal antibody-producing cell line [hereinafter referred to as “anti-HMGB1 antibody (04)]]. ]
(3) Cleaning liquid
Phosphate buffered saline containing 0.05% Tween 20 was used as a washing solution.
(4) Diluent
A 50 mM Tris-HCl buffer solution (pH 8.0) containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide was used as a diluent.
(5) Coloring solution
A 0.045% 3,3 ′, 5,5′-tetramethylbenzidine hydrochloride aqueous solution (pH 2.0) containing 0.2 mM EDTA · disodium was used as a color developing solution.
(6) Substrate solution
A 60 mM disodium phosphate aqueous solution (pH 4.3) containing 5 mM hydrogen peroxide, 41 mM citric acid, and 0.2 mM EDTA · disodium was used as a substrate solution.
(7) Chromogenic substrate
The chromogenic solution and the substrate solution were returned to room temperature before use, and mixed in equal amounts at the time of use to obtain a chromogenic substrate.
(8) Reaction stop solution
0.7N sulfuric acid was used as a reaction stop solution.
2. sample
(1) Sample (the HMGB1 degradation product)
The HMGB1 degradation product prepared in [2] of Reference Example 1 was subjected to a 50 mM Tris-HCl buffer (pH 8.0) containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide. What was prepared so that it might become a concentration of 16 ng / mL was made into the sample (said HMGB1 degradation product).
(2) Sample (HMGB1)
The HMGB1 prepared in [1] of Reference Example 1 was mixed with a 50 mM Tris-HCl buffer solution (pH 8.0) containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide at 16 ng / mL. A sample (HMGB1) was prepared so as to have a concentration of.
(3) Sample (reagent blind test)
50 mM Tris-HCl buffer solution (pH 8.0) containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide for reagent blind (reagent blank) measurement as a control [degradation of HMGB1 The concentration of the product and HMGB1 was 0 ng / mL] was used as a sample (reagent blind test).
3. Measurement
(1) Each well of each of the antibody-immobilized microplates for each of the five types of antibodies (1) (i) to (v) of (1) is 3 with 400 μL of the washing solution of (3) above. Washed twice.
(2) Next, after 100 μL of the sample (1) (degradation product of HMGB1) of 2 above was dispensed into each well, it was allowed to stand overnight at 37 ° C. and contained in this sample (degradation product of HMGB1) The HMGB1 degradation product was allowed to undergo an antigen-antibody reaction with each of the five types of antibodies immobilized on the wells of each antibody-immobilized microplate.
(3) Next, each well was washed 5 times with 400 μL of the washing solution of (1) above.
(4) Next, 100 μL of the POD-labeled antibody solution obtained by peroxidase-labeling the antibody of (1) (2) (A) above was dispensed into each well and allowed to stand at 37 ° C. for 90 minutes. I was allowed to do.
(5) Next, each well was washed 5 times with 400 μL of the washing solution of (1) above.
(6) Next, 100 μL of the chromogenic substrate of (7) above was dispensed into each well and allowed to stand at room temperature for 30 minutes to cause a chromogenic reaction with peroxidase (POD) as a labeling enzyme.
(7) Next, 100 μL of the reaction stop solution of (1) above was dispensed into each well to stop the color reaction.
(8) Next, the absorbance (450 nm) of the liquid in each well was measured, and the absorbance when the sample (the HMGB1 degradation product) was measured was obtained.
(9) Instead of the sample in (2) (degradation product of HMGB1), the sample in (2) (HMGB1) in 2 and the sample in (3) in 2 (reagent blind test) are used as samples. Except for the above, it was carried out as described in the above (1) to (8), and the absorbance when each of these samples was measured was obtained.
(10) In place of “the POD-labeled antibody solution obtained by peroxidase-labeling the antibody of (1) in (2) above” in (4) above, the antibody in (B) of (1) above (2) "An oxidase-labeled POD-labeled antibody solution", "POD-labeled antibody solution obtained by peroxidase-labeling the antibody of (1) in (2) above", "Peroxidase-labeled antibody in (1) of (1) above" Peroxidase-labeled POD-labeled antibody solution ”,“ POD-labeled antibody solution obtained by peroxidase-labeling the antibody of (1) in (2) above ”, and“ peroxidase-labeled antibody of (F) in (1) above ” Except for using each “POD-labeled antibody solution” as the POD-labeled antibody solution, the procedure was as described in (1) to (9) above, and the absorbance when each sample was measured was obtained. [The combination of the antibody-immobilized microplate and the POD-labeled antibody solution used for the measurement is as shown in FIG. 5 and Table 2. ]
4). Measurement result
(1) Measurement results in the above 3, ie, an enzyme immunoassay method (ELISA method) using an antibody that binds to the HMGB1 degradation product obtained in Examples 1 to 4 and an antibody that binds to other HMGB1 FIG. 5 shows the results of measuring the HMGB1 degradation product and HMGB1.
In this figure, the upper part of the horizontal axis shows the letters representing the antibody production cell line of the POD-labeled antibody solution used for the measurement, and the lower part of the horizontal axis shows the production of the antibody on the antibody-immobilized microplate used for the measurement. The letters representing cell lines are shown, and the vertical axis represents the absorbance (450 nm) obtained by measurement.
Further, in this figure (bar graph), each bar represents a measured value (measurement value described above) measured for the sample (HMGB1) in order from the left side for each POD-labeled antibody solution and antibody-immobilized microplate used for the measurement. The measured value when measured for the sample (the HMGB1 degradation product) (the absorbance described above), and the measured value when measured for the sample (reagent blind) are shown.
(2) Table 2 shows the measurement values of the measurement results in 3 above.
Figure JPOXMLDOC01-appb-T000002
 In this table, in order from the left side, letters representing the antibody producing cell line of the antibody-immobilized microplate used for the measurement, letters representing the antibody producing cell line of the POD-labeled antibody solution used for the measurement, sample Measured value when measured for (HMGB1) (absorbance described above), measured value when measured for sample (degraded product of HMGB1) (absorbance described above), measured value when measured for sample (blind reagent) (The above-mentioned absorbance) is shown.
5. Summary
(1) From FIG. 5 and Table 2 which are the results of the measurement by the enzyme immunoassay described above, the following can be understood.
That is, “this anti-HMGB1 degradation product antibody (2H6)” and “anti-HMGB1 degradation product antibody (5D1)”, “anti-HMGB1 degradation product antibody (2A10)” or “anti-HMGB1 degradation product antibody (6H3)” , The measured value (absorbance) of HMGB1 is low, while the measured value (absorbance) of the HMGB1 degradation product is high. Also, in this case, it can be seen that all of the reagent blind tests (reagent blanks) are low.
That is, “the antibody of (i) of (5) in Example 6” (the antibody of (a) in the immunological measurement method and immunological measurement reagent of the HMGB1 degradation product of the present invention), and “ Used in combination with the antibody of (ii) (ii) of Example 6 (the method for immunological measurement of the HMGB1 degradation product of the present invention and the antibody (b) in the immunological measurement reagent) of Example 6 In this case, it can be seen that measurement of HMGB1 contained in the sample can be suppressed, and that the HMGB1 degradation product contained in the sample can be measured with high sensitivity.
(2) On the other hand, when “anti-HMGB1 antibody (MD77)” or “anti-HMGB1 antibody (04)” is used for measurement, the measured value (absorbance) of the degradation product of HMGB1 is extremely low. It can be seen that the measured value (absorbance) of the HMGB1 degradation product and the measured value (absorbance) of HMGB1 are of the same order and have almost no difference.
That is, when “the antibody of (iii) in (5) of Example 6” (antibodies other than (i) and (ii) of (1) of Example 6 is used) is used for the measurement. Shows that the HMGB1 degradation product contained in the sample cannot be measured with high sensitivity and high specificity.
