WO2019167935A1 - Use of anti-cd14 antibody useful for measurement of presepsin - Google Patents

Use of anti-cd14 antibody useful for measurement of presepsin Download PDF

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Publication number
WO2019167935A1
WO2019167935A1 PCT/JP2019/007291 JP2019007291W WO2019167935A1 WO 2019167935 A1 WO2019167935 A1 WO 2019167935A1 JP 2019007291 W JP2019007291 W JP 2019007291W WO 2019167935 A1 WO2019167935 A1 WO 2019167935A1
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preceptin
antibody
sample
presepsin
measurement
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PCT/JP2019/007291
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French (fr)
Japanese (ja)
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白川　嘉門
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持田製薬株式会社
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Priority to JP2020503519A priority Critical patent/JP7288428B2/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • 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
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the present invention relates to a method for suppressing an increase in an apparent preceptin measurement value in a sample due to physical stimulation of the sample in measurement of presepsin in the sample.
  • the CD14 molecule is a glycoprotein expressed on the membrane surface of mononuclear cells and is known to have a function as a receptor for LPS (lipopolysaccharide).
  • LPS lipopolysaccharide
  • CD14 molecules membrane-bound CD14 (mCD14) and soluble CD14 (sCD14) expressed on the cell surface.
  • sCD14 As sCD14, sCD14 having a molecular weight of about 55 kDa and about 49 kDa (hereinafter referred to as “high molecular weight soluble CD14” or “high molecular weight sCD14”) is known, and sepsis (SEPSIS), acquired immune deficiency syndrome (AIDS), It has been reported to show high levels in the blood of patients in many diseases such as acute respiratory distress syndrome (ARDS) and systemic lupus erythematosus (SLE). Therefore, these high molecular weight sCD14 are considered not to be disease-specific markers (Non-Patent Documents 1 and 2).
  • SEPSIS sepsis
  • AIDS acquired immune deficiency syndrome
  • ARDS acute respiratory distress syndrome
  • SLE systemic lupus erythematosus
  • sCD14-ST also called a soluble CD14 antigen subtype, also known as preceptin
  • SCD14-ST (presepsin) is characterized in that it migrates to a molecular weight of 13 ⁇ 2 kDa in SDS-PAGE under non-reducing conditions, and retains the N-terminal part of CD14. Compared with high molecular weight sCD14, it has an amino acid sequence that is largely deleted on the C-terminal side, and unlike high molecular weight sCD14, it does not have LPS binding ability. Moreover, since preceptin shows immunogenicity different from high molecular weight sCD14, both can be distinguished using an antibody. Presepsin specifically increases blood concentration in patients with sepsis (Patent Document 1).
  • Non-patent Document 3 there is a report that it is high in the blood of sepsis patients compared to patients with systemic inflammatory response (SIRS), which is difficult to distinguish from sepsis, and presepsin is a specific diagnostic marker for sepsis.
  • a rabbit-derived polyclonal antibody (S68 antibody) and a rat-derived monoclonal antibody (F1146-17-2) that specifically recognize preceptin are disclosed (Patent Documents 1 and 2).
  • the presepsin measurement kit As described above, with the presepsin measurement kit, if a physical stimulus such as vigorous agitation is applied to the sample to be used, the presepsin value in the sample may not be obtained due to an increase in the measured value of preceptin. There are concerns. For this reason, it is said that the presepsin measurement kit should be handled with care so as not to give vigorous stirring.
  • An object of the present invention is to provide a method useful for measuring presepsin in a sample, which can solve at least one of the conventional problems.
  • the present inventors have found that the phenomenon that the measured value of preceptin increases when a physical stimulus such as vigorous stirring is applied to the specimen is caused by a product derived from sCD14 in the specimen caused by the physical stimulus. I found out. Therefore, the present inventor can eliminate the influence of the product derived from the sCD14 by, for example, bringing the specimen into contact with the anti-CD14 antibody or separating it from preceptin using chromatography. The inventors have conceived that the original preceptin value in the specimen can be measured, thereby completing the present invention.
  • Example 2 it confirmed that the increase in the preceptin value by shaking related with sCD14 in a test substance. Specifically, as a result of analyzing each fraction by gel filtration of the product derived from sCD14 produced by shaking, the elution pattern of the sample shaken by adding the control and anti-CD14 antibody is almost the same. It was confirmed that a higher molecular weight aggregate was generated from sCD14 by shaking (this high molecular weight aggregate is also referred to as “product derived from sCD14” or “pseudopreceptin”). It was also confirmed that the formation of high molecular weight aggregates can be effectively suppressed by allowing the sample to coexist with an anti-CD14 antibody. Furthermore, it was confirmed that the high molecular weight aggregate can be separated by chromatography.
  • the product derived from sCD14 can eliminate the influence of the product by bringing the sample into contact with the anti-CD14 antibody or separating it from preceptin using chromatography. It can be seen that the increase in the presepsin measurement value due to shaking in the measurement of presepsin can be effectively prevented, and the original presepsin measurement value in the sample can be obtained.
  • the measured value may be affected.
  • the cause of the influence on the measured value is sCD14 in blood, and it is separated from preceptin by contacting a specimen with an anti-CD14 antibody (for example, F1024-1-3 antibody) or using chromatography. It was surprising that the effect of the product on the measured value of presepsin can be removed.
  • the present invention can include the following embodiments.
  • a method for stabilizing a preceptin measurement value in a sample comprising a step of bringing the sample into contact with an anti-CD14 antibody immediately after the sample is collected.
  • a method of suppressing pseudopreceptin production in a sample comprising a step of bringing the sample into contact with an anti-CD14 antibody immediately after the sample is collected.
  • a method for inhibiting denaturation of sCD14 in a specimen comprising a step of bringing the specimen into contact with an anti-CD14 antibody immediately after the specimen is collected.
  • a method for quantifying only pseudo-preceptin comprising a step of separating pseudo-preceptin and true pre-ceptin before measuring pre-ceptin.
  • a method for quantifying a true preceptin value comprising measuring a preceptin value of a specimen to calculate a preceptin value, and subtracting the pseudo-preceptin value obtained by the method according to the aspect [11] from the calculated preceptin value .
  • It includes at least one selected from the group consisting of an anti-CD14 antibody, a chromatography column, and a membrane for use in the method according to any one of the above aspects [1] to [12]. Presepsin measurement kit.
  • a preceptin measurement system comprising at least one selected.
  • the measured value of presepsin when measuring presepsin in a sample, even if a physical stimulus is applied to the sample to be used, the measured value of presepsin is hardly increased and the original measured value of presepsin is obtained.
  • the product derived from sCD14 can be excluded from the influence of the product by contacting the sample with the anti-CD14 antibody or separated from preceptin using chromatography.
  • the increase in the presepsin measurement value due to mechanical stimulation can be effectively prevented, and the original presepsin measurement value in the sample can be obtained.
  • the measurement of the presepsin measurement in the sample can be performed. At this time, even when a physical stimulus such as vigorous stirring is applied to the specimen, that is, regardless of how the specimen is handled, a correct preceptin measurement value can be obtained.
  • the present invention relates to a method useful for measuring presepsin in a specimen from which a value is obtained.
  • some other embodiments relate to a method for removing a presepsin-like reaction product (pseudopreceptin) generated by physical stimulation.
  • a presepsin measurement kit when using a presepsin measurement kit, even if a physical stimulus such as vigorous stirring is given to the sample, the apparent preceptin measurement value in the sample does not increase, regardless of how the sample is handled.
  • the present invention relates to a method that enables stable preceptin measurement.
  • a first aspect is a method for stabilizing a preceptin measurement value in a specimen, including a step of bringing the specimen into contact with an anti-CD14 antibody immediately after the specimen is collected.
  • the second aspect is a method for suppressing pseudo-preceptin production in a specimen, including a step of bringing the specimen into contact with an anti-CD14 antibody immediately after the specimen is collected.
  • a third aspect is a method for inhibiting denaturation of sCD14 in a specimen, comprising a step of bringing the specimen into contact with an anti-CD14 antibody immediately after the specimen is collected.
  • the sCD14 in the specimen and the antibody CD14 antibody form a conjugate by the step of bringing the specimen into contact with the anti-CD14 antibody. It is the method as described in any one aspect of these.
  • the sixth aspect is a method for quantifying the amount of true preceptin in a sample, which comprises the step of bringing the sample into contact with an anti-CD14 antibody before measuring presepsin.
  • the seventh aspect is characterized in that the pseudopreceptin is adsorbed and removed by using the anti-CD14 antibody that reacts with the pseudopreceptin in the specimen in the step of bringing the specimen into contact with the anti-CD14 antibody. 6].
  • the eighth aspect is a method for quantifying only true preceptin, comprising a step of separating pseudo-preceptin from true pre-ceptin prior to measuring pre-ceptin.
  • a ninth aspect is the method according to the aspect [8], wherein the fractionation of the pseudo preceptin and the true preceptin is a chromatographic separation.
  • a tenth aspect is the method according to the aspect [8], wherein the separation of the pseudo-preceptin and the true preceptin is separation by a membrane.
  • the eleventh aspect is a method for quantifying only pseudo-preceptin, including a step of separating pseudo-preceptin from true pre-ceptin before measuring pre-sepsin.
  • a twelfth aspect includes a true preceptin value obtained by measuring preceptin of a specimen, and subtracting the pseudo-preceptin value obtained by the method according to the aspect [11] from the calculated preceptin value. This is a method for quantifying preceptin levels.
  • the thirteenth aspect is selected from the group consisting of an anti-CD14 antibody, a chromatography column, and a membrane for use in the method according to any one of the aspects [1] to [12]. It is a presepsin measurement kit containing at least one.
  • a specimen cartridge, a specimen processing section, a reagent cartridge section, a preceptin measurement section, a calculation section for use in the method according to any one of the above aspects [1] to [12]
  • a preceptin measurement system comprising at least one selected from the group consisting of display units.
  • immediate after sample collection means immediately after collecting a sample from a patient (for example, immediately after collecting blood) or simultaneously with collecting a sample (for example, simultaneously with collecting blood).
  • immediately after sample collection “before transport”, that is, before collecting a sample from a patient (eg, collecting blood) and sending it to a specialized center for measurement of various sample parameters (eg, blood parameter measurement). included.
  • it means a period from immediately after collection of a specimen (for example, immediately after blood collection) until it is sent to an examination center.
  • Before measuring presepsin means before starting measurement of the presepsin value, for example, before collecting a sample and starting measurement on the spot, or before measuring the preceptin value of a sample arriving at a test center .
  • the “step of contacting the specimen with the anti-CD14 antibody” means, for example, 1) adding an anti-CD14 antibody to the specimen, 2) An anti-CD14 antibody is previously present in a container such as a blood collection tube (for example, the anti-CD14 antibody is immobilized on beads or the like placed in the container such as a blood collection tube), and the sample collected in the container Containing the process, 3) using a dedicated container such as a dedicated blood collection tube in which the anti-CD 14 is immobilized in advance, and storing the sample collected in the container; 4) A step of adsorbing and removing sCD14 in a specimen using an anti-CD14 antibody (more specifically, for example, using an affinity chromatography column packed with a carrier in which an anti-CD14 antibody is bound to a resin or the like, Process to pass through the column), Any process selected from the group consisting of, etc.
  • sCD14 in the specimen gains tolerance due to physical stimulation, or sCD14 is removed, so that the presepsin measurement value in the specimen is stabilized, pseudopreceptin production in the specimen is suppressed, or sCD14 It becomes possible to suppress the denaturation of.
  • Examples of adding an anti-CD14 antibody to a specimen include adding an anti-CD14 antibody to a specimen collected from a patient. Further, in order to allow the anti-CD14 antibody to coexist in the specimen before transportation, the anti-CD14 antibody can coexist in the specimen by adding the anti-CD14 antibody to the specimen, or the anti-CD14 antibody is present in the blood collection tube in advance. Examples include coexistence of an anti-CD14 antibody in a specimen by putting blood into a blood collection tube.
  • the “pseudopreceptin” means a product or the like that is derived from sCD14 and causes a presepsin-like reaction by aggregation of sCD14, unfolding of a three-dimensional structure, and the like due to vigorous stirring and the like. Thus, presepsin-like reactions of pseudopresepsin will increase the presepsin measurement. “Preventing the production of pseudopreceptin” or “suppressing the production of pseudopreceptin” means inhibiting the production or production of pseudopreceptin derived from sCD14.
  • the “denaturation of sCD14” means that, for example, sCD14 in a specimen aggregates with two molecules to become a higher molecular weight due to vigorous stirring or the like.
  • SCD14 denaturation suppression means that sCD14-derived pseudopreceptin is inhibited from reacting to preceptin measurement.
  • Physical stimulation means vigorous agitation (including gentle agitation for a long time) when handling a specimen, and also means falling agitation of the blood collection tube after shaking or shaking.
  • Truste preceptin value means a correct value indicating the amount of preceptin present in the specimen, for example, this is a measured value of preceptin in the specimen prior to physical stimulation of the specimen.
  • “Stabilization of the preceptin measurement value” means that the specimen exhibits a true preceptin value even when subjected to physical stimulation. That is, for example, by suppressing the aggregation of sCD14, a true preceptin value can be obtained even when subjected to physical stimulation. “Resistant to destabilization by physical stimulation” means that sCD14 aggregates and destabilizes by unfolding the three-dimensional structure by physical stimulation, and shows a preceptin-like reaction in preceptin measurement. In addition to the value, it means to prevent an increase in the apparent preceptin value.
  • true preceptin is “preceptin” described later, that is, sCD14-st (soluble CD14 antigen subtype), and is a substance quantified by a normal preceptin measurement kit.
  • Separatation of pseudopreceptin and true preceptin means the following method: 1) separation by chromatography (for example, separation by gel filtration chromatography (eg, Example 4)), 2) separation by membrane, 3) Separation by an antibody that reacts with pseudopreceptin, Etc.
  • Quantifying the true preceptin value by separating the sample before preceptin measurement by chromatography (eg gel filtration chromatography), obtaining the fraction containing true preceptin, and measuring the preceptin value Is possible.
  • chromatography eg gel filtration chromatography
  • the presepsin measurement method described later is used.
  • a true preceptin is obtained by comparing a standard curve prepared with a standard concentration of preceptin and the amount of preceptin in the fraction containing each preceptin. A value can be obtained.
  • constructing a system for performing the separation and quantification it is possible to construct a system that obtains a true preceptin value simply by directly applying a specimen.
  • the specimen may be passed through a membrane (for example, a 30-kilodalton ultrafiltration membrane) that removes a substance having a higher molecular weight than preceptin.
  • a membrane for example, a 30-kilodalton ultrafiltration membrane
  • the true preceptin value can be obtained by measuring the preceptin value of the specimen from which the pseudopreceptin has been removed by the membrane and the pseudopreceptin has been removed.
  • sCD14-derived pseudopreceptin in order to separate sCD14-derived pseudopreceptin from the specimen, it reacts with sCD14 but does not react with true preceptin, and the pseudoreceptin is contacted with an antibody that reacts with pseudopreceptin to remove the pseudopreceptin. included.
  • the specimen and the antibody are brought into contact with each other, for example, the specimen is allowed to pass through a column on which the antibody is immobilized, and the pseudopreceptin derived from sCD14 is adsorbed and removed by the antibody. included. “Quantifying only the true preceptin value” means separating the sample before preceptin measurement by, for example, gel filtration chromatography and quantifying the preceptin value in the fraction containing true preceptin.
  • a “pseudo preceptin value” can be obtained by performing preceptin measurement on a fraction containing “pseudo preceptin” separated by chromatography. If a pseudo preceptin value is obtained, the prepsin value of the specimen can be measured, and a measured value of the pseudo preceptin can be obtained from the total value (total preceptin amount) of the true preceptin value and the pseudo preceptin value.
