WO2006070732A1 - ブロック化酵素プローブ複合体 - Google Patents
ブロック化酵素プローブ複合体 Download PDFInfo
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- WO2006070732A1 WO2006070732A1 PCT/JP2005/023763 JP2005023763W WO2006070732A1 WO 2006070732 A1 WO2006070732 A1 WO 2006070732A1 JP 2005023763 W JP2005023763 W JP 2005023763W WO 2006070732 A1 WO2006070732 A1 WO 2006070732A1
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
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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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
-
- 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
Definitions
- the present invention relates to a technique for enzyme labeling a probe, and the prepared blocked enzyme probe complex is widely used for immunoassay utilizing an immune reaction such as enzyme immunoassay or immunohistochemistry.
- Enzyme-labeled probes obtained by labeling a probe with an enzyme have been widely used in immunological detection methods or measurement methods.
- probes labeled with enzymes such as horseradish rust peroxidase (HRP), alkaline phosphatase (ALP), ⁇ -galactosidase, and glucose oxidase can be used in the detection stage such as immunohistochemistry and enzyme immunoassay. Is possible.
- Immunohistochemical methods and enzyme immunoassays have been widely used for a long time as methods for detecting self antigens or foreign antigens in vivo. Since detection methods using these immune reactions are highly specific and sensitive, they can be detected without isolating trace amounts of substances present in the body. However, there are many trace amounts of substances that cannot be detected by ordinary immunohistochemical methods or enzyme immunoassays in vivo, and a method for increasing the sensitivity of the measurement method for the purpose of detecting them. Has been studied.
- cancer marker carcinoembryonic antigen (CEA) or monoprotein is a normal human serum concentration of 5-20 ng / ml Human gastrin releasing peptide precursor (ProGRP) is normal The concentration in human serum is about 14 pgZml, and sensitivity about 1000 times is necessary to measure ProGRP.
- C-type hepatitis virus has a very small amount of virus present in the blood, so a highly sensitive antigen detection method has been desired. Sensitivity to detect 100-1000 copies of viral RNA is required to detect the antigen, and a protein concentration of approximately 0.03 to 0.3 pg / ml is required.
- ALP anolecalyph-aged phosphatase
- Non-Patent Document 2 a method has been developed to covalently bind a single enzyme molecule.
- enzyme labels with a total molecular weight of 200,000 or less are often used.
- the inventors describe the molecular weight of the carrier as 5,000 to 500,000, preferably 10,000 to 300,000 forces. In this case, the molecular weight is about 40,000 if the horse mackerel peroxidase is used as an enzyme. Even if the molecular weight of the carrier is 500,000, it is natural that the number of 40,000 molecules that can physically and spatially bind to 500,000 molecules is limited and can be bound to the carrier. There is also a limit to the number of molecules in the enzyme. In other words, if an enzyme and an antibody are bound to a carrier, the functional group in the carrier must be shared between the enzyme and the antibody, so the number of enzymes is reduced and the signal is lowered.
- Patent Document 1 JP 2000-88850
- Non-Patent Document 1 Ultrasensitive Enzyme Immunoassay, Ishikawa Yuji, 1993, Academic Publishing Center
- Patent Document 2 Imagawa Power ⁇ J. Appl. Biochem. 4 ⁇ ; 400, 1982
- Non-Patent Document 3 Butler, (1981) Methods Enzymol., 73 ⁇ ; 482-523
- Non-Patent Document 4 Bobrow, (1989) J. Immunol. Methods, 125, 279-285 Disclosure of the Invention
- an object of the present invention is to provide a highly sensitive enzyme-labeled probe that can be used in a highly sensitive measurement system.
- the present inventors have studied to obtain a highly sensitive enzyme-labeled probe. As a result, two or more molecules of the carrier were bound via the enzyme, and the enzyme was bound to the carrier to form a block body. We succeeded in obtaining the desired highly sensitive blocked enzyme probe complex by binding the probe to the block.
- a blocked enzyme probe complex in which two or more carriers having a molecular weight of 20,000 to 4,000,000 are bound via an enzyme, and a probe molecule is bound to a complex in which the enzyme is bound to the carrier.