(3) Therefore, from the result of the examination in this example, “the antibody in (a)” and “(b) in“ the immunological measurement method and reagent for the HMGB1 degradation product contained in the sample ”of the present invention. It was found that the measurement of HMGB1 contained in the sample can be suppressed and the HMGB1 degradation product contained in the sample can be measured with high sensitivity by using the antibody in the above) for the measurement.
[Example 8] (Method and reagent-1 for immunological measurement of degradation products of HMGB1)
The HMGB1 degradation product and the HMGB1 contained in the sample by the immunological measurement method and immunological measurement reagent of the HMGB1 degradation product of the present invention and the conventional immunological measurement method and immunoassay reagent Was measured.
[1] Measurement using conventional immunological measurement methods and immunological measurement reagents
1. Reagent
“HMGB1 ELISA Kit II” which is a measurement reagent (research reagent) for HMGB1 based on ELISA / sandwich method using solid-phased anti-HMGB1 polyclonal antibody and peroxidase (POD) -labeled anti-HMGB1,2 monoclonal antibody. Sinotest (Japan)) was used as a conventional immunoassay reagent.
2. sample
(1) Sample (the HMGB1 degradation product)
The HMGB1 degradation product prepared in [2] of Reference Example 1 was added to each 2.5 ng / kg of phosphate buffered saline containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide. Samples (the aforementioned HMGB1 degradation products) were prepared so as to have concentrations of mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, and 80 ng / mL, respectively.
In addition, a phosphate buffered saline containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide was used as a sample (the HMGB1 degradation product) having a concentration of the HMGB1 degradation product of 0 ng / mL. .
(2) Sample (HMGB1)
HMGB1 prepared in [1] of Reference Example 1 was added to each of 2.5 ng / mL and 5 ng with phosphate buffered saline containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide. Samples (HMGB1) were prepared to have concentrations of / ng, 10 ng / mL, 20 ng / mL, 40 ng / mL, and 80 ng / mL.
A phosphate buffered saline containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide was used as a sample (HMGB1) having a HMGB1 concentration of 0 ng / mL.
3. Measurement
(1) For each of the sample of (2) (1) (the degradation product of HMGB1) [total 7 concentrations] and each of the sample of (2) (HMGB1) (total 7 concentrations), Using “HMGB1 ELISA Kit II” (Sinotest Inc. [Japan]), which is an immunological measurement reagent, measurement was performed as described in the package insert.
(2) As a result of measurement of each sample, absorbance (450 nm) was obtained by the measurement of (1).
4). Measurement result
(1) The measurement results in 3 above, that is, the results of measurement of the HMGB1 degradation product contained in the sample and HMGB1 contained in the sample by the conventional immunological measurement method and immunological measurement reagent are shown in FIG. It was shown to.
In this figure, the horizontal axis represents the HMGB1 degradation product contained in the sample or the concentration (ng / mL) of HMGB1 contained in the sample, and the vertical axis represents the absorbance (450 nm) obtained by the measurement.
Further, in this figure, “■” indicates a measured value (the absorbance described above) when measured on the sample of (2) (1) (decomposed product of HMGB1) [total 7 concentrations], and “♦” indicates the above 2 The measured value (the above-mentioned absorbance) when measured for the sample (HMGB1) (total 7 concentrations) of (2).
(2) Table 3 shows the measurement values of the measurement results in 3 above.
Figure JPOXMLDOC01-appb-T000003
 In this table, “(i) HMGB1 degradation product contained in the sample or concentration of HMGB1 contained in the sample (ng / mL)”, “(ii) the sample of (2) above, in order from the left side. (HMGB1) [Measured value when measured for a total of 7 concentrations] (absorbance at 450 nm) "," (iii) From the measured value of (ii) above, a reagent blind value [sample with a concentration of HMGB1 of 0 ng / mL ( (Measurement value (absorbance at 450 nm)] in HMGB1)] (subtraction difference) "," (iv) The value (absorbance difference) in the sample (the HMGB1 degradation product) described later (vibration) in (iii) above. Value measured by dividing the value (absorbance difference) in the sample (HMGB1) ”,“ (v) When measuring the sample of (2) (1) (decomposed product of HMGB1) [total 7 concentrations] “Measured value (absorbance at 450 nm)”, “(vi) The value of the reagent blinded from the measured value of (v) [the measured value of the sample having the HMGB1 degradation product concentration of 0 ng / mL (the HMGB1 degradation product) ( (Absorbance at 450 nm)]] is subtracted (absorbance difference) ".
[2] Method of immunoassay of the HMGB1 degradation product of the present invention and measurement using an immunoassay reagent
1. Reagent
(1) Antibody-immobilized microplate (5D1)
The antibody (5D1) such as anti-HMGB1 degradation product of Example 2 was purified using protein A, and diluted with phosphate buffered saline (PBS) to a concentration of 2.5 μg / mL.
Next, 100 μL of this was dispensed into each well of a microtiter plate (microplate) [Nunc, trade name: Maxisorp] and allowed to stand at 25 ° C. overnight, and the above-mentioned anti-HMGB1 degradation product antibody ( 5D1) was immobilized on a well of a microplate.
Next, the liquid in the wells of the microplate was removed, and 250 μL each of Tris-buffered saline (TBS) [pH 8.0] containing 0.5% sodium caseinate and 0.1% sodium azide was added to each well. The aliquots were sealed, and each well was sealed with a plate seal so as not to evaporate and stored refrigerated until use.
This was designated as an antibody-immobilized microplate (5D1).
(2) Biotin-labeled antibody solution (2H6)
The anti-HMGB1 degradation product antibody (2H6) of Example 1 was labeled with biotin using Sulfo-NHS-LC-Biotin (Pierce, product code number: 21335).
This biotin-labeled antibody was mixed with 50 mM Tris-HCl buffer (pH 7.8) containing 100 mM sodium chloride, 0.5% sodium caseinate, 2 mM EDTA.2 sodium, 0.1% sodium azide, and 10% mouse serum. Was dissolved to a concentration of 2 μg / mL, and this was used as a biotin-labeled antibody solution (2H6).
(3) Streptavidin-peroxidase conjugate solution
Streptavidin-PolyHRP40 (Stereospecific Detection Technologies [Germany]; product code number: SP40C), 100 mM sodium chloride, 0.5% casein (vitamin free) and 100 mM Tris-HCl buffer (pH 7) containing 0.5 mM calcium chloride 8) was diluted 10,000 times.
This was used as a streptavidin-peroxidase conjugate solution.
(4) Cleaning fluid
Phosphate buffered saline containing 0.05% Tween 20 was used as a washing solution.
(5) Diluent
A diluted solution was 100 mM CHES buffer (pH 9.5) containing 100 mM sodium chloride, 0.5% sodium caseinate, 2 mM EDTA.2 sodium, 0.1% sodium azide, and 10% mouse serum.
(6) Coloring solution
A 0.045% 3,3 ′, 5,5′-tetramethylbenzidine hydrochloride aqueous solution (pH 2.0) containing 0.2 mM EDTA · disodium was used as a color developing solution.
(7) Substrate solution
A 60 mM disodium phosphate aqueous solution (pH 4.3) containing 5 mM hydrogen peroxide, 41 mM citric acid, and 0.2 mM EDTA · disodium was used as a substrate solution.
(8) Chromogenic substrate
The chromogenic solution and the substrate solution were returned to room temperature before use, and mixed in equal amounts at the time of use to obtain a chromogenic substrate.
(9) Reaction stop solution
0.7N sulfuric acid was used as a reaction stop solution.
2. sample
(1) Sample (the HMGB1 degradation product)
The concentration of the degradation product of HMGB1 is 0 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, and 40 ng / mL, respectively, as described in (1) of (1) -2 above. , And 80 ng / mL sample (the HMGB1 degradation product) was prepared.
(2) Sample (HMGB1)
As described in (2) of [1] above, the concentrations of HMGB1 are 0 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, and 80 ng, respectively. / ML sample (HMGB1) was prepared.