  • “Quantifying only presepsin” means that the sample before presepsin measurement is separated by, for example, gel filtration chromatography and the presepsin value in the fraction containing pseudopresepsin is quantified. More specifically, according to Example 4 described later, the separated relevant fraction is quantified.
  • the “presepsin measurement system” means a device such as a fully automatic device in which elements for performing the preceptin measurement are incorporated in the system.
  • Anti-CD14 antibodies In some embodiments, anti-CD14 antibodies are used.
  • Anti-CD14 antibodies include all antibodies that bind to CD14, preferably human CD14.
  • the anti-CD14 antibody is at least one antibody selected from F1024-1-3 antibody, 3C10 antibody, and MEM-18 antibody, and more preferably F1024-1-3 antibody, 3C10 antibody, or MEM -18 antibody.
  • F1024-1-3 antibody is a hybridoma F1024- obtained by cell fusion of immune cells and myeloma cells immunized with CD14 protein purified from human serum as an antigen, as described in WO2001 / 72993 A1.
  • F1024-1-3 antibody produced by 1-3 More specifically, it can be obtained by the method described in the publication according to a known method of Example 1 “Preparation of anti-human CD14 antibody” in WO2001 / 72993A1.
  • the F1024-1-3 antibody is an anti-CD14 antibody that specifically recognizes an epitope containing 8 or more amino acids in the region from 285 to 315 of human sCD14 described in SEQ ID NO: 3.
  • a human anti-CD14 antibody that controls signal transduction of LPS via human CD14 a 3C10 antibody (Steinman: J. Exp. Med., 158: 126 (1983) and Juan TS, which binds to positions 7 to 14 of human CD14). : J. Biol. Chem., 270: 29, 17237 (1995)) and the 57-64th binding MEM-18 antibody (Bazil: Eur. J. Immunol., 16: 1583 (1986) and Juan TS: J Biol.Chem., 270, 10, 5219 (1995)).
  • the 3C10 antibody and the MEM-18 antibody are available from, for example, abcom.
  • antibody is used in the meaning of “antibody or antigen-binding fragment thereof” unless otherwise specified.
  • An “antigen-binding fragment” refers to a fragment having substantially the same antigen-binding property as the original antibody among partial fragments of an antibody. Examples of the antigen-binding fragment include Fab, Fab ′, F (ab ′) 2 and the like.
  • membranes that can remove pseudopresepsin are used.
  • the membrane is a membrane that can remove a higher molecular weight material than presepsin (eg, a 30 kilodalton membrane).
  • Examples of such a membrane include an ultrafiltration filter (for example, Amicon (trade name) Ultra (Merck Millipore)).
  • a chromatography column eg, a gel filtration chromatography column
  • a chromatography column is used to separate true presepsin and false presepsin in a sample.
  • chromatography columns include Superdex (trade name), Sephacryl (trade name), Superose (trade name), and Sephadex (trade name).
  • Presepsin is also referred to as sCD14-ST (soluble CD14 antigen subtype).
  • CD14 includes soluble CD14 (sCD14) in addition to membrane-bound CD14 (mCD14), and a plurality of soluble CD14 having different molecular weights exist in blood.
  • Preceptin is a soluble fragment of CD14 and refers to a substance having the following properties 1) to 3).
  • the molecular weight is 13 ⁇ 2 kDa. 2) having an amino acid sequence at positions 1 to 11 of the amino acid sequence of SEQ ID NO: 3 (amino acid sequence of human full-length soluble CD14) at the N-terminal sequence; and 3) Specific binding to an antibody prepared using a peptide (S68 peptide) consisting of 16 amino acid residues described in SEQ ID NO: 2 (corresponding to the amino acid sequence of positions 53 to 68 of the amino acid sequence of SEQ ID NO: 3) as an antigen To do.
  • S68 peptide a peptide consisting of 16 amino acid residues described in SEQ ID NO: 2 (corresponding to the amino acid sequence of positions 53 to 68 of the amino acid sequence of SEQ ID NO: 3) as an antigen To do.
  • Preceptin is human preceptin unless otherwise specified.
  • the presepsin is, for example, a presepsin standard product (rsCD14-ST described in Example 16 of WO2005 / 108429).
  • rsCD14-ST described in Example 16 of WO2005 / 108429.
  • a substance obtained by modifying a part of preceptin having binding activity as preceptin may be used.
  • sCD14 As sCD14, sCD14 having a molecular weight of about 55 kDa and about 49 kDa is known. sCD14 is also referred to herein as “high molecular weight soluble CD14” or “high molecular weight sCD14”. sCD14 may be prepared, for example, by adsorbing a 3C10 antibody affinity column of body fluid of a normal person (see Example 23 of WO2005 / 108429).
  • Preceptin measurement is, for example, immunological measurement of preceptin.
  • an anti-preceptin antibody for example, a specific antibody such as S68 polyclonal antibody, P03-recognizing monoclonal antibody, P03-specific polyclonal antibody
  • antibody used for preceptin measurement a specimen containing presepsin (for example, a blood specimen).
  • measurement can be used interchangeably with the terms “detection”, “quantification”, “assay”, and the like, and is used in a sense including quantitative and qualitative determination.
  • the measurement of presepsin is preferably performed in vitro.
  • Method for stabilizing presepsin measurement value in sample method for suppressing production of pseudopreceptin derived from sCD14 in sample, method for suppressing denaturation of sCD14 in sample, method for quantifying true preceptin amount in sample, true preceptin only
  • the method of quantifying the amount, the method of quantifying only the fake preceptin, etc. can be used in the measurement of preceptin.
  • the presepsin measured value in the sample can be obtained with almost no increase in the measured value of presepsin.
  • the product derived from sCD14 can be removed from the effect of the product by bringing the sample into contact with the anti-CD14 antibody or separated from presepsin using chromatography. It is possible to effectively prevent an increase in the presepsin measurement value due to stimulation, and to obtain the original presepsin measurement value in the sample.
  • the pseudo-preceptin in the sample can be removed to measure the preceptin. It is possible to suppress an increase in the apparent preceptin measurement value at.
  • S68 antibody refers to an anti-antibody obtained by purifying a polyclonal antibody obtained from a non-human mammal immunized with the S68 peptide (SEQ ID NO: 2) as an immunogen using a column to which the S68 peptide is immobilized.
  • S68 peptide polyclonal antibody is a peptide consisting of the amino acid sequence of SEQ ID NO: 2 (the amino acid sequence of positions 53 to 68 of the amino acid sequence of SEQ ID NO: 3).
  • a specific method for producing the S68 antibody is as described in Example 1 of WO2004 / 044005.
  • P03-recognizing monoclonal antibody refers to a region corresponding to positions 52 to 61 of the amino acid sequence represented by SEQ ID NO: 1 (krvdadadpr: SEQ ID NO: 3 (human full-length soluble CD14)) in preceptin: P03 sequence Is a monoclonal antibody.
  • the P03-recognizing monoclonal antibody is, for example, a monoclonal antibody described in WO2015 / 129774.
  • P03-specific polyclonal antibody refers to a conventional rabbit-derived anti-preceptin polyclonal antibody (S68 antibody), that is, a polyclonal antibody obtained by immunizing a rabbit with the S68 peptide (SEQ ID NO: 2) and immobilizing the S68 peptide. It is produced by purifying with an affinity column. A polyclonal antibody is purified by an affinity column in which the P03 peptide (SEQ ID NO: 1) is immobilized in place of the S68 peptide-immobilized column, whereby an anti-preceptin polyclonal antibody having high reactivity with preceptin is obtained.
  • a specific method for producing a P03-specific polyclonal antibody is as described in WO2017 / 033181.
  • presepsin is known as a marker used for detecting sepsis
  • the method for measuring presepsin is used in a method for detecting sepsis, which includes a step of contacting an antibody used for presepsin measurement with a specimen containing presepsin. can do.
  • the measurement method of preceptin is (1) a step of measuring a preceptin concentration in a sample of a subject using an antibody used for preceptin measurement; and (2) including a step of determining whether or not the preceptin concentration obtained in (1) is higher than a cutoff value. It can also be called a method of detecting sepsis.
  • the cut-off value when the sample is a blood sample is, for example, 314 to 600 pg / mL, preferably 400 to 580 pg / mL, more preferably 450 to 550 pg / mL, and still more preferably 500 pg / mL.
  • Detection of sepsis may be read as “assisting detection of sepsis” or “assisting diagnosis of sepsis”.
  • the method of measuring presepsin includes, for example, discrimination between sepsis and systemic inflammatory response syndrome (SIRS); risk assessment of severe sepsis; prognosis prediction of sepsis (prediction of mortality); severe sepsis Assessment; detection of postoperative infection; detection of infectious intravascular coagulation (DIC); detection of infectious DIC; detection of heart disease; respiratory infection with bacterial infection, inflammatory bowel disease (Crohn's disease, ulcerative colitis), febrile neutropenia (FN), or hemophagocytic syndrome (HPS) It can be used for detection or evaluation of the disease. That is, the method for measuring presepsin can also be referred to as a method for detecting or evaluating the disease.
  • SIRS systemic inflammatory response syndrome
  • risk assessment of severe sepsis includes, for example, discrimination between sepsis and systemic inflammatory response syndrome (SIRS); risk assessment of severe sepsis; prognosis prediction of sepsis (prediction of mortality); severe seps
  • Postoperative infection is a general term for infections that develop after surgery, and refers to all infections caused by surgery and adjunct therapy required for it.
  • Postoperative infections include all diseases diagnosed as postoperative infections based on Guideline for prevention of surgical site infection, 1999 (CDC).
  • Examples of heart diseases include acute coronary syndrome (ACS), acute heart failure, acute decompensated heart failure (ADHF), chronic heart failure, coronary artery disease, angina, myocardial infarction, ischemic stroke, hemorrhagic stroke and transient Examples include cerebral ischemic attacks.
  • Respiratory tract infections with bacterial infections include lower respiratory tract infections or pneumonia.
  • Lower respiratory tract infections include acute lower respiratory tract infections and chronic lower respiratory tract infections.
  • Acute lower respiratory tract infections include acute tracheitis, acute bronchitis, and acute bronchiolitis, mostly caused by viral infection of the upper respiratory tract that spreads to the lower respiratory tract, but in some cases secondary to bacteria Infection continues. Antibiotics are indicated for signs of secondary bacterial infection.
  • Chronic lower respiratory tract infection is a pathological condition in which persistent infection of bacteria is established in the lower respiratory tract having an organic disorder such as bronchiectasis or chronic obstructive pulmonary disease, and persistent infection and acute ashamed exist.
  • bronchiectasis chronic obstructive pulmonary disease
  • chronic bronchitis diffuse panbronchiolitis
  • old pulmonary tuberculosis pneumoconiosis
  • nontuberculous mycobacterial disease allergy Bronchopulmonary aspergillosis, pulmonary fibrosis, chronic bronchial asthma and the like are included.
  • Antibiotics are indicated for both persistent infection and acute exacerbation.
  • Pneumonia includes community-acquired pneumonia and nosocomial pneumonia. Preferred is community-acquired pneumonia.
  • Examples of a method for immunologically measuring preceptin using an antibody used for preceptin measurement include enzyme immunoassay (hereinafter also referred to as EIA or ELISA), chemiluminescent enzyme immunoassay (CLEIA), chemiluminescent immunity, and the like.
  • Measurement method (CLIA), fluorescent antibody method (FAT), fluorescent enzyme immunoassay method (FEIA), electrochemiluminescence immunoassay method (ECLIA), radioimmunoassay method (RIA), immunochromatography method, aggregation method, competitive method, etc.
  • a direct method or an indirect method may be used, and a sensitizing method in which a biotin-avidin (streptavidin) complex is formed and detected may be used.
  • EIA is one of immunoassays using enzyme-labeled antibodies, and includes direct methods and indirect methods.
  • a preferable example is a sandwich ELISA (enzyme-linked immunosorbent assay).
  • Sandwich ELISA uses two or more types of antibodies with different antigen recognition sites, one of which is immobilized on a solid phase in advance, and the antigen to be detected is sandwiched between the two types of antibodies. It is a method of measuring by forming.
  • the chemiluminescence enzyme immunoassay is a method in which an antigen in a sample is reacted with an antibody immobilized on a magnetic particle or bead, followed by reaction with an enzyme-labeled antibody and washing (B / F separation). ) Thereafter, a chemiluminescent substrate is added, and after the enzyme reaction, the luminescence intensity is measured.
  • an antigen-biotin-bound antibody in a specimen is reacted in a liquid phase, the antibody is trapped on magnetic particles bound with streptavidin, washed (B / F separation), reacted with an enzyme-labeled antibody, The luminescence intensity is measured.
  • the chemiluminescent substrate is preferably CDP-StarTM, AMPPDTM, or CSPDTM.
  • the labeling enzyme is HRP
  • luminol is preferably used as the chemiluminescent substrate.
  • the detection sensitivity is generally said to be higher in the order of chemiluminescence> fluorescence> absorption (coloration), and the measurement method can be selected according to the required sensitivity.
  • chemiluminescence immunoassay an antigen in a sample is reacted with an antibody solid-phased on magnetic particles, followed by reacting an antibody labeled with a chemiluminescent substance and washing (B / F separation). Then, it is a method of measuring luminescence intensity.
  • Acridinium or the like is used as the labeling substance.
  • the antigen in the sample is reacted with the immobilized antibody, the enzyme-labeled antibody is reacted, washed (B / F separation), and then a fluorescent substrate is added.
  • a fluorescent substrate is added.
  • the fluorescence intensity is measured after the enzyme reaction.
  • HRP As a labeling enzyme, HRP, ALP, or the like is used.
  • AmplexTMRed or the like is used when the labeling enzyme is HRP, and 4-MUP (4-Methylumbelliferous phosphate), AttoPhosTM or the like is preferably used when the labeling enzyme is ALP.
  • Electrochemiluminescence immunoassay reacts an antigen in a sample with an antibody solid-phased on a magnetic particle and an antibody labeled with an electrochemiluminescent substance, followed by washing (B / F separation). This is a method for measuring the emission intensity by electric energy. Ruthenium or the like is used as the labeling substance. Ru (bpy) 3 or the like is used as the labeling substance, and excitation light emission is repeated by oxidation due to charging of the electrode and reduction reaction by tripropylamine (TPA) or the like.
  • TPA tripropylamine
  • Radioimmunoassay is a measurement method using a label with a radioisotope. For example, after reacting an antigen in a specimen with an antibody immobilized on a bead or the like, a radioisotope ( 125 I or the like) is reacted, and after washing (B / F separation), the radiation dose of 125 I can be measured.
  • Immunochromatography is an immunoassay method that applies capillary action in which a specimen moves while dissolving a reagent on a test strip.
  • the antigen in the sample forms an immune complex with the labeled antibody and the capture antibody on the test strip, and the color of the label is confirmed.
  • colloidal gold, an enzyme, a fluorescent substance, or the like is used for labeling the antibody. If an enzyme-labeled antibody is used, an enzyme substrate is placed on the test strip and colored.
  • the flow-through method is a method in which an antigen as a test substance forms an antibody-antigen-antibody complex together with a solution in a specimen on a membrane that is an insoluble carrier. At this time, the substances not fixed to the membrane are usually removed vertically from the front and back of the membrane.
  • the agglutination method is a method of observing agglutination by reacting an antigen in a specimen with an antibody in a reagent. Examples thereof include a method not using a solid phase, a particle agglutination (PA) using particles artificially prepared as a solid phase, and a latex agglutination (latex agglutination: LA) using latex particles among PAs. .
  • PA particle agglutination
  • LA latex agglutination
  • an antibody is bound to a solid phase, a test sample and a certain amount of labeled antigen are reacted simultaneously, and the amount of antigen in the sample can be measured from the amount of bound label.