- the carrier is one or more selected from the group consisting of dextran, aminodextran, phyconole, dextrin, agarose, punoleran, various celluloses, chitin, chitosan, ⁇ -galatatosidase, thyroglobulin, hemocyanin, polylysine, polypeptide and DNA.
- the blocked enzyme probe complex according to (1) or (2).
- the probe is one or more selected from the group consisting of an antibody molecule or a functional fragment thereof, protein ⁇ ⁇ ⁇ ⁇ ⁇ , protein G, protein L, lectin, receptor, and avidin, (1) or (2) The blocked enzyme probe complex described.
- the antibody molecule or the functional fragment thereof is at least one selected from the group consisting of an anti-HCV core antigen antibody, an anti-gastrin releasing peptide precursor antibody, and a functional fragment thereof, (5) or (6)
- the blocked enzyme probe complex according to 1.
- An immunoassay kit or nucleic acid detection reagent comprising the blocked enzyme probe complex according to any one of (1) to (8).
- the binding of two or more molecules having a molecular weight of 20,000 to 4,000, 000 in (1) of the present invention includes binding not via an enzyme.
- a linker having a functional group or a protein can be used.
- each of the carrier, the enzyme, and the probe includes a blocked enzyme probe complex via a linker molecule having a functional group.
- the present invention relates to a blocked enzyme probe having many enzyme molecules and probe molecules in one molecule by binding a carrier via an enzyme or a linker and binding the enzyme and the probe to the molecule. It is a complex.
- the blocked enzyme probe complex of the present invention can be produced by forming a block body by binding an enzyme to a carrier and binding a probe molecule to this block body.
- the blocked enzyme probe complex of the present invention can also be produced by forming a conjugate by binding an enzyme and a probe to a carrier and then binding the conjugate to each other to form a block.
- a larger amount of enzyme or probe can be bound to the block body by binding the carrier with an enzyme and increasing the molecular weight.
- the probe since the probe is bonded to the surface of the block body, it can be produced regardless of the probe molecule size in which steric hindrance hardly occurs.
- the final product of the blocked enzyme probe complex is highly effective when the blocked enzyme probe complex having a molecular weight of 440,000 or more is more effective and 668,000 or more.
- the larger the molecular weight of a blocked enzyme probe complex the greater the amount of enzyme bound to one molecule of the complex.
- Sensitivity increases.
- a blocked enzyme probe complex having a large molecular weight can be prepared by the method described in the present invention, a method such as gel filtration can be used to select a blocked enzyme probe complex having a larger molecular weight.
- any molecular weight may be used as long as the blocked enzyme probe complex does not precipitate or precipitate in the liquid.
- no upper limit should be specified.
- Dextran when Dextran is used as a carrier, there is no problem even with 20,000,000 (Examples 1 and 6 described later), and 40, 000, 000 to: 100, 000, 000 will cause a problem. Nare.
- the present invention was originally made in the process of studying an enzyme probe complex that can be used in the field of immunohistochemistry and immunohistochemistry, but there are limitations to its application to other fields. Absent.
- the enzyme-labeled antibody of the present invention makes it possible to detect antigens and proteins that cannot be detected by conventional enzyme-labeled antibodies when detecting antigens and proteins that exist only in trace amounts in the living body. By using this enzyme-labeled antibody, it becomes possible to measure antigens and proteins that could not be measured conventionally.
- the carrier used in the present invention is not particularly limited as long as it has a molecular weight of 20,000 to 4,000,000. However, in order to further improve the sensitivity, it is desirable that a large number of enzymes can bind to it, and therefore it is desirable to have a certain molecular weight.
- Examples of carriers include polysaccharides, high molecular weight proteins and peptide polymers, and their molecular weight is 20,000 to 20,000, 000, preferably 20,000 to 4,000, 000, more preferably 70, 000 to 2,000, 000 is suitable.
- signals having higher molecular weights tend to have higher signals.
- polysaccharide carrier in the present invention examples include dextran, aminodextran, phyconole, dextrin, agarose, punoreran, various celluloses, chitin, chitosan, soluble starch and the like.