3. Measurement
(1) Each well of the antibody-immobilized microplate (5D1) of (1) was washed 3 times with 250 μL of the washing solution of (4).
(2) Next, 100 μL of the diluted solution (1) was dispensed into each well.
(3) Next, 10 μL of each of the samples of 7 concentrations (2) (1) (the degradation product of HMGB1) of 2 above was dispensed into each well, and then the top of each well was sealed with a plate seal, and 5 ° C. Then, the antibody immobilized on the well of this microplate was allowed to undergo an antigen-antibody reaction between the HMGB1 degradation product contained in the sample.
(4) Next, each well was washed 5 times with 400 μL of the washing solution of (1) above.
(5) Next, 100 μL of the biotin-labeled antibody solution (2H6) of (1) above is dispensed into each well, left at 25 ° C. for 2 hours, and the HMGB1 bound to the solid-phased antibody. Antigen-antibody reaction between the degradation product and biotin-labeled antibody was performed.
(6) Next, each well was washed 5 times with 400 μL of the washing solution of (1) above.
(7) Next, 100 μL of the streptavidin-peroxidase conjugate solution of (1) above is dispensed into each well and left at 25 ° C. for 1 hour to perform the “biotin-streptavidin” binding reaction. I did it.
(8) Next, each well was washed 5 times with 400 μL of the washing solution of (1) above.
(9) Next, 100 μL of the chromogenic substrate of (8) above was dispensed into each well, and allowed to stand at room temperature for 20 minutes to cause a color development reaction with peroxidase as a labeling enzyme.
(10) Next, 100 μL of the above (9) reaction stop solution was dispensed into each well to stop the color development reaction.
(11) Next, the absorbance (450 nm) of the liquid in each well was measured, and the absorbance in the case of measuring the above-mentioned total 7 concentration samples (the HMGB1 degradation products) was obtained.
(12) The above (1) to (11) except that the sample (HMGB1) having a total concentration of 7 in (2) is used instead of the sample having a total concentration of 7 (decomposition product of HMGB1) in (3) above. ), And the absorbance in the case of measuring each of the samples having a total concentration of 7 (HMGB1) was obtained.
4). Measurement result
(1) The measurement result in 3 above, that is, the HMGB1 degradation product contained in the sample and the HMGB1 contained in the sample are measured by the immunological measurement method and immunological measurement reagent of the HMGB1 degradation product of the present invention. The results are shown in FIG.
In this figure, the horizontal axis represents the HMGB1 degradation product contained in the sample or the concentration (ng / mL) of HMGB1 contained in the sample, and the vertical axis represents the absorbance (450 nm) obtained by the measurement.
Further, in this figure, “■” indicates a measured value (the absorbance described above) when measured on the sample of (2) (1) (decomposed product of HMGB1) [total 7 concentrations], and “♦” indicates the above 2 The measured value (the above-mentioned absorbance) when measured for the sample (HMGB1) (total 7 concentrations) of (2).
(2) Table 4 shows the measured values of the measurement results in 3 above.
Figure JPOXMLDOC01-appb-T000004
 In this table, “(i) HMGB1 degradation product contained in the sample or concentration of HMGB1 contained in the sample (ng / mL)”, “(ii) the sample of (2) above, in order from the left side. (HMGB1) [Measured value when measured for a total of 7 concentrations] (absorbance at 450 nm) "," (iii) From the measured value of (ii) above, a reagent blind value [sample with a concentration of HMGB1 of 0 ng / mL ( (Measurement value (absorbance at 450 nm)] in HMGB1)] (subtraction difference) "," (iv) The value (absorbance difference) in the sample (the HMGB1 degradation product) described later (vibration) in (iii) above. Value measured by dividing the value (absorbance difference) in the sample (HMGB1) ”,“ (v) When measuring the sample of (2) (1) (decomposed product of HMGB1) [total 7 concentrations] “Measured value (absorbance at 450 nm)”, “(vi) The value of the reagent blinded from the measured value of (v) [the measured value of the sample having the HMGB1 degradation product concentration of 0 ng / mL (the HMGB1 degradation product) ( (Absorbance at 450 nm)]] is subtracted (absorbance difference) ".
[3] Summary
(1) From FIG. 6 and Table 3 which are measurement results in [1], and FIG. 7 and Table 4 which are measurement results in [2], the following can be understood.
That is, in the immunological measurement method and reagent for immunological measurement of the HMGB1 degradation product of the present invention, the sample (the HMGB1 degradation product) [2.5 to 80 ng / mL] in which the measurement was performed The value (absorbance difference) in (the HMGB1 degradation product) divided by the value (absorbance difference) in the sample (HMGB1) was in the range of about 8.5 to 14.9.
That is, in the immunological measurement method and reagent for immunoassay of the HMGB1 degradation product of the present invention, the measured value (absorbance difference) of HMGB1 is very low, and the measurement of HMGB1 contained in the sample is very low. It turns out that it is suppressed.
In contrast, the measured value (absorbance difference) of the HMGB1 degradation product is high, indicating that the HMGB1 degradation product contained in the sample can be measured with high specificity.
Moreover, in the immunological measurement method and immunological measurement reagent of the HMGB1 degradation product of the present invention, from FIG. 7 which is a graph of the calibration curve of the measurement, the calibration curve is a graph of the HMGB1 degradation product contained in the sample. It also shows that the HMGB1 degradation product contained in the sample can be quantitatively measured over a wide range from a low concentration range to a high concentration range, extending linearly in proportion to the concentration.
(2) On the other hand, in the conventional immunological measurement method and immunological measurement reagent, the sample (the HMGB1 degradation product) [2.5 to 80 ng / mL] in which the measurement was performed The value (absorbance difference) in (the HMGB1 degradation product) divided by the value (absorbance difference) in the sample (HMGB1) was in the range of about 0.72 to 0.79.
That is, in the conventional immunological measurement method and immunological measurement reagent, the measured value (absorbance difference) of HMGB1 is higher than the measured value (absorbance difference) of the HMGB1 degradation product at the same concentration at any concentration. Is high.
That is, in the conventional immunological measurement method and immunological measurement reagent, the HMGB1 contained in the sample is measured with higher sensitivity, and the influence of the positive error derived from this HMGB1 is extremely high. It is understood that it is impossible to measure the HMGB1 degradation product contained in the sample with high specificity.
(3) Therefore, from the examination results in this example, the immunological measurement method and the immunological measurement reagent of the HMGB1 degradation product of the present invention have high specificity for the HMGB1 degradation product. Measurement was suppressed, that is, it was suppressed that a positive error derived from HMGB1 was generated, and it was confirmed that only the HMGB1 degradation product can be quantitatively measured with high specificity, accuracy and sensitivity. .
[Example 9] (Method for immunological measurement of HMGB1 degradation product and measurement reagent-2)
By the immunological measurement method and immunological measurement reagent of the HMGB1 degradation product of the present invention, the HMGB1 degradation product contained in the sample, the HMGB1 contained in the sample, the HMGB2 degradation product contained in the sample, and the sample Measurement of HMGB2 was performed.
1. Reagent
(1) Antibody-immobilized microplate (5D1)
Prepared as described in 1 of (2) of Example 8 [1], this was used as an antibody-immobilized microplate (5D1).
(2) Biotin-labeled antibody solution (2H6)
Prepared as described in Example 8, [2], 1 (2), and this was used as a biotin-labeled antibody solution (2H6).
(3) Streptavidin-peroxidase conjugate solution
This was prepared as described in Example 8, [2], 1 (3), and this was used as a streptavidin-peroxidase conjugate solution.
(4) Cleaning fluid
Prepared as described in Example 8, [2], 1 (4), and this was used as a cleaning solution.
(5) Diluent
Prepared as described in Example 8, [2], 1 (5), and this was used as a diluent.
(6) Coloring solution
It was prepared as described in Example 8, [2], 1 (6), and this was used as a color developing solution.