  • the antibody used for preceptin measurement is preferably used in the above-described measurement method.
  • the specimen used for preceptin measurement is collected from a subject.
  • examples of the subject include humans and non-human mammals (eg, rabbits, goats, horses, sheep, pigs, rats, and mice).
  • the subject is a human.
  • the sample is not particularly limited, but an aqueous sample is preferable.
  • blood whole blood, plasma, serum, etc.
  • urine tissue fluid, lymph fluid, joint fluid, milk, cerebrospinal fluid, pus, saliva, tear fluid, mucus
  • Body fluids such as runny nose, sputum, ascites, irrigation fluid, semen, and washing fluid after washing nasal cavity, bronchi, lung, skin, abdominal cavity, various organs, joints, bones, etc., cell culture supernatant, or column elution Liquid and the like.
  • the specimen used for preceptin measurement is preferably a blood specimen, more preferably a human blood specimen.
  • a whole blood sample when used as a sample for preceptin measurement, the whole blood sample is collected within 72 hours, 48 hours, 24 hours, 12 hours, 6 hours, or 4 hours.
  • An analysis may be performed.
  • a whole blood sample may be collected by an EDTA blood collection tube or a heparin blood collection tube.
  • a whole blood sample is collected in an EDTA blood collection tube and analyzed within 6 hours, or a whole blood sample is collected in a heparin blood collection tube and analyzed within 4 hours.
  • Presepsin Measurement Kit Also provided herein is a presepsin measurement kit (also referred to as “measurement kit”) comprising at least one selected from the group consisting of an anti-CD14 antibody, a chromatography column, and a membrane.
  • the measurement kit is preferably a method for stabilizing the preceptin value in the sample, a method for suppressing the production of pseudopreceptin derived from sCD14 in the sample, a method for suppressing the denaturation of sCD14 in the sample, and the determination of the amount of true preceptin in the sample.
  • the measurement kit preferably includes an anti-CD14 antibody, a chromatography column, or a membrane.
  • the “anti-CD14 antibody”, “chromatography column”, or “membrane” is specifically as described above.
  • the measurement kit more preferably contains an auxiliary reagent for preceptin measurement.
  • auxiliary reagents include primary antibodies, secondary antibodies, labeled antibodies, labeled enzymes, labeling substances such as gold colloids, chromogenic substrates, fluorescent substrates (Amplex TM Red, AttoPhos TM, 4-MUP, etc.), chemiluminescent substrates (luminol, CDP-StarTM, AMPPDTM, CSPDTM, etc.), specific binding substances such as biotin-streptavidin, insoluble carriers, blocking agents, diluents, washings, standard substances and the like, but are not limited thereto.
  • Auxiliary reagents for presepsin measurement are used in appropriate combination according to the method of presepsin measurement.
  • the primary antibody is preferably an antibody that binds to preceptin, and more preferably an antibody that recognizes an epitope different from the antibody.
  • Examples thereof include the F1106-13-3 antibody and the F1031-8-3 antibody described in Example 3 of WO2004 / 044005. Reference can also be made to WO 2005/108429.
  • Either the antibody used for preceptin measurement or the primary antibody may be used as the labeled antibody.
  • a labeled secondary antibody may be used.
  • insoluble carriers include magnetic particles, beads, glass, cellulose, nitrocellulose, porous synthetic polymers, glass fibers, polyacrylamide, nylon, polystyrene, polyvinyl chloride, polypropylene, plastic plates, latex particles, non-woven fabric, filter paper, and the like. Can be mentioned.
  • the label of the antibody used for preceptin measurement is preferably used, but is not limited to enzymes such as peroxidase (HRP), alkaline phosphatase (ALP), ⁇ -galactosidase, colloidal gold, and the like.
  • HRP peroxidase
  • ALP alkaline phosphatase
  • ⁇ -galactosidase colloidal gold, and the like.
  • examples of the chromogenic substrate include 3,3 ', 5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (OPD), and the like.
  • TMB 3,3 ', 5,5'-tetramethylbenzidine
  • OPD o-phenylenediamine
  • examples of the chromogenic substrate in the case of using ⁇ -galactosidase include o-nitrophenyl- ⁇ -D-galactopyranoside (o-Nitrophenyl- ⁇ -D-Galactopyranoside: ONPD).
  • the measurement kit for the sandwich ELISA method may contain an antibody and a primary antibody (any antibody may be enzyme-labeled) used for preceptin measurement, a chromogenic substrate, a diluent, a standard substance, and the like.
  • a labeled secondary antibody may be contained.
  • a measurement kit for chemiluminescent enzyme immunoassay can contain, for example, an antibody immobilized on magnetic particles, an enzyme-labeled antibody, a chemiluminescent substrate, a diluent, a washing solution, and the like.
  • a fluorescent enzyme immunoassay (FEIA) measurement kit can contain, for example, an antibody immobilized on magnetic particles, an enzyme-labeled antibody, a fluorescent substrate, a diluent, a washing solution, and the like.
  • the measurement kit for electrochemiluminescence immunoassay can contain, for example, biotinylated antibody, Ru (bpy) 3-labeled antibody, streptavidin-coated magnetic particles, tripropylamine and the like.
  • the measurement kit by immunochromatography is a test strip provided with a sample addition part, a reagent part, a detection part, and an absorption part so that the liquid sample added to the test addition part moves in the above order.
  • an insoluble carrier in which the second antibody labeled is impregnated in the reagent part and the first antibody is bound to the detection part can be installed.
  • the test strip is exemplified by using a porous carrier or the like.
  • the porous carrier include nitrocellulose, cellulose, cellulose derivatives, nylon, nylon fibers, glass fibers, and porous synthetic polymers.
  • the absorption part include an absorption polymer such as a water-absorbing material sponge, cellulose filter paper, filter paper and the like.
  • the presepsin measurement kit is a kit for detecting sepsis or a kit for assisting in the detection or diagnosis of sepsis. Also good.
  • the measurement kit can be used as a sepsis diagnostic agent or as an auxiliary agent for sepsis diagnosis.
  • the preceptin measurement kit can be used for the detection of such sepsis, etc.
  • the subject can be septic when the preceptin concentration in the subject's sample measured using an antibody is higher than the cutoff value. It can be determined that there is sex, and detection or diagnosis can be assisted.
  • the cut-off value is 314 to 600 pg / mL, preferably 400 to 580 pg / mL, more preferably 450 to 550 pg / mL, and still more preferably 500 pg / mL.
  • the kit for measuring presepsin distinguishes between sepsis and systemic inflammatory response syndrome (SIRS); risk assessment of severe sepsis; prognosis prediction of septicemia (mortality rate) Prediction); severity assessment of sepsis; detection of postoperative infection; detection of infectious intravascular coagulation (DIC); detection of infectious DIC; detection of heart disease; respiratory infection with bacterial infection Detection of inflammation, inflammatory bowel disease (Crohn's disease, ulcerative colitis), febrile neutropenia (FN), or hemophagocytic syndrome (HPS) Chosen can be used for detection The evaluation of at least one disease.
  • the kit for measuring presepsin may be a kit for detecting or evaluating at least one disease.
  • the pseudo preceptin and true preceptin differential quantification method in a sample includes using a gel filtration method.
  • “separate quantification of pseudo-presepsin and true pre-sepsin” means that, as in Example 4, sCD14-derived pseudo-preceptin and preceptin are separated.
  • the gel filtration method can be used according to the method according to Example 4, and the preceptin value in each fraction is measured. More specifically, the sample before presepsin measurement is separated by, for example, gel filtration chromatography, the fraction containing true presepsin is obtained, and the presepsin value is measured to quantify the true preceptin value. Is possible.
  • a true preceptin is obtained by comparing the standard curve prepared with standard concentration of preceptin and the amount of preceptin in the fraction containing each preceptin. A value can be obtained.
  • a pseudo preceptin value can be obtained by obtaining a fraction containing a pseudo preceptin instead of a fraction containing a true preceptin and measuring the pseudo preceptin value. If a pseudo preceptin value is obtained, the prepsin value of the specimen can be measured, and a true preceptin measurement value can be obtained from the total value (total preceptin amount) of the true preceptin value and the pseudo preceptin value.
  • Presepsin measurement system In addition, here, a method for stabilizing the preceptin value in the sample, a method for suppressing the production of pseudopreceptin derived from sCD14 in the sample, a method for suppressing degeneration of sCD14 in the sample, the amount of true preceptin in the sample
  • An apparatus such as a fully automatic apparatus in which a quantification method, a method of quantifying only true preceptin, a method of quantifying only a pseudopreceptin value, and the like are incorporated in the system is provided.
  • a system that performs the separation and quantification it is possible to construct a system that obtains a true preceptin value simply by directly applying a specimen.
  • Sample cartridge unit For example, a sample sample used for measurement is put in a microtube, and a portion for holding the microtube corresponds.
  • Specimen processing unit a part that performs specimen sampling, and includes, for example, a part that can be directly mounted with a nozzle for sampling or a disposable chip, and corresponds to a part for collecting a sample;
  • Reagent cartridge part a part containing a reagent necessary for presepsin measurement, for example, a reagent necessary for presepsin measurement described in the above-mentioned presepsin measurement or presepsin measurement kit item;
  • Presepsin measurement unit a part for measuring presepsin in a sample, which corresponds to a part for measuring presepsin according to the above-mentioned preceptin measurement or preceptin measurement kit item;
  • Calculation part The part which calculates a preceptin value based on the measurement in the measurement part of
  • Example 1 Influence of specimen shaking on preceptin measured value
  • Two specimens of normal human plasma (EDTA) (# R255292, # R255287) from TENNESSEE Blood Services were used as specimens for preceptin ELISA kit (Mochida Pharmaceutical Co., Ltd., Lot: 2S015) was used to measure each preceptin value in each shaken sample.
  • the presepsin value of the specimen was measured before shaking (0 minutes) and 10 minutes and 60 minutes after shaking.
  • 2 mL of the sample was dispensed into a microtube, and EYELA CUTE CM-1000 (1800 rpm) was used for 60 seconds with a TAITEC Vortex Mixer until 60 minutes thereafter.
  • Each measurement sample was used by sampling 50 ⁇ L from 2 mL of each sample.
  • the measured values of each preceptin are shown in Table 1.
  • Example 2 Examination of cause of increase in preceptin measurement value Using normal human serum (# STL130145) from Access Biologicals and human serum pretreated with anti-CD14 antibody (hereinafter referred to as CD14-absorbing human serum), each sample was used. In both cases, 100 ⁇ L was dispensed per 1.5 mL microtube and shaken for 10 minutes (the shaking conditions were the same as in Example 1).
  • the CD14-absorbed human serum was prepared by binding F1024-1-3 antibody (prepared by the method described in Examples 1 and 2 of WO2001 / 72993) to a chromatography resin (Sepharose 4FF). Were prepared using affinity chromatography.
  • Example 3 Examination of the effect of anti-CD14 antibody on changes in preceptin concentration
  • F1024-1-3 antibody (anti-CD14 antibody) was added to about 30 ⁇ g of 1 mL of normal human plasma (EDTA), shaken for 10 minutes, The presepsin concentration of the specimen was measured using a preceptin kit (Path First Presepsin, PATHHFAST (trade name)), and the influence of shaking of the anti-CD14 antibody on the change of the preceptin concentration was examined. The results are shown in FIG. When presepsin was measured using normal human plasma in the presence or absence of F1024-1-3 antibody (anti-CD14 antibody), F1024-1-3 antibody suppressed the increase in preceptin level caused by shaking. It was shown that.
  • Example 4 Gel filtration analysis of shaken human plasma Normal human plasma (# STL130145) was dispensed in 0.5 mL aliquots into three tubes, one was allowed to stand as a control, and then gel filtration analysis Used for. The remaining two are After 10 minutes of shaking treatment, gel filtration analysis was performed. At this time, about 200 ⁇ g of F1024-1-3 antibody was added to one, and the mixture was allowed to stand at room temperature for 10 minutes and then shaken. ⁇ Column Superdex 75 10/300 GL (1cm ⁇ ⁇ 30cm) ⁇ Fraction 0.5mL / tube ⁇ Sample addition amount to the column 100 ⁇ L
  • the chromatographic elution pattern of the substance detected by the CD14 measurement kit shown in FIG. 3 is a pattern in which the control sample shows a single peak in the vicinity of the fraction 17 and the plasma is shaken to obtain a vicinity of the void position. It changed to a peak with a shoulder. This suggested that sCD14 having a high molecular weight by shaking was generated. Further, this shoulder portion coincides with the elution position where the pseudopreceptin was detected, suggesting that the pseudopreceptin was obtained by denaturing and aggregating the sCD14 molecule to increase the molecular weight.
  • sCD14-ST preceptin
  • sCD14-ST soluble CD14
  • the true pre-sepsin can be quantified by distinguishing the pseudo-preceptin from the true pre-ceptin. For example, when measuring presepsin, (i) removing pseudopreceptin by passing through a 30 kDa ultrafiltration membrane, (ii) using an antibody that reacts with pseudopreceptin without reacting with true preceptin. It is possible to perform correct preceptin measurement by performing pretreatment such as adsorption removal / separation of pseudopreceptin.
  • Example 4 it is possible to separate and analyze pseudo-presepsin and true pre-sepsin using a chromatographic graph. What is necessary is just to quantify the presepsin value in the fraction containing true presepsin. Moreover, if pseudo preceptin can be quantified by any means, a true preceptin value can be obtained by calculation. For example, a “pseudopreceptin value” can be obtained by performing preceptin measurement on a fraction containing “pseudopreceptin” separated by chromatography.
  • a pseudo preceptin value is obtained, it is also possible to measure the prepsin value of the specimen and obtain a measured value of the pseudo preceptin from the total value (total preceptin amount) of the true preceptin value and the pseudo preceptin value. Furthermore, by screening a measurement system that does not react with pseudo-preceptin but reacts with only true preceptin, only the true preceptin value can be quantified without requiring special treatment.
  • SEQ ID NO: 1 is the amino acid sequence of the P03 peptide.
  • SEQ ID NO: 2 is the amino acid sequence of the S68 peptide.
  • SEQ ID NO: 3 is the amino acid sequence of human full-length soluble CD14.

Abstract

Provided by the present invention is a method for stabilizing measurements of presepsin in specimens, the method comprising a step for bringing a specimen into contact with an anti-CD14 antibody immediately after collection of the specimen. This method enables obtaining of true presepsin levels in specimens without increasing presepsin measurements even when specimens to be used are subjected to physical stimuli such as intensive agitation during specimen measurement.

Description

プレセプシン測定に有用な抗ＣＤ１４抗体の使用Use of anti-CD14 antibody useful for preceptin measurement
　本発明は、検体中のプレセプシン測定において、検体の物理的刺激による検体中の見かけのプレセプシン測定値の増加を抑制するための方法などに関する。 The present invention relates to a method for suppressing an increase in an apparent preceptin measurement value in a sample due to physical stimulation of the sample in measurement of presepsin in the sample.