- high molecular weight protein carrier in the present invention examples include ⁇ -galactosidase, thyroglobulin, hemosinin and the like.
- various peptide polymers can be used as the carrier for the peptide polymer in the present invention.
- the enzyme that can be used in the present invention is not particularly limited, but is commonly used in immunoassays: horseradish saponin peroxidase (HRP), alkaline phosphatase (AL ⁇ ), ⁇ -galactosidase, glucose Oxidase, luciferase, etc. are used in a timely manner. Since an enzyme binds to two or more carriers or a carrier and a probe molecule, two or more functional groups, such as sugar chains and amino groups, or two or more amino groups, an amino group, a carboxy group, and a thiol group An enzyme having an amino group and the like is desirable.
- HRP horseradish saponin peroxidase
- AL ⁇ alkaline phosphatase
- ⁇ -galactosidase glucose Oxidase
- luciferase etc. Since an enzyme binds to two or more carriers or a carrier and a probe molecule, two or more functional groups, such as sugar chains and amino groups, or two
- Any linker or binding mode may be used for binding the carrier and the enzyme, but it is necessary to bind the probe to the block after binding the carrier and the enzyme. There is a need to leave functional groups on the support.
- a hydrazine group present in a carrier having a molecular weight of 20,000 to 4,000, 000, or a hydrazine group introduced into the carrier using an appropriate linker molecule, or bound to an enzyme molecule or A block through the enzyme can be prepared by binding to the carrier via a hydrazine group introduced into the enzyme molecule using a single linker molecule.
- a blocked enzyme probe complex can be prepared by binding a probe molecule via a carrier in the block or a functional group in the enzyme molecule bound to the carrier.
- a suitable linker molecule is used to bind the hydrazine group introduced into the carrier having a molecular weight of 20,000 to 4,000, 000 and the aldehyde group obtained by oxidizing the sugar chain in the enzyme molecule.
- the probe molecule is bound via a linker molecule that is bound to a functional group in the enzyme molecule that is bound to the carrier or carrier in the block body that is blocked via the enzyme molecule. By binding, a blocked enzyme probe complex can be similarly produced.
- the linker molecule having a hydrazine group to be introduced into the carrier or the enzyme may be a hydrazine salt such as hydrazine sulfate or hydrazine hydrochloride having a hydrazine group (one NHNH 2).
- Hydrazides having a hydrazine group (one CO—NHNH) or functional groups and hydrazides
- a substance having a dodecyl group may be used.
- the linker molecule for binding the carrier molecule or the enzyme molecule to the probe molecule can be used as long as it has a functional group such as a maleimide group, a succinimidole group, a carboxyl group, or a thiol group.
- Enzyme-blocky enzyme The enzyme weight relative to the carrier weight when preparing the enzyme is 0.2 to 10 times. A desired value is 0.3 to 5 times.
- the sugar chain of horseradish rust peroxidase is oxidized and bound to Dextran having a molecular weight of 20,000 or more with a hydrazine group introduced to prepare a blocking enzyme via the enzyme.
- a linker molecule such as N— (6—Maleimidocaproyloxy) succinimide (EMCS) is bound to the remaining hydrazine group on the carrier and the remaining amino group of HRP, and a maleimide group is introduced onto the surface of the blocked enzyme, in the probe molecule or in the probe
- the SH group introduced into is reacted to prepare a blocked enzyme probe complex.
- the sugar chain of the enzyme is used to produce a blocked enzyme probe complex, and the residual hydrazine group or residual amino group is used for probe binding.
- any cross-linking agent may be used and any binding mode may be used.
- a heterobifunctional crosslinking agent or a homobifunctional crosslinking agent having high stability in an aqueous solution is desirable.
- antibodies monoclonal antibodies, polyclonal antibodies
- fragments thereof F (ab '), Fab ⁇ Fab, F (abc'), Fabc, etc.
- various receptors various avidins
- the antibody may be any antibody that binds to the target antigen or analyte.
- Antibodies use proteases such as pepsin and papain to obtain fragments such as F (ab ') and Fab.