(7) Substrate solution
Prepared as described in Example 8 [2] 1 (7), this was used as the substrate solution.
(8) Chromogenic substrate
It was prepared as described in Example 8, [2], 1 (8), and this was used as a chromogenic substrate.
(9) Reaction stop solution
Prepared as described in Example 9, [2], 1 (9), and this was used as a reaction stop solution.
2. sample
(1) Sample (the HMGB1 degradation product)
The HMGB1 degradation product prepared in [2] of Reference Example 1 was mixed with a phosphate buffered saline containing 0.5% sodium caseinate, 100 mM sodium chloride, and 0.1% sodium azide, each 1.5 ng / Samples prepared with concentrations of mL, 3.1 ng / mL, 6.2 ng / mL, 12.5 ng / mL, 25 ng / mL, 50 ng / mL, and 100 ng / mL, respectively (the HMGB1 degradation product) It was.
In addition, a phosphate buffered saline containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide was used as a sample (the HMGB1 degradation product) having a concentration of the HMGB1 degradation product of 0 ng / mL. .
(2) Sample (HMGB1)
The HMGB1 prepared in [1] of Reference Example 1 was mixed with phosphate buffered saline containing 0.5% sodium caseinate, 100 mM sodium chloride, and 0.1% sodium azide, 1.5 ng / mL, 3 Samples (HMGB1) were prepared to have concentrations of 0.1 ng / mL, 6.2 ng / mL, 12.5 ng / mL, 25 ng / mL, 50 ng / mL, and 100 ng / mL, respectively.
A phosphate buffered saline containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide was used as a sample (HMGB1) having a HMGB1 concentration of 0 ng / mL.
(3) Sample (said HMGB2 degradation product)
The HMGB2 degradation product prepared in [3] of Reference Example 1 was added to a phosphate buffered saline containing 0.5% sodium caseinate, 100 mM sodium chloride, and 0.1% sodium azide, each 1.5 ng / Samples prepared with concentrations of mL, 3.1 ng / mL, 6.2 ng / mL, 12.5 ng / mL, 25 ng / mL, 50 ng / mL, and 100 ng / mL, respectively (the HMGB2 degradation product) It was.
In addition, a phosphate buffered saline containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide was used as a sample having the HMGB2 degradation product concentration of 0 ng / mL (the HMGB2 degradation product). .
(4) Sample (HMGB2)
The HMGB2 prepared in [1] of Reference Example 1 was mixed with phosphate buffered saline containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide at 1.5 ng / mL, 3 Samples (HMGB2) were prepared to have concentrations of .1 ng / mL, 6.2 ng / mL, 12.5 ng / mL, 25 ng / mL, 50 ng / mL, and 100 ng / mL.
Further, phosphate buffered saline containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide was used as a sample (HMGB2) having a concentration of HMGB2 of 0 ng / mL.
3. Measurement
(1) Each well of the antibody-immobilized microplate (5D1) of (1) was washed 3 times with 250 μL of the washing solution of (4).
(2) Next, 100 μL of the diluted solution (1) was dispensed into each well.
(3) Next, 10 μL of each of the samples of 8 concentrations (2) (1) (the degradation product of HMGB1) of 2 above was dispensed into each well, and then the top of each well was sealed with a plate seal, and 5 ° C. Then, the antibody immobilized on the well of this microplate was allowed to undergo an antigen-antibody reaction between the HMGB1 degradation product contained in the sample.
(4) Next, each well was washed 5 times with 400 μL of the washing solution of (1) above.
(5) Next, 100 μL of the biotin-labeled antibody solution (2H6) of (1) above is dispensed into each well, left at 25 ° C. for 2 hours, and the HMGB1 bound to the solid-phased antibody. Antigen-antibody reaction between the degradation product and biotin-labeled antibody was performed.
(6) Next, each well was washed 5 times with 400 μL of the washing solution of (1) above.
(7) Next, 100 μL of the streptavidin-peroxidase conjugate solution of (1) above is dispensed into each well and left at 25 ° C. for 1 hour to perform the “biotin-streptavidin” binding reaction. I did it.
(8) Next, each well was washed 5 times with 400 μL of the washing solution of (1) above.
(9) Next, 100 μL of the chromogenic substrate of (8) above was dispensed into each well, and allowed to stand at room temperature for 20 minutes to cause a color development reaction with peroxidase as a labeling enzyme.
(10) Next, 100 μL of the above (9) reaction stop solution was dispensed into each well to stop the color development reaction.
(11) Next, the absorbance (primary wavelength: 450 nm, subwavelength: 550 nm) of the liquid in each well was measured, and the absorbance in the case of measuring each of the above-mentioned total 8 concentrations of the sample (the HMGB1 degradation product) was obtained. .
(12) The above (1) to (11) except that the sample (HMGB1) having the total concentration of 8 in (2) above is used instead of the sample having a total concentration of 8 (the degradation product of HMGB1) in (3) above. ), And the absorbance in the case of measuring each of the total 8 concentration samples (HMGB1) was obtained.
(13) The above (1) except that the total 8 concentration sample of (2) (3) (the HMGB2 degradation product) is used instead of the total 8 concentration sample (the HMGB1 degradation product) in (3) above. ~ As described in (11), the absorbance was obtained when each of the samples with the total concentration of 8 (the HMGB2 degradation product) was measured.
(14) The above (1) to (11) except that the sample (HMGB2) having a total concentration of 8 in (2) above is used instead of the sample having a total concentration of 8 in (3) (the degradation product of HMGB1). ), And the absorbance in the case of measuring each of the samples with a total of 8 concentrations (HMGB2) was obtained.
4). Measurement result
(1) The measurement result in 3 above, that is, the HMGB1 degradation product contained in the sample, the HMGB1 contained in the sample, contained in the sample by the immunological measurement method and the immunological measurement reagent of the HMGB1 degradation product of the present invention FIG. 8 shows the measurement results of the HMGB2 degradation product and HMGB2 contained in the sample.
In this figure, letters representing the production cell lines of the immobilized antibody and the labeled antibody used for the measurement are shown above the figure.
In this figure, the horizontal axis represents the concentration (ng / mL) of the HMGB1 degradation product contained in the sample, the HMGB1 contained in the sample, the HMGB2 degradation product contained in the sample, or the HMGB2 contained in the sample. The axis indicates the absorbance obtained by measurement [absorbance at the main wavelength (450 nm) minus the absorbance at the sub-wavelength (550 nm)].
Further, in this figure, “■” indicates a measured value (the absorbance described above) when measured for the sample of (2) (1) (the degradation product of HMGB1) [total 8 concentrations], and “♦” indicates the above 2 (2) sample (HMGB1) [total 8 concentrations] measured value (absorbance described above), “●” represents the sample (3) (decomposition product of HMGB2) [total 8 The measurement value (the absorbance described above) when measured for [Concentration], and “▲” indicates the measurement value (the absorbance described above) measured for the sample (HMGB2) (total 8 concentrations) of (2) above. Show.
(2) Table 5 shows the measured values of the measurement results in 3 above.
Figure JPOXMLDOC01-appb-T000005
 In this table, letters representing the production cell lines of the immobilized antibody and the labeled antibody used for the measurement are shown in the upper left of the table.