　ＣＤ１４分子は、単核球細胞の膜表面に発現している糖タンパク質であり、ＬＰＳ（リポポリサッカライド）のレセプターとしての機能を有することが知られている。ＣＤ１４分子には、細胞表面上に発現している膜結合型ＣＤ１４（ｍＣＤ１４）と、可溶型ＣＤ１４（ｓＣＤ１４）の２種類が存在する。ｓＣＤ１４として、分子量約５５ｋＤａおよび約４９ｋＤａのｓＣＤ１４（以下「高分子量可溶型ＣＤ１４」または「高分子量ｓＣＤ１４」という。）が知られており、敗血症（ＳＥＰＳＩＳ）、後天性免疫不全症候群（ＡＩＤＳ）、急性呼吸促進症候群（ＡＲＤＳ）、全身性エリテマトーデス（ＳＬＥ）等、多くの疾患における患者の血中で高値を示す事が報告されている。そのため、これらの高分子量ｓＣＤ１４は疾患特異的なマーカーではないと考えられている（非特許文献１～２）。 The CD14 molecule is a glycoprotein expressed on the membrane surface of mononuclear cells and is known to have a function as a receptor for LPS (lipopolysaccharide). There are two types of CD14 molecules, membrane-bound CD14 (mCD14) and soluble CD14 (sCD14) expressed on the cell surface. As sCD14, sCD14 having a molecular weight of about 55 kDa and about 49 kDa (hereinafter referred to as “high molecular weight soluble CD14” or “high molecular weight sCD14”) is known, and sepsis (SEPSIS), acquired immune deficiency syndrome (AIDS), It has been reported to show high levels in the blood of patients in many diseases such as acute respiratory distress syndrome (ARDS) and systemic lupus erythematosus (SLE). Therefore, these high molecular weight sCD14 are considered not to be disease-specific markers (Non-Patent Documents 1 and 2).
　一方、敗血症患者において特徴的に血中濃度が上昇する、新たなｓＣＤ１４の分子種として、ｓＣＤ１４－ＳＴ（可溶性ＣＤ１４抗原サブタイプ，プレセプシンともいう）が存在することが報告されている。 On the other hand, it has been reported that sCD14-ST (also called a soluble CD14 antigen subtype, also known as preceptin) exists as a new sCD14 molecular species whose blood concentration is characteristically increased in septic patients.
　ｓＣＤ１４－ＳＴ（プレセプシン）とは、ｓＣＤ１４のうち、非還元条件下ＳＤＳ－ＰＡＧＥにおいて分子量１３±２ｋＤａに泳動されることを特徴とし、ＣＤ１４のＮ端部を保持しているものである。高分子量ｓＣＤ１４と比べると、Ｃ端側が大きく欠失したアミノ酸配列を有しており、高分子量ｓＣＤ１４とは異なりＬＰＳ結合能を有していない。また、プレセプシンは高分子量ｓＣＤ１４とは異なる免疫原性を示すため、抗体を用いて両者を区別できる。プレセプシンは敗血症患者において特異的に血中濃度が上昇する（特許文献１）。また、敗血症との判別が難しい、全身性炎症反応（ＳＩＲＳ）を示す患者と比較しても、敗血症患者の血中で高値を示すという報告があり、プレセプシンは敗血症の特異的な診断マーカーであると考えられている（非特許文献３）。 SCD14-ST (presepsin) is characterized in that it migrates to a molecular weight of 13 ± 2 kDa in SDS-PAGE under non-reducing conditions, and retains the N-terminal part of CD14. Compared with high molecular weight sCD14, it has an amino acid sequence that is largely deleted on the C-terminal side, and unlike high molecular weight sCD14, it does not have LPS binding ability. Moreover, since preceptin shows immunogenicity different from high molecular weight sCD14, both can be distinguished using an antibody. Presepsin specifically increases blood concentration in patients with sepsis (Patent Document 1). In addition, there is a report that it is high in the blood of sepsis patients compared to patients with systemic inflammatory response (SIRS), which is difficult to distinguish from sepsis, and presepsin is a specific diagnostic marker for sepsis (Non-patent Document 3).
　プレセプシンを特異的に認識するウサギ由来ポリクローナル抗体（Ｓ６８抗体）及びラット由来モノクローナル抗体（Ｆ１１４６－１７－２）が開示されている（特許文献１、２）。 A rabbit-derived polyclonal antibody (S68 antibody) and a rat-derived monoclonal antibody (F1146-17-2) that specifically recognize preceptin are disclosed (Patent Documents 1 and 2).
　現在、プレセプシンの測定には、プレセプシンに対する特異抗体としてウサギ由来ポリクローナル抗体を用いた測定系が実用化され、測定キットが欧州及び日本で上市されている（ＰＡＴＨＦＡＳＴ（商品名）　Ｐｒｅｓｅｐｓｉｎ，（株）ＬＳＩメディエンス）。 Currently, for the measurement of preceptin, a measurement system using a rabbit-derived polyclonal antibody as a specific antibody for preceptin has been put into practical use, and measurement kits have been put on the market in Europe and Japan (PATHAST (trade name) Presepsin, LSI) Medience).
　また、ウサギモノクローナル抗体を用いたプレセプシンの全自動測定装置も実用化されている（例えば、ＳＴＡＣＩＡ（商品名）（（株）ＬＳＩメディエンス）、ＡＩＡ（商品名）（東ソー（株））など）。 In addition, a fully automatic preceptin measurement apparatus using a rabbit monoclonal antibody has been put into practical use (for example, STACIA (trade name) (LSI Medience Corporation), AIA (trade name) (Tosoh Corporation), etc.).
　プレセプシン測定キットについては、使用する検体に激しい撹拌などの物理的刺激を与えると、検体中の見かけのプレセプシン測定値が増加し、検体中の本来のプレセプシン値が得られなくなってしまう場合があると言われている。 For presepsin measurement kits, if physical stimulation such as vigorous agitation is applied to the sample to be used, the apparent preceptin measurement value in the sample may increase, and the original preceptin value in the sample may not be obtained. It is said.
国際公開ＷＯ２００５／１０８４２９International Publication WO2005 / 108429 国際公開ＷＯ２００４／０４４００５International Publication WO2004 / 044005
　前述の通り、プレセプシン測定キットについては、使用する検体に激しい撹拌などの物理的刺激を与えると、プレセプシンの測定値が増加することにより、検体中の本来のプレセプシン値が得られなくなってしまうことが懸念されている。このため、プレセプシン測定キットについては、激しい撹拌を与えないように検体を扱うよう注意すべきであると言われている。 As described above, with the presepsin measurement kit, if a physical stimulus such as vigorous agitation is applied to the sample to be used, the presepsin value in the sample may not be obtained due to an increase in the measured value of preceptin. There are concerns. For this reason, it is said that the presepsin measurement kit should be handled with care so as not to give vigorous stirring.
　このような状況において、検体中のプレセプシン測定の際、使用する検体に物理的刺激を与えても、プレセプシンの測定値がほとんど増加することなく、検体中の本来のプレセプシン測定値を得るための方法が求められていた。 In such a situation, when measuring presepsin in a sample, even if a physical stimulus is applied to the sample to be used, the method for obtaining the original preceptin measured value in the sample is hardly increased even if the measured value of presepsin is hardly increased. Was demanded.
　あるいは、検体中のプレセプシン測定の際、物理的刺激を検体に与えても、検体中の見かけのプレセプシン測定値がほとんど増加せず、検体の扱い方によらず安定したプレセプシン測定を可能とする方法が求められていた。 Alternatively, when measuring presepsin in a sample, even if a physical stimulus is applied to the sample, the apparent preceptin measurement value in the sample hardly increases, and it enables stable preceptin measurement regardless of how the sample is handled Was demanded.
　本発明の目的は、従来の課題の少なくとも１つを解決することができる、検体中のプレセプシン測定に有用な方法などを提供することである。 An object of the present invention is to provide a method useful for measuring presepsin in a sample, which can solve at least one of the conventional problems.
　本発明者は、鋭意検討した結果、検体に激しい撹拌などの物理的刺激を与えたときにプレセプシンの測定値が増加する現象は、物理的刺激により生じた検体中のｓＣＤ１４由来の生成物が原因であることを見出した。そこで、本発明者は、このｓＣＤ１４由来の生成物は、例えば、検体と抗ＣＤ１４抗体とを接触させること、或いはクロマトグラフィーを用いてプレセプシンと分離できること等によって、その生成物による影響を排除でき、検体中の本来のプレセプシン値が測定できることなどに想到し、本発明を完成した。 As a result of intensive studies, the present inventors have found that the phenomenon that the measured value of preceptin increases when a physical stimulus such as vigorous stirring is applied to the specimen is caused by a product derived from sCD14 in the specimen caused by the physical stimulus. I found out. Therefore, the present inventor can eliminate the influence of the product derived from the sCD14 by, for example, bringing the specimen into contact with the anti-CD14 antibody or separating it from preceptin using chromatography. The inventors have conceived that the original preceptin value in the specimen can be measured, thereby completing the present invention.
　後述の実施例では、正常ヒト血漿（ＥＤＴＡ）を用い、検体の振とうによるプレセプシンＥＬＡＩＳＡキットの測定値への影響を確認し、検体の振とうによって、プレセプシンの測定値が増加することを確認した。
　また、実施例では、正常人のヒト血清およびＣＤ１４吸収血清を用いて、振とうによるプレセプシン値の増加が、血中ｓＣＤ１４を含むサンプルでのみ増加し、血清中のｓＣＤ１４に由来することも確認した。
　また、実施例では、正常人の血漿を用い、Ｆ１０２４－１－３抗体（抗ＣＤ１４抗体）の存在下・非存在下でプレセプシンを測定したところ、振とうによって生じるプレセプシン値の増加をＦ１０２４－１－３抗体が抑制することを確認した。
　また、実施例では、振とうによるプレセプシン値の増加は、検体中のｓＣＤ１４が関連することを確認した。具体的には、振とうによって生成するｓＣＤ１４由来の生成物をゲル濾過による各フラクションを分析した結果、コントロールと抗ＣＤ１４抗体を添加して振とうした検体の溶出パターンはほぼ同じであり、検体の振とうによりｓＣＤ１４からより高分子量の凝集体が生成（この高分子量の凝集体を「ｓＣＤ１４由来の生成物」または「偽プレセプシン」とも言う。）することを確認した。そして、その高分子量の凝集体の生成は、検体に抗ＣＤ１４抗体を共存させることによって効果的に抑制できることも確認した。さらに、当該高分子量の凝集体は、クロマトグラフィーにより分離できることを確認した。 In Examples described later, normal human plasma (EDTA) was used, and the influence of the shaking of the specimen on the measured value of the preceptin ELISA kit was confirmed. It was confirmed that the measured value of preceptin increased by shaking the specimen. .
Further, in the Examples, it was confirmed that the increase in preceptin level due to shaking was increased only in the sample containing blood sCD14 and derived from sCD14 in the serum using normal human serum and CD14-absorbed serum. .
In the examples, when presepsin was measured using normal human plasma in the presence / absence of F1024-1-3 antibody (anti-CD14 antibody), the increase in preceptin level caused by shaking was observed in F1024-1 -3 antibody was confirmed to be suppressed.
Moreover, in the Example, it confirmed that the increase in the preceptin value by shaking related with sCD14 in a test substance. Specifically, as a result of analyzing each fraction by gel filtration of the product derived from sCD14 produced by shaking, the elution pattern of the sample shaken by adding the control and anti-CD14 antibody is almost the same. It was confirmed that a higher molecular weight aggregate was generated from sCD14 by shaking (this high molecular weight aggregate is also referred to as “product derived from sCD14” or “pseudopreceptin”). It was also confirmed that the formation of high molecular weight aggregates can be effectively suppressed by allowing the sample to coexist with an anti-CD14 antibody. Furthermore, it was confirmed that the high molecular weight aggregate can be separated by chromatography.
　以上の実施例から、例えば、ｓＣＤ１４由来の生成物は、検体と抗ＣＤ１４抗体とを接触させること、或いはクロマトグラフィーを用いてプレセプシンと分離すること等によって、その生成物による影響を排除でき、検体中のプレセプシン測定における振とうによるプレセプシン測定値の増加を効果的に防ぐことが可能であり、検体中の本来のプレセプシン測定値を得ることができることなどが分かる。 From the above examples, for example, the product derived from sCD14 can eliminate the influence of the product by bringing the sample into contact with the anti-CD14 antibody or separating it from preceptin using chromatography. It can be seen that the increase in the presepsin measurement value due to shaking in the measurement of presepsin can be effectively prevented, and the original presepsin measurement value in the sample can be obtained.
　従来、検体中のプレセプシン測定の際、検体を撹拌（これには、例えば激しい短時間撹拌、緩やかな長時間撹拌が含まれる）した場合、測定値に影響を与えることがあるとの注意がされているが、この測定値に影響を与える原因が血中のｓＣＤ１４であり、検体と抗ＣＤ１４抗体（例えば、Ｆ１０２４－１－３抗体）とを接触させること、或いはクロマトグラフィーを用いてプレセプシンと分離すること等によって、その生成物によるプレセプシンの測定値への影響を除去できることは驚くべきことであった。 Conventionally, when measuring presepsin in a sample, it has been noted that if the sample is agitated (this includes, for example, vigorous short time agitation and gentle long time agitation), the measured value may be affected. However, the cause of the influence on the measured value is sCD14 in blood, and it is separated from preceptin by contacting a specimen with an anti-CD14 antibody (for example, F1024-1-3 antibody) or using chromatography. It was surprising that the effect of the product on the measured value of presepsin can be removed.
　本発明は以下の態様を包含し得る。
［１］　検体採取直後に検体と抗ＣＤ１４抗体とを接触させる工程を含む、検体中のプレセプシン測定値を安定化させる方法。
［２］　検体採取直後に検体と抗ＣＤ１４抗体とを接触させる工程を含む、検体中の偽プレセプシン産生を抑制させる方法。
［３］　検体採取直後に検体と抗ＣＤ１４抗体とを接触させる工程を含む、検体中のｓＣＤ１４の変性抑制方法。
［４］　検体と抗ＣＤ１４抗体とを接触させる工程により、検体中のｓＣＤ１４を吸着除去することを含む、前記態様［１］ないし［３］のいずれか１態様に記載の方法。
［５］　検体と抗ＣＤ１４抗体とを接触させる工程により、検体中のｓＣＤ１４と抗体ＣＤ１４抗体とが結合物をつくることを特徴とする、前記態様［１］ないし［３］のいずれか１態様に記載の方法。
［６］　プレセプシン測定前に、検体と抗ＣＤ１４抗体とを接触させる工程を含む、検体中の真のプレセプシン量の定量方法。
［７］　検体と抗ＣＤ１４抗体とを接触させる工程により、検体中の偽プレセプシンと反応する抗ＣＤ１４抗体を用いて偽レセプシンを吸着除去することを特徴とする、前記態様［６］に記載の方法。
［８］　プレセプシン測定前に、偽プレセプシンと真のプレセプシンとを分別する工程を含む、真のプレセプシンのみを定量する方法。
［９］　偽プレセプシンと真のプレセプシンとの分別が、クロマトグラフィーによる分離であることを特徴とする、前記態様［８］に記載の方法。
［１０］　偽プレセプシンと真のプレセプシンとの分別が、膜による分離であることを特徴とする、前記態様［８］に記載の方法。
［１１］　プレセプシン測定前に、偽プレセプシンと真のプレセプシンとを分別する工程を含む、偽プレセプシンのみを定量する方法。
［１２］　検体のプレセプシンを測定してプレセプシン値を算出し、算出したプレセプシン値から前記態様［１１］に記載の方法で得た偽プレセプシン値を減じることを含む、真のプレセプシン値を定量する方法。
［１３］　前記態様［１］～［１２］のいずれか１態様に記載の方法に用いるための、抗ＣＤ１４抗体、クロマトグラフィー用カラム、および膜からなる群から選択される少なくとも１つを含む、プレセプシン測定キット。
［１４］　前記態様［１］～［１２］のいずれか１態様に記載の方法に用いるための、検体カートリッジ、検体処理部、試薬カートリッジ部、プレセプシン測定部、計算部及び表示部からなる群から選択される少なくとも１つを含む、プレセプシン測定システム。 The present invention can include the following embodiments.
[1] A method for stabilizing a preceptin measurement value in a sample, comprising a step of bringing the sample into contact with an anti-CD14 antibody immediately after the sample is collected.