- H chains heavy chains of antibodies are bound to each other by S—S bonds, and the bonds are cleaved by a reducing agent.
- the reducing agent include cysteamine and mercaptoethanol, and F (ab ') is cleaved into Fab' by such a reducing agent.
- a new (SH) group is generated.
- the present invention includes these fragments of antibodies (F (ab '), Fab ⁇ Fa
- Dextran T2000 (average molecular weight 2,000, 000; Amersham) 0.3g is weighed and dissolved in 6ml of 0.1M phosphate buffer (PH7.0). Add 3 ml of sodium periodate solution, allow to stand at room temperature for 2 hours, perform gel filtration (Sephadex G25, Amersham), fractionate the void fraction, and add hydrazine hydrochloride (Wako Pure Chemicals). He introduced hydrazine to Dextran. Theoretically, dextran with a molecular weight of 2,000,000 is introduced. Up to 22,000 hydrazine is introduced.
- HRP Peroxidase
- Toyobo lOOmg was weighed, mixed and dissolved in 3 ml of 0.1 M sodium bicarbonate solution, mixed with 1.5 ml of sodium periodate solution, and allowed to stand at room temperature for 2 hours. After desalting by gel filtration (Sephadex G25), add about 150 mg of the previously obtained dextran hydrazide, mix and react at room temperature for 2 hours, and after reduction, add glycine to 0.1 M. Dialysis was performed 3 times for 4 hours at 4 ° C, and a blocked carrier HRP conjugate was obtained.
- Dextran T70 (average molecular weight 70, 000; Amersham) 0.3 g was weighed, and HRP-Fab complex was obtained in the same manner as in Example 1, and the absorbance at 403 nm was measured. The HRP concentration was determined.
- Dextran T500 (average molecular weight 500, 000; Amersham) 0 ⁇ 3g was weighed, and the same carrier (150mg): enzyme (lOOmg) weight ratio (0 ⁇ 66) as in Example 1; 3 times the weight ratio (300mg) (2.00) and 5 times the weight ratio (500mg) (3.33)
- a carrier-enzyme conjugate was prepared in the same manner as described above, and then an anti-HCV core antigen monoclonal antibody was bound in the same manner as in Example 1.
- the absorbance at 403 nm was measured, and the HRP concentration in the complex was determined from the molecular absorbance coefficient of HRP at 403 nm.
- Dextran T500 (average molecular weight 500, 000; Amersham) 0.3 g was weighed, and a carrier HRP conjugate was obtained in the same manner as in Example 1. 1 mg of EMCS dissolved in DMF was added to 5 mg of this conjugate, and reacted at room temperature for 2 hours. Excess EMC S was removed by gel filtration (Sephadex G25), and maleimide was introduced into the carrier HRP conjugate. 0.1 F cysteamine in F (ab ') solution of anti-ProGRP monoclonal antibody (2B10) in 1M phosphate buffer (pH 6.0)
- the enzyme anti-HCV core antigen monoclonal antibody complex prepared in Example 1 was subjected to gel filtration using a Sephacryl S-500 Superfine (Amersham) (1.6 ⁇ 60) column.
- This Sephacryl S-500 Superfine column is mainly used to separate large molecular weight molecules and small particles. According to Falmacia Gel filtration theory and practice, this column fractionation range is 4 ⁇ 10 4 to 2 ⁇ 10 7 for polysaccharides.
- PBS was used for the carrier, and gel filtration was performed at a flow rate of lmlZmin.
- each fraction in 4mlZ2min Separate each fraction in 4mlZ2min, measure the absorbance at 403nm, obtain the HRP concentration ( ⁇ g / ml) from the molecular extinction coefficient of HRP, and then dilute the reactivity of each fraction to 1 ng / ml in terms of HRP concentration. And then examined.
- the test method is as follows.
- Anti-HCV core antigen monoclonal antibody (cl 1 3 & cl 1-7 etc.) in 96-well microplate 200 ⁇ l was added at a concentration of 4 ⁇ g / ml and incubated at 4 ° C. After washing with 10 mM phosphate buffer ( ⁇ 7.3), 350% of 0.5% casein was added and incubated for 2 hours.