In this table, in order from the left side, “(i) the concentration of the HMGB1 degradation product contained in the sample, the HMGB1 contained in the sample, the HMGB2 degradation product contained in the sample, or the concentration of HMGB2 contained in the sample (ng / mL) ”,“ (ii) Sample (HMGB1) of 2 above (2) (total 8 concentrations) measured value [absorbance at sub-wavelength (550 nm) subtracted from absorbance at main wavelength (450 nm) Things] ”,“ (iii) From the measurement value of (ii) above, the value of the reagent blind test [the measurement value in the sample (HMGB1) having a concentration of HMGB1 of 0 ng / mL [from the absorbance at the main wavelength (450 nm) to the subwavelength ( Value obtained by subtracting the absorbance at 550 nm)] (subtraction difference) "," (iv) Sample (vi) described later (the HMGB) 1 (degradation product) value (absorbance difference) divided by (iii) sample (HMGB1) value (absorbance difference) "," (v) 2 (1) sample (the HMGB1 degradation product) " Measured values when measuring [total 8 concentrations] [absorbance at main wavelength (450 nm) minus absorbance at sub-wavelength (550 nm)] ”,“ (vi) blinded reagent from measured value of (v) [The measured value of the sample having the HMGB1 degradation product concentration of 0 ng / mL (the HMGB1 degradation product) [the absorbance at the main wavelength (450 nm) minus the absorbance at the subwavelength (550 nm)]] Value (absorbance difference) ”,“ (vii) Measured value when measuring the sample (HMGB2) of 2 above (4) [total 8 concentrations] [main wavelength (450 nm The value obtained by subtracting the absorbance at the sub-wavelength (550 nm) from the absorbance in the above] ”,“ (viii) The value measured in the above (vii) and the value of the reagent blind test (the measurement in the sample (HMGB2) where the concentration of HMGB2 is 0 ng / mL) Value (absorbance difference) obtained by subtracting the value [absorbance at the main wavelength (450 nm) minus the absorbance at the subwavelength (550 nm)] ”,“ (ix) in the sample of (vi) (the HMGB1 degradation product) The value (absorbance difference) divided by the value (absorbance difference) in the sample (HMGB2) of (viii) "," (x) the sample of (3) of (2) above (the HMGB2 degradation product) [total 8 concentrations] Measured value when measured for [absorbance at the main wavelength (450 nm) minus the absorbance at the sub-wavelength (550 nm)] ”,“ xi) From the measured value of (x) above, the value of the reagent blind test [the measured value of the sample having the concentration of the HMGB2 degradation product of 0 ng / mL (the HMGB2 degradation product) [from the absorbance at the main wavelength (450 nm) to the subwavelength ( Value obtained by subtracting the absorbance at 550 nm)] (absorbance difference) "," (xii) The value (absorbance difference) in the sample of (vi) (the degradation product of HMGB1) (the absorbance difference) of the sample of (xi) ( The value divided by the value (absorbance difference) in the HMGB2 degradation product) is shown.
5. Summary
(1) The following can be understood from FIG.
That is, in the immunological measurement method and reagent for immunological measurement of the HMGB1 degradation product of the present invention, the sample (the HMGB1 degradation product) in the sample (the HMGB1 degradation product) [concentration: 12.5 to 100 ng / mL]. ) (The difference in absorbance) divided by the value (absorbance difference) in the sample (HMGB1) was in the range of about 7-30.
Further, in the immunological measurement method and immunological measurement reagent for the HMGB1 degradation product of the present invention, the sample (the HMGB1 degradation product) in the sample (the HMGB1 degradation product) [concentration: 12.5 to 100 ng / mL]. ) (The difference in absorbance) divided by the value (absorbance difference) in the sample (HMGB2) was in the range of about 17-39.
In the immunological measurement method and the immunological measurement reagent for the HMGB1 degradation product of the present invention, the sample (the HMGB1 degradation product) in the sample (the HMGB1 degradation product) [concentration: 12.5 to 100 ng / mL]. ) Divided by the value (absorbance difference) in the sample (the HMGB2 degradation product) was in the range of about −33 to 76.
That is, in the immunological measurement method and immunological measurement reagent of the HMGB1 degradation product of the present invention, the measured value (absorbance difference) of HMGB1, the measured value (absorbance difference) of the HMGB2 degradation product, and the measured value of HMGB2 (Absorbance difference) are all very low, and it can be seen that the measurement of HMGB1, the HMGB2 degradation product, and HMGB2 contained in the sample is extremely suppressed.
In contrast, the measured value (absorbance difference) of the HMGB1 degradation product is high, indicating that the HMGB1 degradation product contained in the sample can be measured with high specificity.
Moreover, in the immunological measurement method and immunological measurement reagent of the HMGB1 degradation product of the present invention, from FIG. 8 which is a graph of the calibration curve of the measurement, the calibration curve is a graph of the HMGB1 degradation product contained in the sample. It also shows that the HMGB1 degradation product contained in the sample can be quantitatively measured over a wide range from a low concentration range to a high concentration range, extending linearly in proportion to the concentration.
(2) Therefore, also from the examination results in this example, the HMGB1 degradation product immunological measurement method and immunological measurement reagent of the present invention have high specificity for the HMGB1 degradation product, and HMGB1 Measurement is suppressed, that is, the occurrence of a positive error derived from HMGB1 or the like is suppressed, and only the HMGB1 degradation product can be quantitatively measured with high specificity and accuracy with high sensitivity. It was confirmed.
[Example 10] (Method for immunological measurement of HMGB1 degradation product and measurement reagent-3)
By the immunological measurement method and immunological measurement reagent of the HMGB1 degradation product of the present invention, the HMGB1 degradation product contained in the sample, the HMGB1 contained in the sample, the HMGB2 degradation product contained in the sample, and the sample Measurement of HMGB2 was performed.
1. Reagent
(1) Antibody-immobilized microplate
(I) Antibody-immobilized microplate (2A10)
Example 1 except that the anti-HMGB1 degradation product antibody (2A10) of Example 3 is used instead of the anti-HMGB1 degradation product antibody (5D1) in 1 (1) of Example 8 [2]. This was prepared as described in 8 [2] 1 (1), and this was used as an antibody-immobilized microplate (2A10).
(Ii) Antibody-immobilized microplate (6H3)
Example 1 except that the anti-HMGB1 degradation product antibody (6H3) of Example 4 is used instead of the anti-HMGB1 degradation product antibody (5D1) in 1 (1) of Example 8 [2]. This was prepared as described in 8 [2] 1 (1), and this was used as an antibody-immobilized microplate (6H3).
(2) Biotin-labeled antibody solution (2H6)
Prepared as described in Example 8, [2], 1 (2), and this was used as a biotin-labeled antibody solution (2H6).
(3) Streptavidin-peroxidase conjugate solution
This was prepared as described in Example 8, [2], 1 (3), and this was used as a streptavidin-peroxidase conjugate solution.
(4) Cleaning fluid
Prepared as described in Example 8, [2], 1 (4), and this was used as a cleaning solution.
(5) Diluent
Prepared as described in Example 8, [2], 1 (5), and this was used as a diluent.
(6) Coloring solution
It was prepared as described in Example 8, [2], 1 (6), and this was used as a color developing solution.
(7) Substrate solution
Prepared as described in Example 8 [2] 1 (7), this was used as the substrate solution.
(8) Chromogenic substrate
It was prepared as described in Example 8, [2], 1 (8), and this was used as a chromogenic substrate.
(9) Reaction stop solution
Prepared as described in Example 9, [2], 1 (9), and this was used as a reaction stop solution.
2. sample
(1) Sample (the HMGB1 degradation product)
The HMGB1 degradation product prepared in [2] of Reference Example 1 was added to each 2.5 ng / kg of phosphate buffered saline containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide. Samples (the aforementioned HMGB1 degradation products) were prepared so as to have concentrations of mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, and 80 ng / mL, respectively.
In addition, a phosphate buffered saline containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide was used as a sample (the HMGB1 degradation product) having a concentration of the HMGB1 degradation product of 0 ng / mL. .
(2) Sample (HMGB1)
HMGB1 prepared in [1] of Reference Example 1 was added to each of 2.5 ng / mL and 5 ng with phosphate buffered saline containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide. Samples (HMGB1) were prepared to have concentrations of / ng, 10 ng / mL, 20 ng / mL, 40 ng / mL, and 80 ng / mL.
A phosphate buffered saline containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide was used as a sample (HMGB1) having a HMGB1 concentration of 0 ng / mL.