[2] A method of suppressing pseudopreceptin production in a sample, comprising a step of bringing the sample into contact with an anti-CD14 antibody immediately after the sample is collected.
[3] A method for inhibiting denaturation of sCD14 in a specimen, comprising a step of bringing the specimen into contact with an anti-CD14 antibody immediately after the specimen is collected.
[4] The method according to any one of the above-described embodiments [1] to [3], comprising adsorbing and removing sCD14 in the sample by a step of bringing the sample into contact with the anti-CD14 antibody.
[5] In any one of the above embodiments [1] to [3], the step of bringing the sample into contact with the anti-CD14 antibody forms a conjugate between the sCD14 and the antibody CD14 antibody in the sample. The method described.
[6] A method for quantifying the amount of true preceptin in a sample, comprising a step of bringing the sample into contact with an anti-CD14 antibody before measuring presepsin.
[7] The method according to the above aspect [6], wherein the pseudo-receptin is adsorbed and removed by using the anti-CD14 antibody that reacts with the pseudopreceptin in the sample by the step of contacting the sample with the anti-CD14 antibody. .
[8] A method for quantifying only true preceptin, comprising a step of separating pseudo-preceptin and true preceptin before measuring preceptin.
[9] The method according to the above aspect [8], wherein the fractionation between the pseudo preceptin and the true preceptin is a chromatographic separation.
[10] The method according to the above aspect [8], wherein the separation between the pseudo-presepsin and the true pre-sepsin is separation by a membrane.
[11] A method for quantifying only pseudo-preceptin, comprising a step of separating pseudo-preceptin and true pre-ceptin before measuring pre-ceptin.
[12] A method for quantifying a true preceptin value, comprising measuring a preceptin value of a specimen to calculate a preceptin value, and subtracting the pseudo-preceptin value obtained by the method according to the aspect [11] from the calculated preceptin value .
[13] It includes at least one selected from the group consisting of an anti-CD14 antibody, a chromatography column, and a membrane for use in the method according to any one of the above aspects [1] to [12]. Presepsin measurement kit.
[14] From the group consisting of a specimen cartridge, a specimen processing section, a reagent cartridge section, a preceptin measurement section, a calculation section, and a display section for use in the method according to any one of the above aspects [1] to [12]. A preceptin measurement system comprising at least one selected.
　いくつかの態様によれば、検体中のプレセプシン測定の際、使用する検体に物理的刺激を与えても、プレセプシンの測定値がほとんど増加することなく、検体中の本来のプレセプシン測定値を得ることができる。例えば、ｓＣＤ１４由来の生成物は、検体と抗ＣＤ１４抗体とを接触させること、或いはクロマトグラフィーを用いてプレセプシンと分離すること等によって、その生成物による影響を排除でき、検体中のプレセプシン測定における物理的刺激によるプレセプシン測定値の増加を効果的に防ぐことが可能であり、検体中の本来のプレセプシン測定値を得ることができる
　また、別のいくつかの態様によれば、検体中のプレセプシン測定の際、激しい撹拌などの物理的刺激を検体に与えても、すなわち検体の扱い方によらず、正しいプレセプシン測定値を得ることができる。 According to some embodiments, when measuring presepsin in a sample, even if a physical stimulus is applied to the sample to be used, the measured value of presepsin is hardly increased and the original measured value of presepsin is obtained. Can do. For example, the product derived from sCD14 can be excluded from the influence of the product by contacting the sample with the anti-CD14 antibody or separated from preceptin using chromatography. The increase in the presepsin measurement value due to mechanical stimulation can be effectively prevented, and the original presepsin measurement value in the sample can be obtained. According to another aspect, the measurement of the presepsin measurement in the sample can be performed. At this time, even when a physical stimulus such as vigorous stirring is applied to the specimen, that is, regardless of how the specimen is handled, a correct preceptin measurement value can be obtained.
抗ＣＤ１４抗体添加のプレセプシン濃度への影響を示す図である。It is a figure which shows the influence on the presepsin density | concentration of anti-CD14 antibody addition. 実施例４におけるゲル濾過の各フラクションをプレセプシンＥＬＡＩＳＡキットで測定し、当該測定液の４５０－６５０ｎｍの吸光度を測定した結果を示した図である。It is the figure which showed the result of having measured each fraction of the gel filtration in Example 4 with the preceptin ELISA kit, and measuring the 450-650 nm light absorbency of the said measurement liquid. 実施例４におけるゲル濾過の各フラクションをＣＤ１４ＥＬＡＩＳＡキットで測定し、当該測定液の４５０－６５０ｎｍの吸光度を測定した結果を示した図である。It is the figure which showed the result of having measured each fraction of gel filtration in Example 4 with the CD14ELAISA kit, and measuring the 450-650 nm light absorbency of the said measurement liquid.
　いくつかの態様は、検体中のプレセプシン測定の際、使用する検体に激しい撹拌などの物理的刺激を与えても、検体中のプレセプシンの測定値がほとんど増加することなく、検体中の本来のプレセプシン値が得られる、検体中のプレセプシン測定に有用な方法に関する。 In some embodiments, when measuring presepsin in a sample, even if a physical stimulus such as vigorous agitation is applied to the sample to be used, the measured value of presepsin in the sample is hardly increased, and the original preceptin in the sample is not increased. The present invention relates to a method useful for measuring presepsin in a specimen from which a value is obtained.
　また、別のいくつかの態様は、物理的刺激により生成されるプレセプシン様に反応する生成物（偽プレセプシン）を除去する方法に関する。さらに別のいくつかの態様は、プレセプシン測定キットの使用に当り、激しい撹拌などの物理的刺激を検体に与えても、検体中の見かけのプレセプシン測定値が増加しない、検体の扱い方によらず、安定したプレセプシン測定を可能とする方法に関する。 Further, some other embodiments relate to a method for removing a presepsin-like reaction product (pseudopreceptin) generated by physical stimulation. In still another embodiment, when using a presepsin measurement kit, even if a physical stimulus such as vigorous stirring is given to the sample, the apparent preceptin measurement value in the sample does not increase, regardless of how the sample is handled. The present invention relates to a method that enables stable preceptin measurement.
　ここでは、以下の態様［１］～［１４］が提供される。
［１］　第１の態様は、検体採取直後に検体と抗ＣＤ１４抗体とを接触させる工程を含む、検体中のプレセプシン測定値を安定化させる方法である。
［２］　第２の態様は、検体採取直後に検体と抗ＣＤ１４抗体とを接触させる工程を含む、検体中の偽プレセプシン産生を抑制させる方法である。
［３］　第３の態様は、検体採取直後に検体と抗ＣＤ１４抗体とを接触させる工程を含む、検体中のｓＣＤ１４の変性抑制方法である。
［４］　第４の態様は、検体と抗ＣＤ１４抗体とを接触させる工程により、検体中のｓＣＤ１４を吸着除去することを含む、前記態様［１］ないし［３］のいずれか１態様に記載の方法である。
［５］　第５の態様は、検体と抗ＣＤ１４抗体とを接触させる工程により、検体中のｓＣＤ１４と抗体ＣＤ１４抗体とが結合物をつくることを特徴とする、前記態様［１］ないし［３］のいずれか１態様に記載の方法である。
［６］　第６の態様は、プレセプシン測定前に、検体と抗ＣＤ１４抗体とを接触させる工程を含む、検体中の真のプレセプシン量の定量方法である。
［７］　第７の態様は、検体と抗ＣＤ１４抗体とを接触させる工程により、検体中の偽プレセプシンと反応する抗ＣＤ１４抗体を用いて偽プレセプシンを吸着除去することを特徴とする、前記態様［６］に記載の方法である。
［８］　第８の態様は、プレセプシン測定前に、偽プレセプシンと真のプレセプシンとを分別する工程を含む、真のプレセプシンのみを定量する方法である。
［９］　第９の態様は、偽プレセプシンと真のプレセプシンとの分別が、クロマトグラフィーによる分離であることを特徴とする、前記態様［８］に記載の方法である。
［１０］　第１０の態様は、偽プレセプシンと真のプレセプシンとの分別が、膜による分離であることを特徴とする、前記態様［８］に記載の方法である。
［１１］　第１１の態様は、プレセプシン測定前に、偽プレセプシンと真のプレセプシンとを分別する工程を含む、偽プレセプシンのみを定量する方法である。
［１２］　第１２の態様は、検体のプレセプシンを測定してプレセプシン値を算出し、算出したプレセプシン値から前記態様［１１］に記載の方法で得た偽プレセプシン値を減じることを含む、真のプレセプシン値を定量する方法である。
［１３］　第１３の態様は、前記態様［１］～［１２］のいずれか１態様に記載の方法に用いるための、抗ＣＤ１４抗体、クロマトグラフィー用カラム、および膜からなる群から選択される少なくとも１つを含む、プレセプシン測定キットである。
［１４］　第１４の態様は、前記態様［１］～［１２］のいずれか１態様に記載の方法に用いるための、検体カートリッジ、検体処理部、試薬カートリッジ部、プレセプシン測定部、計算部、及び表示部からなる群から選択される少なくとも１つを含む、プレセプシン測定システムである。 Here, the following modes [1] to [14] are provided.
[1] A first aspect is a method for stabilizing a preceptin measurement value in a specimen, including a step of bringing the specimen into contact with an anti-CD14 antibody immediately after the specimen is collected.
[2] The second aspect is a method for suppressing pseudo-preceptin production in a specimen, including a step of bringing the specimen into contact with an anti-CD14 antibody immediately after the specimen is collected.
[3] A third aspect is a method for inhibiting denaturation of sCD14 in a specimen, comprising a step of bringing the specimen into contact with an anti-CD14 antibody immediately after the specimen is collected.
[4] A fourth aspect according to any one of the aspects [1] to [3], wherein the sCD14 in the specimen is adsorbed and removed by the step of contacting the specimen with the anti-CD14 antibody. Is the method.
[5] In the fifth aspect, the sCD14 in the specimen and the antibody CD14 antibody form a conjugate by the step of bringing the specimen into contact with the anti-CD14 antibody. It is the method as described in any one aspect of these.
[6] The sixth aspect is a method for quantifying the amount of true preceptin in a sample, which comprises the step of bringing the sample into contact with an anti-CD14 antibody before measuring presepsin.
[7] The seventh aspect is characterized in that the pseudopreceptin is adsorbed and removed by using the anti-CD14 antibody that reacts with the pseudopreceptin in the specimen in the step of bringing the specimen into contact with the anti-CD14 antibody. 6].
[8] The eighth aspect is a method for quantifying only true preceptin, comprising a step of separating pseudo-preceptin from true pre-ceptin prior to measuring pre-ceptin.
[9] A ninth aspect is the method according to the aspect [8], wherein the fractionation of the pseudo preceptin and the true preceptin is a chromatographic separation.
[10] A tenth aspect is the method according to the aspect [8], wherein the separation of the pseudo-preceptin and the true preceptin is separation by a membrane.
[11] The eleventh aspect is a method for quantifying only pseudo-preceptin, including a step of separating pseudo-preceptin from true pre-ceptin before measuring pre-sepsin.
[12] A twelfth aspect includes a true preceptin value obtained by measuring preceptin of a specimen, and subtracting the pseudo-preceptin value obtained by the method according to the aspect [11] from the calculated preceptin value. This is a method for quantifying preceptin levels.
[13] The thirteenth aspect is selected from the group consisting of an anti-CD14 antibody, a chromatography column, and a membrane for use in the method according to any one of the aspects [1] to [12]. It is a presepsin measurement kit containing at least one.
[14] In a fourteenth aspect, a specimen cartridge, a specimen processing section, a reagent cartridge section, a preceptin measurement section, a calculation section, for use in the method according to any one of the above aspects [1] to [12] And a preceptin measurement system comprising at least one selected from the group consisting of display units.
　本明細書における用語等について、以下に説明する。
　「検体採取直後」とは、患者から検体を採取した直後（例えば、採血した直後）、もしくは検体を採取するのと同時（例えば、採血と同時）にとの意味である。ここで、検体採取直後には、「輸送前」、すなわち、患者から検体を採取し（例えば、採血し）、種々の検体パラメーター測定（例えば血液パラメーター測定）の為、専門のセンターへ送る前も含まれる。とりわけ、検体を採取した直後（例えば、採血直後）から検査センターに送る迄の間を意味する。
　「プレセプシン測定前」とは、プレセプシン値測定を始める前との意味で、例えば、検体採取し、その場で測定をはじめる前、あるいは検査センターに到着した検体のプレセプシン値の測定前等が該当する。 Terms and the like in this specification will be described below.
The term “immediately after sample collection” means immediately after collecting a sample from a patient (for example, immediately after collecting blood) or simultaneously with collecting a sample (for example, simultaneously with collecting blood). Here, immediately after sample collection, “before transport”, that is, before collecting a sample from a patient (eg, collecting blood) and sending it to a specialized center for measurement of various sample parameters (eg, blood parameter measurement). included. In particular, it means a period from immediately after collection of a specimen (for example, immediately after blood collection) until it is sent to an examination center.
“Before measuring presepsin” means before starting measurement of the presepsin value, for example, before collecting a sample and starting measurement on the spot, or before measuring the preceptin value of a sample arriving at a test center .
　「検体と抗ＣＤ１４抗体とを接触させる工程」とは、例えば、
　　１）検体に抗ＣＤ１４抗体を添加する工程、
　　２）採血管等の容器中に抗ＣＤ１４抗体を予め存在させておき（例えば、採血管等の容器中にいれたビーズ等に抗ＣＤ１４抗体を固定化しておく等）、当該容器に採取した検体を収容する工程、
　　３）抗ＣＤ１４を予め固定化した専用採血管等の専用容器を用い、当該容器に採取した検体を収容する工程、
　　４）検体中のｓＣＤ１４を抗ＣＤ１４抗体を用いて吸着除去する工程（より具体的には、例えば、樹脂等に抗ＣＤ１４抗体を結合させた担体を充填したアフィニティークロマトグラフィー用カラムを用いて、検体がカラムを通過するようにさせる工程）、
　等からなる群から選択されるいずれかの工程である。これらの工程を経ることで、検体中のｓＣＤ１４が物理的刺激による耐性を得る、もしくはｓＣＤ１４が除去されることから、検体中のプレセプシン測定値の安定化、検体中の偽プレセプシン産生抑制、またはｓＣＤ１４の変性抑制をすることが可能となる。 The “step of contacting the specimen with the anti-CD14 antibody” means, for example,
1) adding an anti-CD14 antibody to the specimen,
2) An anti-CD14 antibody is previously present in a container such as a blood collection tube (for example, the anti-CD14 antibody is immobilized on beads or the like placed in the container such as a blood collection tube), and the sample collected in the container Containing the process,
3) using a dedicated container such as a dedicated blood collection tube in which the anti-CD 14 is immobilized in advance, and storing the sample collected in the container;
4) A step of adsorbing and removing sCD14 in a specimen using an anti-CD14 antibody (more specifically, for example, using an affinity chromatography column packed with a carrier in which an anti-CD14 antibody is bound to a resin or the like, Process to pass through the column),
Any process selected from the group consisting of, etc. Through these steps, sCD14 in the specimen gains tolerance due to physical stimulation, or sCD14 is removed, so that the presepsin measurement value in the specimen is stabilized, pseudopreceptin production in the specimen is suppressed, or sCD14 It becomes possible to suppress the denaturation of.
　検体に抗ＣＤ１４抗体を添加することとしては、例えば、患者から採取した検体に抗ＣＤ１４抗体を加えることが挙げられる。
　また、輸送前に検体に抗ＣＤ１４抗体を共存させることには、検体に抗ＣＤ１４抗体を添加することで検体に抗ＣＤ１４抗体を共存させることや、予め採血管に抗ＣＤ１４抗体を存在させ、当該採血管に血液を入れることで検体に抗ＣＤ１４抗体を共存させること、などが含まれる。 Examples of adding an anti-CD14 antibody to a specimen include adding an anti-CD14 antibody to a specimen collected from a patient.