- Recombinant HCV core antigen (cl l) adjusted to a concentration of 0 fmol / L and 1200 fmol / L was added as a sample and incubated at room temperature for 60 minutes with stirring.
- each fraction was diluted to 1 ⁇ gZml as HRP concentration in terms of HRP. Then, the enzyme antibody prepared by the conventional method was added at 200 ⁇ l at an HRP concentration of 1 ⁇ g / ml and incubated for 30 minutes. Further, the plate was washed 6 times with a washing solution, 200 ⁇ l of a substrate solution (orthodienediamine, hydrogen peroxide) was added and incubated for 30 minutes.
- a substrate solution orthodienediamine, hydrogen peroxide
- the enzyme reaction was stopped by adding 50 ⁇ l of 5% sulfuric acid, and the absorbance at 492 nm (reference wavelength: 630 nm) was measured with a microplate reader (Corona 32).
- Table 1 and Fig. 1 show the HRP concentration of each gel filtration fraction and the above-described assay results (difference between the absorbance of the core antigen 1200 fmol ZL and the absorbance of Ofmol ZL).
- the molecular weights of the three kinds of molecular weight markers are Thyroglobulin; 668,000, Ferritin; 440,000, BSA; 68,000.
- the absorbance at the core antigens Ofmol / L and 1200 fmol / L in the enzyme antibody 1 ⁇ g / ml prepared by the conventional method measured simultaneously was 0.000 and 0.050, respectively. Therefore, the difference between the absorbance at 1200 fmol / L and the absorbance at Ofmol / L by the conventional method was 0.050. From FIG. 1, it was confirmed that the absorbance at the core antigen of 1200 fmol / L tends to increase as the molecular weight increases.
- the signal increase was less than 3 times that of the conventional method, but in fractions 2 to 18 the signal increase was more than 3 times.
- Thyroglobulin molecular weight 668,000
- Ferritin molecular weight 440,000
- the molecular weight was about 440,000 or more, and about 5.9 times more than the conventional method.
- An excellent effect of about 7.7 times higher than that of the conventional method was confirmed at a molecular weight of 668,000 or more.
- the carrier Dextran T2000 in this example has an average molecular weight of 2,000,000, 14 molecules of HRP having a molecular weight of 40,000 are bound to this carrier, and the molecular weight of this blocked carrier-enzyme is 46,000.
- the molecular weight of the enzyme probe complex in which 8 molecules of Fab 'are bound is about 2,900,000. This is understood to be the basic unit of the enzyme probe complex in this example.
- the separation range of the Sephacryl S-500 Superfine column used for the gel filtration in this example is a force of 4 ⁇ 10 4 to 2 ⁇ 10 7.
- Including The first fraction also shows high activity.
- the first fraction containing this void fraction is
- the enzyme probe complex existing in the first fraction contains at least two enzyme probe complexes, which are the smallest units, are further covalently bound to form a larger molecular weight enzyme block complex, that is, a blocked enzyme probe complex. It can be understood to form a body.
- the carrier Dextran T2000 used in this example includes Dextran having an average molecular weight of less than 2,000,000 and degradation products thereof. It is clear that these are also involved in the formation of the enzyme probe complex or blocked enzyme probe complex of the present invention. Therefore, in this example, the average molecular weight of the basic unit of the enzyme probe complex is 2,900, Enzyme probe complexes having a molecular weight of 000 or less are also included, and these are none other than the present invention.
- each enzyme anti-HCV core antigen monoclonal antibody complex was diluted to 2 ⁇ g / ml in terms of enzyme concentration. Was added and incubated for 30 minutes. Wash 6 times with the washing solution, and then add the substrate solution (orthophenylenediamine, excess Hydrogen oxide) 200 ⁇ 1 was added and incubated for 30 minutes. The enzyme reaction was stopped by adding 50 ⁇ l of 5% sulfuric acid, and the absorbance at 492 nm (reference wavelength 630 nm) was measured with a microplate reader (Corona 32).