(3) Sample (said HMGB2 degradation product)
The HMGB2 degradation product prepared in [3] of Reference Example 1 was added to each 2.5 ng / kg of phosphate buffered saline containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide. Samples (the aforementioned HMGB2 degradation products) were prepared to have concentrations of mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, and 80 ng / mL, respectively.
In addition, a phosphate buffered saline containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide was used as a sample having the HMGB2 degradation product concentration of 0 ng / mL (the HMGB2 degradation product). .
(4) Sample (HMGB2)
The HMGB2 prepared in [1] of Reference Example 1 was mixed with phosphate buffered saline containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide, 2.5 ng / mL and 5 ng, respectively. Samples (HMGB2) were prepared to have concentrations of / ng, 10 ng / mL, 20 ng / mL, 40 ng / mL, and 80 ng / mL.
Further, phosphate buffered saline containing 0.5% sodium caseinate, 100 mM sodium chloride and 0.1% sodium azide was used as a sample (HMGB2) having a concentration of HMGB2 of 0 ng / mL.
3. Measurement
(1) Each well of the antibody-immobilized microplate (2A10) of (1) (1) of (1) was washed three times with 400 μL of the washing solution of (1) (4).
(2) Next, 100 μL of the diluted solution (1) was dispensed into each well.
(3) Next, 10 μL of each of the samples of 7 concentrations (2) (1) (the degradation product of HMGB1) of 2 above was dispensed into each well, and then the top of each well was sealed with a plate seal, and 5 ° C. Then, the antibody immobilized on the well of this microplate was allowed to undergo an antigen-antibody reaction between the HMGB1 degradation product contained in the sample.
(4) Next, each well was washed 5 times with 400 μL of the washing solution of (1) above.
(5) Next, 100 μL of the biotin-labeled antibody solution (2H6) of (1) above is dispensed into each well, left at 25 ° C. for 2 hours, and the HMGB1 bound to the solid-phased antibody. Antigen-antibody reaction between the degradation product and biotin-labeled antibody was performed.
(6) Next, each well was washed 5 times with 400 μL of the washing solution of (1) above.
(7) Next, 100 μL of the streptavidin-peroxidase conjugate solution of (1) above is dispensed into each well and left at 25 ° C. for 1 hour to perform the “biotin-streptavidin” binding reaction. I did it.
(8) Next, each well was washed 5 times with 400 μL of the washing solution of (1) above.
(9) Next, 100 μL of the chromogenic substrate of (8) above was dispensed into each well, and allowed to stand at room temperature for 20 minutes to cause a color development reaction with peroxidase as a labeling enzyme.
(10) Next, 100 μL of the above (9) reaction stop solution was dispensed into each well to stop the color development reaction.
(11) Next, the absorbance (primary wavelength: 450 nm, subwavelength: 550 nm) of the liquid in each well was measured, and the absorbance in the case of measuring each of the above-mentioned total 7 concentrations of the sample (the HMGB1 degradation product) was obtained. .
(12) The above (1) to (11) except that the sample (HMGB1) having a total concentration of 7 in (2) is used instead of the sample having a total concentration of 7 (decomposition product of HMGB1) in (3) above. ), And the absorbance in the case of measuring each of the samples having a total concentration of 7 (HMGB1) was obtained.
(13) The above (1) except that the total 7 concentration sample (the HMGB2 degradation product) of (2) (3) above is used instead of the total 7 concentration sample (the HMGB1 degradation product) in (3) above. ~ As described in (11), the absorbance in the case of measuring each of the above seven concentrations of the sample (the HMGB2 degradation product) was obtained.
(14) The above (1) to (11) except that the total 7 concentration sample (HMGB2) of (2) above (4) is used instead of the total 7 concentration sample (the HMGB1 degradation product) in (3) above. ), And the absorbance was measured when each of the samples having a total concentration of 7 (HMGB2) was measured.
(15) The above (1) except that the antibody-immobilized microplate (6H3) of (1) (ii) is used instead of the antibody-immobilized microplate (2A10) of (1). ~ (14), the sample (the HMGB1 degradation product) [total 7 concentration], the sample (HMGB1) [total 7 concentration], the sample (the HMGB2 degradation product) [total 7 concentration], and Absorbance was measured when each sample (HMGB2) [total 7 concentrations] was measured.
4). Measurement result
(1) The measurement result in 3 above, that is, the HMGB1 degradation product contained in the sample, the HMGB1 contained in the sample, contained in the sample by the immunological measurement method and the immunological measurement reagent of the HMGB1 degradation product of the present invention FIG. 9 [Results of measurement using an antibody-immobilized microplate (2A10) as the antibody-immobilized microplate] and the results of the measurement of the HMGB2 degradation product and the HMGB2 contained in the sample. FIG. 10 [Results of measurement when antibody-immobilized microplate (6H3) is used as the antibody-immobilized microplate].
In these figures, letters representing the production cell lines of the immobilized antibody and the labeled antibody used for the measurement are shown above the figures.
In these figures, the horizontal axis indicates the concentration (ng / mL) of the HMGB1 degradation product contained in the sample, the HMGB1 contained in the sample, the HMGB2 degradation product contained in the sample, or the HMGB2 contained in the sample. The vertical axis represents the absorbance obtained by measurement [the absorbance at the main wavelength (450 nm) minus the absorbance at the sub-wavelength (550 nm)].
Further, in these figures, “■” indicates the measured value (the absorbance described above) when measured for the sample of (2) (1) (decomposition product of HMGB1) [total 7 concentrations], and “♦” indicates the above 2 (2) sample (HMGB1) [total 7 concentrations] measured value (the absorbance described above), “●” indicates the sample of (3) (decomposition product of HMGB2) [total 7 (concentration) indicates the measured value (absorbance), and “▲” indicates the measured value (absorbance) when measured for the sample (HMGB2) (total 7 concentrations) in (2) above. Indicates.
(2) Also, the measurement values of the measurement results in 3 above are shown in Table 6 [Results of measurement when antibody-immobilized microplate (2A10) is used as the antibody-immobilized microplate] and Table 7 [Antibody solid-phase. Results of measurement using antibody-immobilized microplate (6H3) as the modified microplate].
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000007
 In these tables, letters representing the production cell lines of the immobilized antibody and the labeled antibody used for the measurement are shown at the upper left of the table.
In these tables, in order from the left side, “(i) the concentration (ng) of the HMGB1 degradation product contained in the sample, the HMGB1 contained in the sample, the HMGB2 degradation product contained in the sample, or the HMGB2 contained in the sample. / (ML) ”,“ (ii) Sample (HMGB1) of 2 above (2) (total 7 concentrations) measured value [absorbance at sub-wavelength (550 nm) subtracted from absorbance at main wavelength (450 nm) "(Iii) From the measured value of (ii) above, the value of the blinded reagent [the measured value in the sample (HMGB1) having a concentration of HMGB1 of 0 ng / mL [absorbance at the main wavelength (450 nm) to the subwavelength] Value obtained by subtracting the absorbance at (550 nm)] (absorbance difference) "," (iv) Sample of (vi) described later (the HM Value obtained by dividing the value (absorbance difference) in GB1 degradation product) by the value (absorbance difference) in the sample (HMGB1) of (iii) "," (v) the sample of (1) of (2) (degradation product of HMGB1) " Measured value when measuring [total 7 concentrations] [absorbance at the main wavelength (450 nm) minus the absorbance at the sub-wavelength (550 nm)] ”,“ (vi) blinded reagent from the measured value of (v) [The measured value of the sample having the HMGB1 degradation product concentration of 0 ng / mL (the HMGB1 degradation product) [the absorbance at the main wavelength (450 nm) minus the absorbance at the subwavelength (550 nm)]] Value (absorbance difference) ”,“ (vii) Measured value when measuring the sample (HMGB2) of 2 above (4) [total 7 concentrations] [main wavelength (450 m) minus absorbance at sub-wavelength (550 nm)] ”,“ (viii) In the sample (HMGB2) where the concentration of HMGB2 is 0 ng / mL from the measured value of (vii) above. The value obtained by subtracting the measured value [absorbance at the main wavelength (450 nm) minus the absorbance at the sub-wavelength (550 nm)] ”,“ (ix) sample of (vi) (the HMGB1 degradation product) ) (Value obtained by dividing the value (absorbance difference) by the value (absorbance difference) in the sample (HMGB2) of (viii) ”,“ (x) sample of (3) of (2) above (decomposition product of HMGB2) [total 7 Measured value when measuring [concentration] [absorbance at the main wavelength (450 nm) minus the absorbance at the sub-wavelength (550 nm)] “(Xi) From the measured value of (x), the value of the reagent blinded [the measured value of the sample having the HMGB2 degradation product concentration of 0 ng / mL (the HMGB2 degradation product) [from the absorbance at the dominant wavelength (450 nm) The value obtained by subtracting the absorbance at the wavelength (550 nm)] ”(absorbance difference)”, “(xii) The value (absorbance difference) in the sample of (vi) (the degradation product of HMGB1) of the above (xi) The values divided by the value (absorbance difference) in the sample (the HMGB2 degradation product) are shown.