Further, in order to allow the anti-CD14 antibody to coexist in the specimen before transportation, the anti-CD14 antibody can coexist in the specimen by adding the anti-CD14 antibody to the specimen, or the anti-CD14 antibody is present in the blood collection tube in advance. Examples include coexistence of an anti-CD14 antibody in a specimen by putting blood into a blood collection tube.
　「偽プレセプシン」とは、ｓＣＤ１４に由来し、激しい撹拌等により、ｓＣＤ１４が凝集、立体構造のアンフォールディング等が起こり、プレセプシン様の反応を示してしまう生成物等を意味する。従って、偽プレセプシンのプレセプシン様の反応により、プレセプシン測定値が見かけ上増加することになる。
　「偽プレセプシンの生成を阻止」又は「偽プレセプシンの産生抑制」とは、ｓＣＤ１４に由来する偽プレセプシンの生成もしくは産生を阻害することを意味する。
　「ｓＣＤ１４の変性」とは、激しい撹拌等により、検体中のｓＣＤ１４が、例えば、２分子で凝集し、より高分子量になること等を意味する。当該凝集によりプレセプシン測定に反応する部位が分子表面にでてきてしまい、プレセプシンを測定する抗体に反応してしまう状態となる。
　「ｓＣＤ１４の変性抑制」とは、ｓＣＤ１４由来の偽プレセプシンがプレセプシン測定に反応してしまうことを阻害することを意味する。
　「物理的刺激」とは、検体を扱う際の、激しい攪拌（緩やかな長時間攪拌を含む）等を意味し、採決後の採血管の転倒撹拌、もしくは振とうをも意味する。
　「真のプレセプシン値」とは、検体中に存在するプレセプシン量を示す正しい値を意味し、例えば、これは、検体に物理的刺激を与える前の検体中のプレセプシンの測定値である。ｓＣＤ１４が凝集し、プレセプシン測定に反応してしまうことで見かけ上のプレセプシン値が増加してしまう現象がある。例えば、ｓＣＤ１４が凝集しないようにすることで検体中に存在するプレセプシン値そのものを知ることができ、振とうなどの影響があっても安定して正しいプレセプシン値が得られる。但し、検体に物理的刺激を与える前の検体中のプレセプシンの測定値と完全に一致することまでを意味するものではない。 The “pseudopreceptin” means a product or the like that is derived from sCD14 and causes a presepsin-like reaction by aggregation of sCD14, unfolding of a three-dimensional structure, and the like due to vigorous stirring and the like. Thus, presepsin-like reactions of pseudopresepsin will increase the presepsin measurement.
“Preventing the production of pseudopreceptin” or “suppressing the production of pseudopreceptin” means inhibiting the production or production of pseudopreceptin derived from sCD14.
The “denaturation of sCD14” means that, for example, sCD14 in a specimen aggregates with two molecules to become a higher molecular weight due to vigorous stirring or the like. Due to the aggregation, a site that reacts with preceptin measurement appears on the surface of the molecule, resulting in a state of reacting with an antibody that measures preceptin.
“SCD14 denaturation suppression” means that sCD14-derived pseudopreceptin is inhibited from reacting to preceptin measurement.
“Physical stimulation” means vigorous agitation (including gentle agitation for a long time) when handling a specimen, and also means falling agitation of the blood collection tube after shaking or shaking.
“True preceptin value” means a correct value indicating the amount of preceptin present in the specimen, for example, this is a measured value of preceptin in the specimen prior to physical stimulation of the specimen. There is a phenomenon in which the apparent preceptin value increases due to aggregation of sCD14 and reaction to preceptin measurement. For example, by preventing sCD14 from aggregating, it is possible to know the preceptin value itself present in the specimen, and a stable and correct preceptin value can be obtained even if there is an influence such as shaking. However, this does not mean that the measured value of presepsin in the sample before giving a physical stimulus to the sample completely coincides.
　「プレセプシン測定値の安定化」とは、検体が物理的刺激を受けても真の値のプレセプシン値を示すようにすることを意味する。すなわち、例えば、ｓＣＤ１４が凝集することを抑制することで物理的刺激を受けても真のプレセプシン値が得られるようになる。
　「物理的刺激による不安定化に抵抗」とは、物理的刺激によりｓＣＤ１４が凝集、立体構造のアンフォールディングする不安定化を抑制することで、プレセプシン測定においてプレセプシン様の反応を示し、真のプレセプシン値に加えて、見かけ上のプレセプシン値の増加を阻止することを意味する。 “Stabilization of the preceptin measurement value” means that the specimen exhibits a true preceptin value even when subjected to physical stimulation. That is, for example, by suppressing the aggregation of sCD14, a true preceptin value can be obtained even when subjected to physical stimulation.
“Resistant to destabilization by physical stimulation” means that sCD14 aggregates and destabilizes by unfolding the three-dimensional structure by physical stimulation, and shows a preceptin-like reaction in preceptin measurement. In addition to the value, it means to prevent an increase in the apparent preceptin value.
　「真のプレセプシン」とは、後述する「プレセプシン」、すなわちｓＣＤ１４－ｓｔ（可溶性ＣＤ１４抗原サブタイプ）であり、通常のプレセプシン測定キットで定量される物質である。
　「偽プレセプシンと真のプレセプシンとを分別」とは、以下の方法、
　　１）クロマトグラフィーによる分離（例えば、ゲル濾過クロマトグラフィーによる分離（例：実施例４））、
　　２）膜による分離、
　　３）偽プレセプシンに反応する抗体による分離、
等が挙げられる。 The “true preceptin” is “preceptin” described later, that is, sCD14-st (soluble CD14 antigen subtype), and is a substance quantified by a normal preceptin measurement kit.
“Separation of pseudopreceptin and true preceptin” means the following method:
1) separation by chromatography (for example, separation by gel filtration chromatography (eg, Example 4)),
2) separation by membrane,
3) Separation by an antibody that reacts with pseudopreceptin,
Etc.
　プレセプシン測定前の検体中を、クロマトグラフィー（例えば、ゲル濾過クロマトグラフィー）で分離し、真のプレセプシンを含む分画中を得て、プレセプシン値を測定することで、真のプレセプシン値を定量することが可能である。定量にあたっては、後述するプレセプシン測定方法を用いるが、例えば、実施例４で言えば、標準濃度のプレセプシンによって作成した検量線と各プレセプシンを含む分画のプレセプシン量を比較することで、真のプレセプシン値を得ることができる。また、当該分離と定量を行うシステムを構築することで、検体を直接アプライするだけで真のプレセプシン値を得るシステムを構築することも可能である。 Quantifying the true preceptin value by separating the sample before preceptin measurement by chromatography (eg gel filtration chromatography), obtaining the fraction containing true preceptin, and measuring the preceptin value Is possible. For the quantification, the presepsin measurement method described later is used. For example, in the case of Example 4, a true preceptin is obtained by comparing a standard curve prepared with a standard concentration of preceptin and the amount of preceptin in the fraction containing each preceptin. A value can be obtained. In addition, by constructing a system for performing the separation and quantification, it is possible to construct a system that obtains a true preceptin value simply by directly applying a specimen.
　膜によって検体中の偽プレセプシンを除去するには、例えば、検体を、プレセプシンより高分子量の物質を除ける膜（例えば、３０キロダルトンの限外濾過膜）を通過させてもよい。当該膜によって偽プレセプシンを除去され、偽プレセプシンが除去された検体のプレセプシン値を測定することで真のプレセプシン値を得ることができる。 In order to remove the pseudo-presepsin in the specimen by the membrane, for example, the specimen may be passed through a membrane (for example, a 30-kilodalton ultrafiltration membrane) that removes a substance having a higher molecular weight than preceptin. The true preceptin value can be obtained by measuring the preceptin value of the specimen from which the pseudopreceptin has been removed by the membrane and the pseudopreceptin has been removed.
　また、検体からｓＣＤ１４由来の偽プレセプシンを分離するには、ｓＣＤ１４には反応するが、真のプレセプシンには反応せず、偽プレセプシンに反応する抗体と検体を接触させ、偽プレセプシンを除去することが含まれる。偽プレセプシンを抗体で除去するには、検体と抗体とを接触させ、例えば、当該抗体を固定化したカラムを検体が通過するようにさせ、当該抗体にｓＣＤ１４由来の偽プレセプシンを吸着除去することが含まれる。
　「真のプレセプシン値のみを定量する」とは、プレセプシン測定前の検体中を、例えば、ゲル濾過クロマトグラフィーで分離し、真のプレセプシンを含む分画中のプレセプシン値を定量することを意味する。より具体的には、後述する実施例４に従い、分離した該当分画を定量することである。
　また、クロマトグラフィーによって分離した「偽プレセプシン」を含む分画でプレセプシン測定を行うことによって、「偽プレセプシン値」を得ることができる。偽プレセプシン値が得られれば、検体のプレプシン値を測定し、真のプレセプシン値と偽プレセプシン値の合計値（総プレセプシン量）から偽プレセプシンの測定値を得ることが可能である。
　「偽プレセプシンのみを定量する」とは、プレセプシン測定前の検体中を、例えば、ゲル濾過クロマトグラフィーで分離し、偽プレセプシンを含む分画中の偽プレセプシン値を定量することを意味する。より具体的には、後述する実施例４に従い、分離した該当分画を定量することである。
　「プレセプシン測定システム」とは、プレセプシン測定を実施するための各要素をシステム中に組み込んだ全自動装置等の装置を意味する。 Further, in order to separate sCD14-derived pseudopreceptin from the specimen, it reacts with sCD14 but does not react with true preceptin, and the pseudoreceptin is contacted with an antibody that reacts with pseudopreceptin to remove the pseudopreceptin. included. In order to remove the pseudopreceptin with an antibody, the specimen and the antibody are brought into contact with each other, for example, the specimen is allowed to pass through a column on which the antibody is immobilized, and the pseudopreceptin derived from sCD14 is adsorbed and removed by the antibody. included.
“Quantifying only the true preceptin value” means separating the sample before preceptin measurement by, for example, gel filtration chromatography and quantifying the preceptin value in the fraction containing true preceptin. More specifically, according to Example 4 described later, the separated relevant fraction is quantified.
Moreover, a “pseudo preceptin value” can be obtained by performing preceptin measurement on a fraction containing “pseudo preceptin” separated by chromatography. If a pseudo preceptin value is obtained, the prepsin value of the specimen can be measured, and a measured value of the pseudo preceptin can be obtained from the total value (total preceptin amount) of the true preceptin value and the pseudo preceptin value.
“Quantifying only presepsin” means that the sample before presepsin measurement is separated by, for example, gel filtration chromatography and the presepsin value in the fraction containing pseudopresepsin is quantified. More specifically, according to Example 4 described later, the separated relevant fraction is quantified.
The “presepsin measurement system” means a device such as a fully automatic device in which elements for performing the preceptin measurement are incorporated in the system.
　以下、本発明について詳細に説明する。以下の実施の形態は、本発明を説明するための例示であり、本発明をこの実施の形態のみに限定する趣旨ではない。本発明は、その要旨を逸脱しない限り、様々な形態で実施をすることができる。 Hereinafter, the present invention will be described in detail. The following embodiment is an example for explaining the present invention, and is not intended to limit the present invention to this embodiment alone. The present invention can be implemented in various forms without departing from the gist thereof.
１．抗ＣＤ１４抗体、膜およびクロマトグラフィー用カラム
１．１．抗ＣＤ１４抗体
　いくつかの態様では、抗ＣＤ１４抗体を用いる。抗ＣＤ１４抗体とは、ＣＤ１４、好ましくはヒトＣＤ１４に結合する抗体全般を含むものである。好ましくは、抗ＣＤ１４抗体は、Ｆ１０２４－１－３抗体、３Ｃ１０抗体、及びＭＥＭ－１８抗体から選択される少なくとも１つの抗体であり、より好ましくは、Ｆ１０２４－１－３抗体、３Ｃ１０抗体、又はＭＥＭ－１８抗体である。 1. Anti-CD14 antibody, membrane and chromatography column 1.1. Anti-CD14 antibodies In some embodiments, anti-CD14 antibodies are used. Anti-CD14 antibodies include all antibodies that bind to CD14, preferably human CD14. Preferably, the anti-CD14 antibody is at least one antibody selected from F1024-1-3 antibody, 3C10 antibody, and MEM-18 antibody, and more preferably F1024-1-3 antibody, 3C10 antibody, or MEM -18 antibody.
　“Ｆ１０２４－１－３抗体”とは、ＷＯ２００１／７２９９３　Ａ１に記載の通り、ラットにヒト血清中より精製したＣＤ１４蛋白質を抗原として免疫した免疫細胞とミエローマ細胞を細胞融合して取得したハイブリドーマＦ１０２４－１－３が産生するＦ１０２４－１－３抗体である。より具体的には、ＷＯ２００１／７２９９３ Ａ１の実施例１「抗ヒトＣＤ１４抗体の作製」の公知の方法に準じ、当該公報記載の方法で取得することができる。 “F1024-1-3 antibody” is a hybridoma F1024- obtained by cell fusion of immune cells and myeloma cells immunized with CD14 protein purified from human serum as an antigen, as described in WO2001 / 72993 A1. F1024-1-3 antibody produced by 1-3. More specifically, it can be obtained by the method described in the publication according to a known method of Example 1 “Preparation of anti-human CD14 antibody” in WO2001 / 72993A1.
　Ｆ１０２４－１－３抗体は、配列番号３に記載のヒトｓＣＤ１４の２８５番から３１５番までの領域の８個以上のアミノ酸を含むエピトープを特異的に認識する抗ＣＤ１４抗体である。 The F1024-1-3 antibody is an anti-CD14 antibody that specifically recognizes an epitope containing 8 or more amino acids in the region from 285 to 315 of human sCD14 described in SEQ ID NO: 3.
　また、ＬＰＳのヒトＣＤ１４を介するシグナル伝達を制御するヒト抗ＣＤ１４抗体として、ヒトＣＤ１４の７～１４番目に結合する３Ｃ１０抗体（Ｓｔｅｉｎｍａｎ：Ｊ．Ｅｘｐ．Ｍｅｄ．，１５８：１２６（１９８３）およびＪｕａｎ　ＴＳ：Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．，２７０：２９，１７２３７（１９９５））および５７～６４番目に結合するＭＥＭ－１８抗体（Ｂａｚｉｌ：Ｅｕｒ．Ｊ．Ｉｍｍｕｎｏｌ．，１６：１５８３（１９８６）およびＪｕａｎ　ＴＳ：Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．，２７０，１０，５２１９（１９９５））が知られている。
　３Ｃ１０抗体、ＭＥＭ－１８抗体は、例えば、ａｂｃｏｍ社より入手可能である。 Further, as a human anti-CD14 antibody that controls signal transduction of LPS via human CD14, a 3C10 antibody (Steinman: J. Exp. Med., 158: 126 (1983) and Juan TS, which binds to positions 7 to 14 of human CD14). : J. Biol. Chem., 270: 29, 17237 (1995)) and the 57-64th binding MEM-18 antibody (Bazil: Eur. J. Immunol., 16: 1583 (1986) and Juan TS: J Biol.Chem., 270, 10, 5219 (1995)).
The 3C10 antibody and the MEM-18 antibody are available from, for example, abcom.