- the reactivity of the anti-HCV core antigen monoclonal antibody complex prepared using the carrier-enzyme conjugate prepared at a carrier: enzyme weight ratio of 0.66, 2.00, and 3.33 is expressed as follows: Compared with the weight ratio of 0.66, the weight ratio of 2.00 is 77.6% and the weight ratio of 3.33 is 70.5%. The higher the amount of enzyme, the lower the reactivity. It was. Even if the carrier: enzyme weight ratio is 2.00, 3.33 or more, a sufficiently sensitive complex can be obtained.
- the enzyme probe complex of the present invention has a molecular weight higher than that of the enzyme probe complex, which is at least two minimum units, further bound by a covalent bond. It can be seen that by forming a large enzyme probe complex, ie, a blocked enzyme probe complex, the reactivity has increased.
- Example 1 or 2 two types of anti-HCV core antigen monoclonal antibodies ⁇ cl 1 10F (ab ') and cl l-14F (ab') ⁇ were reacted together with a blocked carrier enzyme. Also these 2
- [0050] Add 200 ⁇ l of anti-HCV core antigen monoclonal antibody (cl 1 _3 & cl 1 _7 equivalent mixture) to a 96-well microplate at a concentration of 4 ⁇ gZml. Incubate at C. After washing with 10 mM phosphate buffer pH 7.3 (PBS), 350 ⁇ l of 0.5% casein was added and incubated for 2 hours. After removing 0.5% casein by aspiration, prepare a 3-fold dilution series of recombinant HCV core antigen (cl l) from 2 1870 fmol / L, add it as a sampnore, and mix at room temperature with stirring. Incubated for minutes.
- anti-HCV core antigen monoclonal antibody cl 1 _3 & cl 1 _7 equivalent mixture
- HRP has very few amino groups, and Ishikawa et al. Studied the introduction of maleimide groups into the HRP using amino groups. From 1 to no more than 3 [Eishi Ishikawa, Biochemistry Experimental Method 27, Enzyme labeling method, Academic Publishing Center]. After blocking the amino group with low HRP using 2-methylmaleic anhydride, the same method as in Example 3 was used, and Dextran T500 was used as the carrier. After preparation, anti-HCV core antigen monoclonal antibody was bound. The absorbance at 403 nm was measured, and the HRP concentration in the complex was determined from the molecular extinction coefficient of HRP at 403 nm.
- [0053] Add 200 ⁇ l of anti-HCV core antigen monoclonal antibody (cl 1 _3 & cl 1 _ 7 equivalent volume) to a 96-well microplate at a concentration of 4 ⁇ gZml. Incubate at C. After washing with 10 mM phosphate buffer pH 7.3 (PBS), 350% 1 of 0.5% casein was added and incubated for 2 hours. After removing 0.5% casein by aspiration, prepare a 3-fold dilution series of recombinant HCV core antigen (cl l) from 2 1870 fmol / L, add it as a sample, and mix at room temperature while stirring. Incubated for minutes.
- PBS mM phosphate buffer pH 7.3
- the labeled antibody was converted to HRP concentration, and 200 ⁇ l was added at a total concentration of 2 ⁇ g / ml and incubated for 20 minutes. More The plate was washed 6 times with a washing solution, and 200 ⁇ l of a substrate solution (10 mg orthophenylenediamine, hydrogen peroxide) was incubated for 30 minutes. The enzyme reaction was stopped by adding 50 ⁇ l of 5% sulfuric acid, and the absorbance at 492 nm (reference wavelength 630 nm) was measured with a microplate reader (Corona 32). For comparison, as in Example 3, a carrier-enzyme conjugate was prepared at a carrier: enzyme weight ratio of 0.66 and then bound with an anti-HCV core antigen monoclonal antibody. [Figure 4].
- the reactivity of the enzyme-antibody complex blocked with an amino group shows about 88.1% of its activity compared to the non-blocked one, and it is sufficient even if the antibody binds only to the carrier. It was confirmed that it showed a good reactivity. Since the blocked enzyme-antibody complex of the present invention uses a large excess of enzyme, the antibody can bind to the carrier / enzyme. Even if an antibody as a probe binds only on the carrier, it seems to be sufficiently reactive.