5. Summary
[A] When using an antibody-immobilized microplate (2A10)
(1) From FIG. 9 and Table 6 [both results of measurement using an antibody-immobilized microplate (2A10) as an antibody-immobilized microplate], which are measurement results in 3 above, the following can be understood. .
That is, in the immunological measurement method and reagent for immunological measurement of the HMGB1 degradation product of the present invention, the sample (the HMGB1 degradation product) [2.5 to 80 ng / mL] in which the measurement was performed The value (absorbance difference) in (the HMGB1 degradation product) divided by the value (absorbance difference) in the sample (HMGB1) was in the range of about 3.6-15.
Further, in the immunological measurement method and reagent for immunological measurement of the HMGB1 degradation product of the present invention, any of the measured samples (the HMGB1 degradation product) [2.5 to 80 ng / mL] The value (absorbance difference) in (the HMGB1 degradation product) divided by the value (absorbance difference) in the sample (HMGB2) was in the range of about −78 to 29.
And in the immunological measurement method and immunological measurement reagent of the HMGB1 degradation product of the present invention, any of the measured samples (the HMGB1 degradation product) [2.5 to 80 ng / mL] The value (absorbance difference) in (the HMGB1 degradation product) divided by the value (absorbance difference) in the sample (the HMGB2 degradation product) was in the range of about −5 to 29.
That is, in the immunological measurement method and immunological measurement reagent of the HMGB1 degradation product of the present invention, the measured value (absorbance difference) of HMGB1, the measured value (absorbance difference) of the HMGB2 degradation product, and the measured value of HMGB2 (Absorbance difference) are all very low, and it can be seen that the measurement of HMGB1, the HMGB2 degradation product, and HMGB2 contained in the sample is extremely suppressed.
In contrast, the measured value (absorbance difference) of the HMGB1 degradation product is high, indicating that the HMGB1 degradation product contained in the sample can be measured with high specificity.
Moreover, in the immunological measurement method and immunological measurement reagent of the HMGB1 degradation product of the present invention, from FIG. 9 which is a graph of the calibration curve of the measurement, the calibration curve is a graph of the HMGB1 degradation product contained in the sample. It also shows that the HMGB1 degradation product contained in the sample can be quantitatively measured over a wide range from a low concentration range to a high concentration range, extending linearly in proportion to the concentration.
(2) Therefore, the immunology of the HMGB1 degradation product of the present invention is also based on the results of the examination in this example [results of measurement using the antibody-immobilized microplate (2A10) as the antibody-immobilized microplate]. The measurement method and the immunological measurement reagent have high specificity for the HMGB1 degradation product, and the measurement of HMGB1 and the like is suppressed, that is, the occurrence of a positive error derived from HMGB1 and the like is suppressed, It was confirmed that only the HMGB1 degradation product can be quantitatively measured with high specificity, accuracy and sensitivity.
[B] When using antibody-immobilized microplate (6H3)
(1) From the measurement results in [1] above and FIG. 10 and Table 7 [both are the results of measurement using the antibody-immobilized microplate (6H3) as the antibody-immobilized microplate] I understand.
That is, in the immunological measurement method and reagent for immunological measurement of the HMGB1 degradation product of the present invention, the sample (the HMGB1 degradation product) [2.5 to 80 ng / mL] in which the measurement was performed The value (absorbance difference) in (the HMGB1 degradation product) divided by the value (absorbance difference) in the sample (HMGB1) was in the range of about 3.1 to 5.8.
Further, in the immunological measurement method and reagent for immunological measurement of the HMGB1 degradation product of the present invention, any of the measured samples (the HMGB1 degradation product) [2.5 to 80 ng / mL] The value (absorbance difference) in (the HMGB1 degradation product) divided by the value (absorbance difference) in the sample (HMGB2) was in the range of about −47 to 227.
And in the immunological measurement method and immunological measurement reagent of the HMGB1 degradation product of the present invention, any of the measured samples (the HMGB1 degradation product) [2.5 to 80 ng / mL] The value (absorbance difference) in (the HMGB1 degradation product) divided by the value (absorbance difference) in the sample (the HMGB2 degradation product) was in the range of about −680 to 77.
That is, in the immunological measurement method and immunological measurement reagent of the HMGB1 degradation product of the present invention, the measured value (absorbance difference) of HMGB1, the measured value (absorbance difference) of the HMGB2 degradation product, and the measured value of HMGB2 (Absorbance difference) are all very low, and it can be seen that the measurement of HMGB1, the HMGB2 degradation product, and HMGB2 contained in the sample is extremely suppressed.
In contrast, the measured value (absorbance difference) of the HMGB1 degradation product is high, indicating that the HMGB1 degradation product contained in the sample can be measured with high specificity.
Moreover, in the immunological measurement method and immunological measurement reagent of the HMGB1 degradation product of the present invention, from FIG. 10 which is a graph of the calibration curve of the measurement, the calibration curve is a graph of the HMGB1 degradation product contained in the sample. It also shows that the HMGB1 degradation product contained in the sample can be quantitatively measured over a wide range from a low concentration range to a high concentration range, extending linearly in proportion to the concentration.
(2) Accordingly, the immunology of the HMGB1 degradation product of the present invention is also based on the results of the examination in this example [results of measurement using an antibody-immobilized microplate (6H3) as an antibody-immobilized microplate]. The measurement method and the immunological measurement reagent have high specificity for the HMGB1 degradation product, and the measurement of HMGB1 and the like is suppressed, that is, the occurrence of a positive error derived from HMGB1 and the like is suppressed, It was confirmed that only the HMGB1 degradation product can be quantitatively measured with high specificity, accuracy and sensitivity.

Claims (13)

  1. 　ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物に対する親和性が、ＨＭＧＢ１に対する親和性と比較して、少なくとも１．５倍である、ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物に結合する抗体。 An antibody that binds to the degradation product of HMGB1 by thrombin or thrombin-thrombomodulin complex, wherein the affinity for degradation product by thrombin or thrombin-thrombomodulin complex of HMGB1 is at least 1.5 times that of HMGB1 .
  2. 　ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物に対する親和性が、ＨＭＧＢ２に対する親和性及びＨＭＧＢ２のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物に対する親和性とそれぞれ比較して、各々少なくとも１０倍である、請求項１記載のＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物に結合する抗体。 The affinity of HMGB1 for the degradation product by thrombin or thrombin-thrombomodulin complex is at least 10 times each compared to the affinity for HMGB2 and the degradation product of HMGB2 by thrombin or thrombin-thrombomodulin complex, respectively. An antibody that binds to a degradation product of thrombin or thrombin-thrombomodulin complex of HMGB1 according to claim 1.