　「抗体」の語は、特に断りのない限り、「抗体又はその抗原結合性断片」の意味で用いられる。「抗原結合性断片」は、抗体の部分断片の中で、元の抗体と実質的に同じ抗原結合性を有する断片のことをいう。抗原結合性断片は、例えば、Ｆａｂ、Ｆａｂ’、Ｆ（ａｂ’）２などが挙げられる。 The term “antibody” is used in the meaning of “antibody or antigen-binding fragment thereof” unless otherwise specified. An “antigen-binding fragment” refers to a fragment having substantially the same antigen-binding property as the original antibody among partial fragments of an antibody. Examples of the antigen-binding fragment include Fab, Fab ′, F (ab ′) 2 and the like.
１．２．膜
　いくつかの態様では、偽プレセプシンを除去することのできる膜を用いる。好ましくは、膜は、プレセプシンより高分子量の物質を除くことができる膜（例えば、３０キロダルトンの膜）である。そのような膜としては、例えば、限外ろ過フィルター（例えば、Ａｍｉｃｏｎ（商品名）　Ｕｌｔｒａ（Ｍｅｒｃｋ　Ｍｉｌｌｉｐｏｒｅ）等）が挙げられる。 1.2. Membranes In some embodiments, membranes that can remove pseudopresepsin are used. Preferably, the membrane is a membrane that can remove a higher molecular weight material than presepsin (eg, a 30 kilodalton membrane). Examples of such a membrane include an ultrafiltration filter (for example, Amicon (trade name) Ultra (Merck Millipore)).
１．３．クロマトグラフィー用カラム
　いくつかの態様では、検体中の真のプレセプシンと偽のプレセプシンを分離するのに、クロマトグラフィー用カラム（例えば、ゲルろ過クロマトグラフィー用カラム）を用いる。そのようなクロマトグラフィー用カラムとしては、例えば、Ｓｕｐｅｒｄｅｘ（商品名）、Ｓｅｐｈａｃｒｙｌ（商品名）、Ｓｕｐｅｒｏｓｅ（商品名）、およびＳｅｐｈａｄｅｘ（商品名）などの種類が挙げられる。 1.3. Chromatography Column In some embodiments, a chromatography column (eg, a gel filtration chromatography column) is used to separate true presepsin and false presepsin in a sample. Examples of such chromatography columns include Superdex (trade name), Sephacryl (trade name), Superose (trade name), and Sephadex (trade name).
２．プレセプシン
　「プレセプシン」は、ｓＣＤ１４－ＳＴ（可溶性ＣＤ１４抗原サブタイプ）ともいう。ＣＤ１４には、膜結合型ＣＤ１４（ｍＣＤ１４）のほかに可溶型ＣＤ１４（ｓＣＤ１４）があり、血中には分子量の異なる複数の可溶型ＣＤ１４が存在する。プレセプシンは、ＣＤ１４の可溶型の断片であり、以下の１）～３）の性質を有する物質をいう。 2. Presepsin “Presepsin” is also referred to as sCD14-ST (soluble CD14 antigen subtype). CD14 includes soluble CD14 (sCD14) in addition to membrane-bound CD14 (mCD14), and a plurality of soluble CD14 having different molecular weights exist in blood. Preceptin is a soluble fragment of CD14 and refers to a substance having the following properties 1) to 3).
　　１）　非還元条件下ＳＤＳ－ＰＡＧＥでは、分子量１３±２ｋＤａである、
　　２）　Ｎ末端配列に配列番号３のアミノ酸配列（ヒト全長可溶型ＣＤ１４のアミノ酸配列）の１位～１１位のアミノ酸配列を有する、及び、
　　３）　配列番号２に記載の１６アミノ酸残基（配列番号３のアミノ酸配列の５３位から６８位のアミノ酸配列に相当する）からなるペプチド（Ｓ６８ペプチド）を抗原として作製した抗体に特異的に結合する。 1) In SDS-PAGE under non-reducing conditions, the molecular weight is 13 ± 2 kDa.
2) having an amino acid sequence at positions 1 to 11 of the amino acid sequence of SEQ ID NO: 3 (amino acid sequence of human full-length soluble CD14) at the N-terminal sequence; and
3) Specific binding to an antibody prepared using a peptide (S68 peptide) consisting of 16 amino acid residues described in SEQ ID NO: 2 (corresponding to the amino acid sequence of positions 53 to 68 of the amino acid sequence of SEQ ID NO: 3) as an antigen To do.
　プレセプシンとは、特に説明しない限り、ヒトプレセプシンである。プレセプシンは、例えば、プレセプシン標準品（ＷＯ２００５／１０８４２９の実施例１６に記載のｒｓＣＤ１４－ＳＴ）である。あるいは、プレセプシンとしての結合活性を有する、プレセプシンの一部を改変した物質が用いられてもよい。 Preceptin is human preceptin unless otherwise specified. The presepsin is, for example, a presepsin standard product (rsCD14-ST described in Example 16 of WO2005 / 108429). Alternatively, a substance obtained by modifying a part of preceptin having binding activity as preceptin may be used.
３．ｓＣＤ１４
　ｓＣＤ１４として、分子量約５５ｋＤａおよび約４９ｋＤａのｓＣＤ１４が知られている。ｓＣＤ１４を、本明細書においては「高分子量可溶型ＣＤ１４」または「高分子量ｓＣＤ１４」ともいう。
　ｓＣＤ１４は、例えば、正常人の体液の３Ｃ１０抗体アフィニティーカラム吸着により作製してもよい（ＷＯ２００５／１０８４２９の実施例２３参照）。 3. sCD14
As sCD14, sCD14 having a molecular weight of about 55 kDa and about 49 kDa is known. sCD14 is also referred to herein as “high molecular weight soluble CD14” or “high molecular weight sCD14”.
sCD14 may be prepared, for example, by adsorbing a 3C10 antibody affinity column of body fluid of a normal person (see Example 23 of WO2005 / 108429).
４．プレセプシンの測定方法
　「プレセプシン測定」とは、例えば、プレセプシンを免疫学的に測定することである。プレセプシンを免疫学的に測定することには、例えば、抗プレセプシン抗体（例えば、Ｓ６８ポリクローナル抗体、Ｐ０３認識モノクローナル抗体、Ｐ０３特異的ポリクローナル抗体等の特定の抗体）（以下、「プレセプシン測定に使用する抗体」と記載する）とプレセプシンを含有する検体（例えば、血液検体）とを接触させる工程が含まれる。「測定」の語は、「検出」「定量」「アッセイ」等の語と相互に変換して用いることができ、定量的及び定性的な決定を含む意味で用いられる。プレセプシンの測定は、好ましくは、ｉｎ　ｖｉｔｒｏで行う。 4). Method for Measuring Preceptin “Preceptin measurement” is, for example, immunological measurement of preceptin. For immunological measurement of preceptin, for example, an anti-preceptin antibody (for example, a specific antibody such as S68 polyclonal antibody, P03-recognizing monoclonal antibody, P03-specific polyclonal antibody) (hereinafter referred to as “antibody used for preceptin measurement”). And a specimen containing presepsin (for example, a blood specimen). The term “measurement” can be used interchangeably with the terms “detection”, “quantification”, “assay”, and the like, and is used in a sense including quantitative and qualitative determination. The measurement of presepsin is preferably performed in vitro.
　検体中のプレセプシン測定値を安定化させる方法、検体中のｓＣＤ１４由来の偽プレセプシン産生を抑制させる方法、検体中のｓＣＤ１４の変性抑制方法、検体中の真のプレセプシン量の定量方法、真のプレセプシンのみを定量する方法、偽のプレセプシンのみを定量する方法などは、プレセプシン測定において用いることができる。 Method for stabilizing presepsin measurement value in sample, method for suppressing production of pseudopreceptin derived from sCD14 in sample, method for suppressing denaturation of sCD14 in sample, method for quantifying true preceptin amount in sample, true preceptin only The method of quantifying the amount, the method of quantifying only the fake preceptin, etc. can be used in the measurement of preceptin.
　検体中のプレセプシン測定の際、使用する検体に物理的刺激を与えても、プレセプシンの測定値がほとんど増加することなく、検体中の本来のプレセプシン測定値を得ることができる。例えば、ｓＣＤ１４由来の生成物は、検体と抗ＣＤ１４抗体とを接触させること、或いはクロマトグラフィーを用いてプレセプシンと分離できること等によって、その生成物による影響を排除でき、検体中のプレセプシン測定における物理的刺激によるプレセプシン測定値の増加を効果的に防ぐことが可能で、検体中の本来のプレセプシン測定値を得ることができる
　また、膜を用いることなどで、検体中の偽プレセプシンを除去し、プレセプシン測定における見かけ上のプレセプシン測定値が増加することを抑制できる。 When measuring presepsin in a sample, even if a physical stimulus is applied to the sample to be used, the presepsin measured value in the sample can be obtained with almost no increase in the measured value of presepsin. For example, the product derived from sCD14 can be removed from the effect of the product by bringing the sample into contact with the anti-CD14 antibody or separated from presepsin using chromatography. It is possible to effectively prevent an increase in the presepsin measurement value due to stimulation, and to obtain the original presepsin measurement value in the sample. Also, by using a membrane or the like, the pseudo-preceptin in the sample can be removed to measure the preceptin. It is possible to suppress an increase in the apparent preceptin measurement value at.
　「Ｓ６８抗体」とは、Ｓ６８ペプチド（配列番号２）を免疫原として用いて免疫した非ヒト哺乳動物から得られたポリクローナル抗体を、Ｓ６８ペプチドを固定化したカラムを用いて精製して得られる抗Ｓ６８ペプチドポリクローナル抗体である。「Ｓ６８ペプチド」は、配列番号２のアミノ酸配列（配列番号３のアミノ酸配列の５３位から６８位のアミノ酸配列）からなるペプチドである。具体的なＳ６８抗体の作製方法は、ＷＯ２００４／０４４００５の実施例１に記載の通りである。 “S68 antibody” refers to an anti-antibody obtained by purifying a polyclonal antibody obtained from a non-human mammal immunized with the S68 peptide (SEQ ID NO: 2) as an immunogen using a column to which the S68 peptide is immobilized. S68 peptide polyclonal antibody. “S68 peptide” is a peptide consisting of the amino acid sequence of SEQ ID NO: 2 (the amino acid sequence of positions 53 to 68 of the amino acid sequence of SEQ ID NO: 3). A specific method for producing the S68 antibody is as described in Example 1 of WO2004 / 044005.
　「Ｐ０３認識モノクローナル抗体」とは、プレセプシンにおける、配列番号１で表されるアミノ酸配列（ｋｒｖｄａｄａｄｐｒ：配列番号３（ヒト全長可溶型ＣＤ１４）の５２位～６１位に相当する領域：Ｐ０３配列ともいう）を認識する、モノクローナル抗体である。Ｐ０３認識モノクローナル抗体は、例えば、ＷＯ２０１５／１２９７７４に記載のモノクローナル抗体である。 “P03-recognizing monoclonal antibody” refers to a region corresponding to positions 52 to 61 of the amino acid sequence represented by SEQ ID NO: 1 (krvdadadpr: SEQ ID NO: 3 (human full-length soluble CD14)) in preceptin: P03 sequence Is a monoclonal antibody. The P03-recognizing monoclonal antibody is, for example, a monoclonal antibody described in WO2015 / 129774.
　「Ｐ０３特異的ポリクローナル抗体」とは、従来のウサギ由来抗プレセプシンポリクローナル抗体（Ｓ６８抗体）、すなわちＳ６８ペプチド（配列番号２）でウサギを免疫して得られたポリクローナル抗体を、Ｓ６８ペプチドを固定化したアフィニティーカラムで精製して作製される。Ｓ６８ペプチド固定化したカラムに替えて、Ｐ０３ペプチド（配列番号１）を固定化したアフィニティーカラムにより、ポリクローナル抗体を精製することにより、プレセプシンとの反応性の高い抗プレセプシンポリクローナル抗体が得られる。具体的なＰ０３特異的ポリクローナル抗体の作製方法は、ＷＯ２０１７／０３３２８１に記載の通りである。 “P03-specific polyclonal antibody” refers to a conventional rabbit-derived anti-preceptin polyclonal antibody (S68 antibody), that is, a polyclonal antibody obtained by immunizing a rabbit with the S68 peptide (SEQ ID NO: 2) and immobilizing the S68 peptide. It is produced by purifying with an affinity column. A polyclonal antibody is purified by an affinity column in which the P03 peptide (SEQ ID NO: 1) is immobilized in place of the S68 peptide-immobilized column, whereby an anti-preceptin polyclonal antibody having high reactivity with preceptin is obtained. A specific method for producing a P03-specific polyclonal antibody is as described in WO2017 / 033181.
　プレセプシンは敗血症の検出に用いられるマーカーとして知られているため、プレセプシンの測定方法は、プレセプシン測定に使用する抗体とプレセプシンを含有する検体を接触させる工程を含む、敗血症を検出するための方法に使用することができる。 Since presepsin is known as a marker used for detecting sepsis, the method for measuring presepsin is used in a method for detecting sepsis, which includes a step of contacting an antibody used for presepsin measurement with a specimen containing presepsin. can do.
　すなわち、プレセプシンの測定方法は、
　　（１）プレセプシン測定に使用する抗体を用いて、被験者の検体中のプレセプシン濃度を測定する工程、及び、
　　（２）上記（１）で得られたプレセプシン濃度がカットオフ値と比較して高値であるか否かを判定する工程を含む、
　敗血症を検出する方法ということもできる。 That is, the measurement method of preceptin is
(1) a step of measuring a preceptin concentration in a sample of a subject using an antibody used for preceptin measurement; and
(2) including a step of determining whether or not the preceptin concentration obtained in (1) is higher than a cutoff value.
It can also be called a method of detecting sepsis.
　検体が血液検体である場合のカットオフ値は、例えば、３１４～６００ｐｇ／ｍＬであり、好ましくは４００～５８０ｐｇ／ｍＬ、より好ましくは４５０～５５０ｐｇ／ｍＬ、さらに好ましくは、５００ｐｇ／ｍＬである。 The cut-off value when the sample is a blood sample is, for example, 314 to 600 pg / mL, preferably 400 to 580 pg / mL, more preferably 450 to 550 pg / mL, and still more preferably 500 pg / mL.
　「敗血症の検出」は、「敗血症の検出の補助」あるいは「敗血症の診断の補助」に読み替えて用いられてもよい。 “Detection of sepsis” may be read as “assisting detection of sepsis” or “assisting diagnosis of sepsis”.
　また、プレセプシンの測定方法は、例えば、敗血症と全身性炎症反応症候群（ｓｙｓｔｅｍｉｃ　ｉｎｆｌａｍｍａｔｏｒｙ　ｒｅｓｐｏｎｓｅ　ｓｙｎｄｒｏｍｅ、ＳＩＲＳ）との判別；敗血症の重症化のリスク評価；敗血症の予後予測（死亡率予測）；敗血症の重症度評価；術後感染症の検出；感染性血管内凝固症候群（ｄｉｓｓｅｍｉｎａｔｅｄ　ｉｎｔｒａｖａｓｃｕｌａｒ　ｃｏａｇｕｌａｔｉｏｎ、ＤＩＣ）の検出；感染性ＤＩＣの検出；心疾患の検出；細菌感染を伴う呼吸器感染症、炎症性腸疾患（クローン病、潰瘍性大腸炎）、発熱性好中球減少症（ｆｅｂｒｉｌｅ　ｎｅｕｔｒｏｐｅｎｉａ、ＦＮ）、又は血球貪食症候群（ｈｅｍｏｐｈａｇｏｃｙｔｉｃ　ｓｙｎｄｒｏｍｅ、ＨＰＳ）の検出から選ばれる少なくとも１つの疾患の検出又は評価のために使用することができる。すなわち、プレセプシンの測定方法は、前記疾患の検出又は評価方法ということもできる。 The method of measuring presepsin includes, for example, discrimination between sepsis and systemic inflammatory response syndrome (SIRS); risk assessment of severe sepsis; prognosis prediction of sepsis (prediction of mortality); severe sepsis Assessment; detection of postoperative infection; detection of infectious intravascular coagulation (DIC); detection of infectious DIC; detection of heart disease; respiratory infection with bacterial infection, inflammatory bowel disease (Crohn's disease, ulcerative colitis), febrile neutropenia (FN), or hemophagocytic syndrome (HPS) It can be used for detection or evaluation of the disease. That is, the method for measuring presepsin can also be referred to as a method for detecting or evaluating the disease.