- the horizontal axis represents the ProGRP concentration and the vertical axis represents the absorbance at 492 nm.
- Antibody complex by the conventional method It was found that the signal was remarkably high compared to the prepared enzyme-labeled antibody. Assuming the difference between Opg / ml and ODO. 020 as the detection limit, about 115 pg / ml can be detected with the conventional enzyme antibody, and about 7 pg / ml with the enzyme-antibody complex of Example 3, that is, compared with the conventional method. 16. The sensitivity increased 4 times.
- the serum ProGRP concentration in healthy subjects is approximately 14 pgZml and the cutoff value is around 50 pg / ml. Ipn. J.
- ProGRP cannot be detected in some cancer patient samples.
- ProGRP can be detected in the samples of patients and healthy individuals that could not be detected by the conventional method, and its usefulness was confirmed.
- FIG. 1 shows the results of molecular weight analysis by gel filtration of the enzyme anti-HCV core antigen monoclonal antibody complex of the present invention.
- FIG. 2 shows the results of comparison of detection sensitivity of core antigen using enzyme anti-HCV core antigen monoclonal antibody complex and conventional enzyme antibody.
- FIG. 3 shows the reactivity when one type of monoclonal antibody is bound to a carrier-enzyme complex and when two types of monoclonal antibodies are bound to a carrier-enzyme complex.
- FIG. 4 shows the reactivity of the enzyme with the amino group blocked and the probe bound, and the probe with the amino group unblocked.
- FIG. 5 shows the results of comparison of detection sensitivity of ProGRP using enzyme anti-ProGRP antigen monoclonal antibody complex and conventional enzyme antibody.
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Priority Applications (8)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AT05819496T ATE482396T1 (de) | 2004-12-28 | 2005-12-26 | Blockierter enzymsondenkomplex |
KR1020077015334A KR101181364B1 (ko) | 2004-12-28 | 2005-12-26 | 블록화 효소 프로브 복합체 |
EP05819496A EP1837655B1 (en) | 2004-12-28 | 2005-12-26 | Blocked enzyme probe complex |
CA2592274A CA2592274C (en) | 2004-12-28 | 2005-12-26 | Blocked enzyme-probe complex |
DE602005023784T DE602005023784D1 (de) | 2004-12-28 | 2005-12-26 | Blockierter enzymsondenkomplex |
JP2006550753A JP3961559B2 (ja) | 2004-12-28 | 2005-12-26 | ブロック化酵素プローブ複合体 |
US11/722,867 US20080153089A1 (en) | 2004-12-28 | 2005-12-26 | Blocked Enzyme-Probe Complex |
CN2005800446647A CN101088009B (zh) | 2004-12-28 | 2005-12-26 | 嵌段酶-探针复合物 |
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CN (1) | CN101088009B (ja) |
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2011129357A1 (ja) | 2010-04-14 | 2011-10-20 | 栄研化学株式会社 | 標識化プローブ-水溶性担体複合体 |
WO2021045061A1 (ja) | 2019-09-02 | 2021-03-11 | 富士レビオ株式会社 | レクチン結合性物質測定方法及びレクチン結合性物質測定キット、並びに、これらに用いるブロック化標識レクチン |
WO2021045065A1 (ja) * | 2019-09-02 | 2021-03-11 | 富士レビオ株式会社 | レクチン結合性物質測定方法及びレクチン結合性物質測定キット、並びに、これらに用いる捕捉担体 |