  3. 　ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物に結合する抗体がモノクローナル抗体である、請求項１又は２記載のＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物に結合する抗体。 The antibody that binds to the degradation product of HMGB1 thrombin or thrombin-thrombomodulin complex according to claim 1 or 2, wherein the antibody that binds to the degradation product of HMGB1 thrombin or thrombin-thrombomodulin complex is a monoclonal antibody.
  4. 　試料に含まれるＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物の免疫学的測定方法であって、次の（ａ）の抗体及び（ｂ）の抗体を使用する測定方法。
    　（ａ）　請求項１~３のいずれか１項に記載の抗体。
    　（ｂ）　ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物に結合する抗体であって、ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物に対する親和性が高い抗体。     A method for immunoassay of a degradation product of HMGB1 contained in a sample by thrombin or thrombin / thrombomodulin complex, which comprises the following antibodies (a) and (b):
    (A) The antibody according to any one of claims 1 to 3.
    (B) An antibody that binds to a degradation product of HMGB1 by a thrombin or thrombin-thrombomodulin complex, and has a high affinity for a degradation product of HMGB1 by a thrombin or thrombin-thrombomodulin complex.
  5. 　請求項４における（ｂ）の抗体が、当該抗体の濃度が０．６２５~２．５ｎｇ／ｍＬの範囲の少なくともいずれかの濃度において、ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物を固相化したＥＬＩＳＡ法で、
    （イ）当該分解産物に結合する当該抗体の量を測定するときに得られる吸光度の値を当該抗体の濃度の値で除したときの値が０．５以上となる抗体、又は
    （ロ）当該分解産物に結合する当該抗体の量を測定するときに得られる吸光度の値が、基準とする抗体産生細胞であるハイブリドーマＭＤ７８（ＦＥＲＭ　Ｐ−１８４０５）より産生される抗体における当該吸光度の値の６倍以上となる抗体
    である、請求項４記載の測定方法。     The antibody (b) according to claim 4, wherein the degradation product of HMGB1 thrombin or thrombin-thrombomodulin complex is immobilized on a solid phase at least in the concentration range of 0.625 to 2.5 ng / mL. In the ELISA method,
    (B) an antibody whose value is 0.5 or more when the absorbance value obtained when measuring the amount of the antibody bound to the degradation product is divided by the concentration value of the antibody, or (b) the antibody The absorbance value obtained when measuring the amount of the antibody bound to the degradation product is 6 times the absorbance value in the antibody produced from the hybridoma MD78 (FERM P-18405), which is the standard antibody-producing cell. The measurement method according to claim 4, wherein the antibody is as described above.
  6. 　（ａ）の抗体が、請求項２記載の抗体である、請求項４又は５記載の測定方法。 The measurement method according to claim 4 or 5, wherein the antibody of (a) is the antibody according to claim 2.
  7. 　（ａ）の抗体及び（ｂ）の抗体がモノクローナル抗体である、請求項４~６のいずれか１項に記載の測定方法。 The method according to any one of claims 4 to 6, wherein the antibody (a) and the antibody (b) are monoclonal antibodies.
  8. 　固相化抗体及び標識抗体を用いるものであって、（ａ）の抗体及び（ｂ）の抗体のうち、いずれか一方の抗体が固相化抗体として使用され、他の一方の抗体が標識抗体として使用される、請求項４~７のいずれか１項に記載の測定方法。 A solid-phase antibody and a labeled antibody are used, and one of the antibodies (a) and (b) is used as a solid-phase antibody, and the other antibody is a labeled antibody. 8. The measuring method according to claim 4, wherein the measuring method is used as:
  9. 　試料に含まれるＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物の免疫学的測定試薬であって、次の（ａ）の抗体及び（ｂ）の抗体を含有する測定試薬。
    　（ａ）　請求項１~３のいずれか１項に記載の抗体。
    　（ｂ）　ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物に結合する抗体であって、ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物に対する親和性が高い抗体。     A reagent for immunological measurement of degradation products of HMGB1 thrombin or thrombin-thrombomodulin complex contained in a sample, comprising the following antibody (a) and antibody (b):
    (A) The antibody according to any one of claims 1 to 3.
    (B) An antibody that binds to a degradation product of HMGB1 by a thrombin or thrombin-thrombomodulin complex, and has a high affinity for a degradation product of HMGB1 by a thrombin or thrombin-thrombomodulin complex.
  10. 　請求項９における（ｂ）の抗体が、当該抗体の濃度が０．６２５~２．５ｎｇ／ｍＬの範囲の少なくともいずれかの濃度において、ＨＭＧＢ１のトロンビン又はトロンビン・トロンボモジュリン複合体による分解産物を固相化したＥＬＩＳＡ法で、
    （イ）当該分解産物に結合する当該抗体の量を測定するときに得られる吸光度の値を当該抗体の濃度の値で除したときの値が０．５以上となる抗体、又は
    （ロ）当該分解産物に結合する当該抗体の量を測定するときに得られる吸光度の値が、基準とする抗体産生細胞であるハイブリドーマＭＤ７８（ＦＥＲＭ　Ｐ−１８４０５）より産生される抗体における当該吸光度の値の６倍以上となる抗体
    である、請求項９記載の測定試薬。     The antibody of (b) in claim 9, wherein the degradation product of HMGB1 thrombin or thrombin-thrombomodulin complex is immobilized on a solid phase at least in the concentration range of 0.625 to 2.5 ng / mL. In the ELISA method,
    (B) an antibody having a value of 0.5 or more when the absorbance value obtained by measuring the amount of the antibody bound to the degradation product is divided by the concentration value of the antibody, or (b) the The absorbance value obtained when measuring the amount of the antibody bound to the degradation product is 6 times the absorbance value of the antibody produced from the hybridoma MD78 (FERM P-18405), which is the standard antibody-producing cell. The measurement reagent according to claim 9, which is an antibody as described above.
  11. 　（ａ）の抗体が、請求項２記載の抗体である、請求項９又は１０記載の測定試薬。 The measurement reagent according to claim 9 or 10, wherein the antibody of (a) is the antibody according to claim 2.
  12. 　（ａ）の抗体及び（ｂ）の抗体がモノクローナル抗体である、請求項９~１１のいずれか１項に記載の測定試薬。 The measurement reagent according to any one of claims 9 to 11, wherein the antibody (a) and the antibody (b) are monoclonal antibodies.
  13. 　固相化抗体及び標識抗体を含有するものであって、（ａ）の抗体及び（ｂ）の抗体のうち、いずれか一方の抗体が固相化抗体として使用され、他の一方の抗体が標識抗体として使用される、請求項９~１２のいずれか１項に記載の測定試薬。 An antibody comprising a solid-phased antibody and a labeled antibody, wherein either one of the antibodies (a) and (b) is used as a solid-phased antibody, and the other one is labeled. The measuring reagent according to any one of claims 9 to 12, which is used as an antibody.
PCT/JP2013/076177 2013-03-19 2013-09-19 Antibody that binds specifically with hmgb1 decomposition product, and method and reagent for assaying hmgb1 decomposition product WO2014147873A1 (en)

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WO2018079393A1 (en) * 2016-10-26 2018-05-03 扶桑薬品工業株式会社 Disulfide-type hmgb1-specific antibody, method for measuring disulfide-type hmgb1 and kit for said measurement, and measurement method capable of quantitating all of hmgb1 molecules including reduced hmgb1, disulfide-type hmgb1 and thrombin-cleavable hmgb1 and kit for said measurement
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JP2020148557A (en) * 2019-03-12 2020-09-17 株式会社シノテスト Method for measuring hmgb1 in sample and measurement reagent
JP7313659B2 (en) 2019-03-12 2023-07-25 株式会社シノテスト Method and reagent for measuring HMGB1 in sample
JP7425459B2 (en) 2019-10-11 2024-01-31 株式会社シノテスト Stabilized HMGB1-containing solution

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