JP2021038980A (ja) * | 2019-09-02 | 2021-03-11 | 富士レビオ株式会社 | Afp−l3測定方法及びafp−l3測定キット、並びに、これらに用いるブロック化標識レクチン |
WO2021132470A1 (ja) | 2019-12-25 | 2021-07-01 | 富士レビオ株式会社 | イムノクロマト用ストリップ、イムノクロマト用装置、イムノクロマト用キット、及び被検物質検出方法 |
Families Citing this family (1)
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CN113358863A (zh) * | 2021-06-10 | 2021-09-07 | 武汉原谷生物科技有限责任公司 | 一种多聚酶标记的制作方法 |
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- 2005-12-26 EP EP05819496A patent/EP1837655B1/en active Active
- 2005-12-26 CN CN2005800446647A patent/CN101088009B/zh not_active Expired - Fee Related
- 2005-12-26 AT AT05819496T patent/ATE482396T1/de not_active IP Right Cessation
- 2005-12-26 DE DE602005023784T patent/DE602005023784D1/de active Active
- 2005-12-26 CA CA2592274A patent/CA2592274C/en not_active Expired - Fee Related
- 2005-12-26 WO PCT/JP2005/023763 patent/WO2006070732A1/ja active Application Filing
- 2005-12-26 JP JP2006550753A patent/JP3961559B2/ja active Active
- 2005-12-26 US US11/722,867 patent/US20080153089A1/en not_active Abandoned
- 2005-12-26 KR KR1020077015334A patent/KR101181364B1/ko active IP Right Grant
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Cited By (10)
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WO2011129357A1 (ja) | 2010-04-14 | 2011-10-20 | 栄研化学株式会社 | 標識化プローブ-水溶性担体複合体 |
JPWO2011129357A1 (ja) * | 2010-04-14 | 2013-07-18 | 栄研化学株式会社 | 標識化プローブ−水溶性担体複合体 |
JP5675782B2 (ja) * | 2010-04-14 | 2015-02-25 | 栄研化学株式会社 | 標識化プローブ−水溶性担体複合体 |
WO2021045061A1 (ja) | 2019-09-02 | 2021-03-11 | 富士レビオ株式会社 | レクチン結合性物質測定方法及びレクチン結合性物質測定キット、並びに、これらに用いるブロック化標識レクチン |
WO2021045065A1 (ja) * | 2019-09-02 | 2021-03-11 | 富士レビオ株式会社 | レクチン結合性物質測定方法及びレクチン結合性物質測定キット、並びに、これらに用いる捕捉担体 |
JP2021038980A (ja) * | 2019-09-02 | 2021-03-11 | 富士レビオ株式会社 | Afp−l3測定方法及びafp−l3測定キット、並びに、これらに用いるブロック化標識レクチン |
JP7361543B2 (ja) | 2019-09-02 | 2023-10-16 | 富士レビオ株式会社 | Afp-l3測定方法及びafp-l3測定キット、並びに、これらに用いるブロック化標識レクチン |
US11988666B2 (en) | 2019-09-02 | 2024-05-21 | Fujirebio Inc. | Lectin-binding substance measurement method, lectin-binding substance measurement kit, and blocked labeled lectin for use in these |
WO2021132470A1 (ja) | 2019-12-25 | 2021-07-01 | 富士レビオ株式会社 | イムノクロマト用ストリップ、イムノクロマト用装置、イムノクロマト用キット、及び被検物質検出方法 |
EP4083625A4 (en) * | 2019-12-25 | 2024-01-03 | Fujirebio Inc. | IMMUNOCHROMATOGRAPHIC STRIP, IMMUNOCHROMATOGRAPHIC APPARATUS, IMMUNOCHROMATOGRAPHIC KIT AND METHOD FOR DETECTING A TEST SUBSTANCE |
Also Published As
Publication number | Publication date |
---|---|
CA2592274A1 (en) | 2006-07-06 |
DE602005023784D1 (de) | 2010-11-04 |
JPWO2006070732A1 (ja) | 2008-06-12 |
EP1837655A4 (en) | 2008-09-10 |
ATE482396T1 (de) | 2010-10-15 |
US20080153089A1 (en) | 2008-06-26 |
CN101088009A (zh) | 2007-12-12 |
JP3961559B2 (ja) | 2007-08-22 |
EP1837655B1 (en) | 2010-09-22 |
EP1837655A1 (en) | 2007-09-26 |
KR101181364B1 (ko) | 2012-09-11 |
CN101088009B (zh) | 2013-05-22 |
KR20070090997A (ko) | 2007-09-06 |
CA2592274C (en) | 2014-09-09 |
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