　術後感染症は、術後に発症した感染症を総称し、手術およびそれに必要な補助療法による全ての感染症を意味する。また、術後感染症は、Ｇｕｉｄｅｌｉｎｅ　ｆｏｒ　ｐｒｅｖｅｎｔｉｏｎ　ｏｆ　ｓｕｒｇｉｃａｌ　ｓｉｔｅ　ｉｎｆｅｃｔｉｏｎ，１９９９　（ＣＤＣ）に基づき術後感染症と診断される疾患全てを含むものである。
　心疾患としては、例えば、急性冠症候群（ＡＣＳ）、急性心不全、急性非代償性心不全（ＡＤＨＦ）、慢性心不全、冠動脈疾患、狭心症、心筋梗塞、虚血性脳卒中、出血性脳卒中及び一過性脳虚血発作が挙げられる。 Postoperative infection is a general term for infections that develop after surgery, and refers to all infections caused by surgery and adjunct therapy required for it. Postoperative infections include all diseases diagnosed as postoperative infections based on Guideline for prevention of surgical site infection, 1999 (CDC).
Examples of heart diseases include acute coronary syndrome (ACS), acute heart failure, acute decompensated heart failure (ADHF), chronic heart failure, coronary artery disease, angina, myocardial infarction, ischemic stroke, hemorrhagic stroke and transient Examples include cerebral ischemic attacks.
　細菌感染を伴う呼吸器感染症としては、下気道感染症又は肺炎が挙げられる。下気道感染症には急性下気道感染症と慢性下気道感染症が含まれる。急性下気道感染症には急性気管炎、急性気管支炎、急性細気管支炎が含まれ、多くは上気道へのウイルス感染が下気道に波及することにより発症するが、一部で細菌による二次感染が続発する。細菌二次感染の兆候が見られた場合は抗生剤投与の適応となる。慢性下気道感染症は、気管支拡張症や慢性閉塞性肺疾患などで器質的障害を有する下気道に細菌の持続的な感染が成立した病態であり、持続感染と急性憎悪が存在する。下気道の器質的障害を発生させる疾患には、気管支拡張症、慢性閉塞性肺疾患、慢性気管支炎、びまん性汎細気管支炎、陳旧性肺結核、じん肺、非結核性抗酸菌症、アレルギー性気管支肺アスペルギルス症、肺線維症、慢性気管支喘息などが含まれる。持続感染、急性憎悪ともに抗生剤投与の適応となる。肺炎には市中肺炎、院内肺炎が含まれる。好ましくは市中肺炎である。 ∙ Respiratory tract infections with bacterial infections include lower respiratory tract infections or pneumonia. Lower respiratory tract infections include acute lower respiratory tract infections and chronic lower respiratory tract infections. Acute lower respiratory tract infections include acute tracheitis, acute bronchitis, and acute bronchiolitis, mostly caused by viral infection of the upper respiratory tract that spreads to the lower respiratory tract, but in some cases secondary to bacteria Infection continues. Antibiotics are indicated for signs of secondary bacterial infection. Chronic lower respiratory tract infection is a pathological condition in which persistent infection of bacteria is established in the lower respiratory tract having an organic disorder such as bronchiectasis or chronic obstructive pulmonary disease, and persistent infection and acute hatred exist. Diseases that cause lower airway organic disorders include bronchiectasis, chronic obstructive pulmonary disease, chronic bronchitis, diffuse panbronchiolitis, old pulmonary tuberculosis, pneumoconiosis, nontuberculous mycobacterial disease, allergy Bronchopulmonary aspergillosis, pulmonary fibrosis, chronic bronchial asthma and the like are included. Antibiotics are indicated for both persistent infection and acute exacerbation. Pneumonia includes community-acquired pneumonia and nosocomial pneumonia. Preferred is community-acquired pneumonia.
　プレセプシン測定に使用する抗体を用いて、プレセプシンを免疫学的に測定する方法としては、例えば、エンザイムイムノアッセイ（（以下、ＥＩＡ又はＥＬＩＳＡとも記す）、化学発光酵素免疫測定法（ＣＬＥＩＡ）、化学発光免疫測定法（ＣＬＩＡ），蛍光抗体法（ＦＡＴ）、蛍光酵素免疫測定法（ＦＥＩＡ）、電気化学発光免疫測定法（ＥＣＬＩＡ）、放射免疫測定法（ＲＩＡ）、イムノクロマト法、凝集法、競合法等が挙げられるが、これらに限定されない。直接法と間接法のいずれが用いられてもよい。ビオチン－アビジン（ストレプトアビジン）複合体を形成させて検出する増感法が用いられてもよい。 Examples of a method for immunologically measuring preceptin using an antibody used for preceptin measurement include enzyme immunoassay (hereinafter also referred to as EIA or ELISA), chemiluminescent enzyme immunoassay (CLEIA), chemiluminescent immunity, and the like. Measurement method (CLIA), fluorescent antibody method (FAT), fluorescent enzyme immunoassay method (FEIA), electrochemiluminescence immunoassay method (ECLIA), radioimmunoassay method (RIA), immunochromatography method, aggregation method, competitive method, etc. Although not limited to these, either a direct method or an indirect method may be used, and a sensitizing method in which a biotin-avidin (streptavidin) complex is formed and detected may be used.
　ＥＩＡは、酵素標識抗体を用いた免疫測定法の一つであり、直接法、間接法等が挙げられる。好ましい例としては、サンドイッチＥＬＩＳＡ（ｅｎｚｙｍｅ－ｌｉｎｋｅｄ　ｉｍｍｕｎｏｓｏｒｂｅｎｔ　ａｓｓａｙ）である。 EIA is one of immunoassays using enzyme-labeled antibodies, and includes direct methods and indirect methods. A preferable example is a sandwich ELISA (enzyme-linked immunosorbent assay).
　サンドイッチＥＬＩＳＡとは、抗原認識部位の異なる２種類以上の抗体を用いて、あらかじめ一方の抗体は固相に固定し、検出したい抗原を２種類の抗体で挟んで、抗体－抗原－抗体複合体を形成させることにより測定する方法である。 Sandwich ELISA uses two or more types of antibodies with different antigen recognition sites, one of which is immobilized on a solid phase in advance, and the antigen to be detected is sandwiched between the two types of antibodies. It is a method of measuring by forming.
　化学発光酵素免疫測定法（ＣＬＥＩＡ：Ｃｈｅｍｉｌｕｍｉｎｅｓｃｅｎｔ　Ｅｎｚｙｍｅ　Ｉｍｍｕｎｏａｓｓａｙ）は、検体中の抗原と、磁性粒子やビーズ等に固相した抗体を反応させた後、酵素標識抗体を反応させ、洗浄（Ｂ／Ｆ分離）後、化学発光基質を加えて酵素反応後、発光強度を測定する方法である。 The chemiluminescence enzyme immunoassay (CLEIA) is a method in which an antigen in a sample is reacted with an antibody immobilized on a magnetic particle or bead, followed by reaction with an enzyme-labeled antibody and washing (B / F separation). ) Thereafter, a chemiluminescent substrate is added, and after the enzyme reaction, the luminescence intensity is measured.
　例えば、検体中の抗原とビオチンを結合させた抗体を液相で反応させ、ストレプトアビジンを結合させた磁性粒子へ抗体をトラップし、洗浄（Ｂ／Ｆ分離）後、酵素標識抗体を反応させ、発光強度を測定する。 For example, an antigen-biotin-bound antibody in a specimen is reacted in a liquid phase, the antibody is trapped on magnetic particles bound with streptavidin, washed (B / F separation), reacted with an enzyme-labeled antibody, The luminescence intensity is measured.
　標識酵素としてアルカリホスファターゼ（ＡＬＰ）を用いるとき、化学発光基質は、ＣＤＰ－ＳｔａｒＴＭ，ＡＭＰＰＤＴＭ，ＣＳＰＤＴＭが用いられることが好ましい。標識酵素がＨＲＰのときは、化学発光基質は、ルミノールが用いられることが好ましい。 When alkaline phosphatase (ALP) is used as the labeling enzyme, the chemiluminescent substrate is preferably CDP-StarTM, AMPPDTM, or CSPDTM. When the labeling enzyme is HRP, luminol is preferably used as the chemiluminescent substrate.
　検出感度は、一般的に、化学発光＞蛍光＞吸光（呈色）の順に高いといわれ、求める感度に応じて測定法を選択しうる。 The detection sensitivity is generally said to be higher in the order of chemiluminescence> fluorescence> absorption (coloration), and the measurement method can be selected according to the required sensitivity.
　化学発光免疫測定法（ＣＬＩＡ：Ｃｈｅｍｉｌｕｍｉｎｅｓｃｅｎｔ　Ｉｍｍｕｎｏａｓｓａｙ）は、検体中の抗原と磁性粒子などに固相した抗体を反応させた後、化学発光物質で標識した抗体を反応させ、洗浄（Ｂ／Ｆ分離）後、発光強度を測定する方法である。標識物質は、アクリジニウム等が用いられる。 In chemiluminescence immunoassay (CLIA), an antigen in a sample is reacted with an antibody solid-phased on magnetic particles, followed by reacting an antibody labeled with a chemiluminescent substance and washing (B / F separation). Then, it is a method of measuring luminescence intensity. Acridinium or the like is used as the labeling substance.
　蛍光酵素免疫測定法（ＦＥＩＡ：Ｆｌｕｏｒｅｓｃｅｎｔ　Ｅｎｚｙｍｅ　Ｉｍｍｕｎｏａｓｓａｙ）は、検体中の抗原と固相化した抗体を反応させた後、酵素標識抗体を反応させ、洗浄（Ｂ／Ｆ分離）後、蛍光基質を加えて酵素反応後、蛍光強度を測定する方法である。標識酵素には、ＨＲＰやＡＬＰ等が用いられる。蛍光基質は、標識酵素がＨＲＰのときは、ＡｍｐｌｅｘＴＭＲｅｄ等が用いられ、標識酵素がＡＬＰのときは、４－ＭＵＰ（４－Ｍｅｔｈｙｌｕｍｂｅｌｌｉｐｈｅｎｙｌ　ｐｈｏｓｐｈａｔｅ）、ＡｔｔｏＰｈｏｓＴＭ等が用いられることが好ましい。 In the fluorescent enzyme immunoassay (FEIA: Fluorescent Enzyme Immunoassay), the antigen in the sample is reacted with the immobilized antibody, the enzyme-labeled antibody is reacted, washed (B / F separation), and then a fluorescent substrate is added. In this method, the fluorescence intensity is measured after the enzyme reaction. As a labeling enzyme, HRP, ALP, or the like is used. As the fluorescent substrate, AmplexTMRed or the like is used when the labeling enzyme is HRP, and 4-MUP (4-Methylumbelliferous phosphate), AttoPhosTM or the like is preferably used when the labeling enzyme is ALP.
　電気化学発光免疫測定法（ＥＣＬＩＡ：Ｅｌｅｃｔｒｏ　Ｃｈｅｍｉｌｕｍｉｎｅｓｃｅｎｃｅ　Ｉｍｍｕｎｏａｓｓａｙ）は、検体中の抗原と磁性粒子に固相した抗体及び電気化学発光物質で標識した抗体を反応させた後、洗浄（Ｂ／Ｆ分離）し、電気エネルギーによる発光強度を測定する方法である。標識物質にはルテニウム等が用いられる。標識物質には、Ｒｕ（ｂｐｙ）３等が用いられ、電極への荷電による酸化と、トリプロピルアミン（ＴＰＡ）等による還元反応により励起発光を繰り返す。 Electrochemiluminescence immunoassay (ECLIA: Electro Chemiluminescence Immunoassay) reacts an antigen in a sample with an antibody solid-phased on a magnetic particle and an antibody labeled with an electrochemiluminescent substance, followed by washing (B / F separation). This is a method for measuring the emission intensity by electric energy. Ruthenium or the like is used as the labeling substance. Ru (bpy) 3 or the like is used as the labeling substance, and excitation light emission is repeated by oxidation due to charging of the electrode and reduction reaction by tripropylamine (TPA) or the like.
　放射免疫測定法（（ＲＩＡ：Ｒａｄｉｏｉｍｍｕｎｏａｓｓａｙ）は、放射性同位元素による標識体を用いた測定方法である。例えば、検体中の抗原とビーズ等に固相した抗体を反応された後、放射性同位元素（１２５Ｉ等）で標識した抗体を反応させ、洗浄（Ｂ／Ｆ分離）後、１２５Ｉの放射線量を測定することができる。 Radioimmunoassay (RIA: Radioimmunoassay) is a measurement method using a label with a radioisotope. For example, after reacting an antigen in a specimen with an antibody immobilized on a bead or the like, a radioisotope ( 125 I or the like) is reacted, and after washing (B / F separation), the radiation dose of 125 I can be measured.
　イムノクロマト法は、被検体が、試験ストリップ上を試薬を溶解しながら移動する毛細管現象を応用した免疫測定法である。検体中の抗原が、試験ストリップ上の標識抗体及びキャプチャー抗体の２者と免疫複合体を形成し、標識物の色を確認する方法である。抗体の標識は、金コロイド、酵素、蛍光物質等が用いられる。酵素標識抗体を用いる場合は、試験ストリップ上に酵素基質を配置し発色させる。 Immunochromatography is an immunoassay method that applies capillary action in which a specimen moves while dissolving a reagent on a test strip. In this method, the antigen in the sample forms an immune complex with the labeled antibody and the capture antibody on the test strip, and the color of the label is confirmed. For labeling the antibody, colloidal gold, an enzyme, a fluorescent substance, or the like is used. If an enzyme-labeled antibody is used, an enzyme substrate is placed on the test strip and colored.
　フロースルー法は、不溶性担体であるメンブレン上で、検体中の溶液と共に被験物質である抗原が、抗体－抗原－抗体複合体を形成させる方法である。このとき、メンブレンに固定されなかった物質は、通常は垂直にメンブレンの表から裏を通って除去される。 The flow-through method is a method in which an antigen as a test substance forms an antibody-antigen-antibody complex together with a solution in a specimen on a membrane that is an insoluble carrier. At this time, the substances not fixed to the membrane are usually removed vertically from the front and back of the membrane.
　凝集法は、検体中の抗原と試薬中の抗体を反応させ、凝集を観察する方法である。固相を用いない方法、固相として人工的に作製された粒子を用いる粒子凝集法（ｐａｒｔｉｃｌｅ　ａｇｇｌｕｔｉｎａｔｉｏｎ：ＰＡ）、ＰＡの中でもラテックス粒子を用いたラテックス凝集法（ｌａｔｅｘ　ａｇｇｌｕｔｉｎａｔｉｏｎ：ＬＡ）等が挙げられる。
　競合法は、例えば、固相に抗体を結合させ、被検試料と一定量の標識抗原を同時に反応させ、結合した標識物の量から試料中の抗原の量を測定することができる。
　プレセプシン測定に使用する抗体は、上述の測定方法に好ましく用いられる。 The agglutination method is a method of observing agglutination by reacting an antigen in a specimen with an antibody in a reagent. Examples thereof include a method not using a solid phase, a particle agglutination (PA) using particles artificially prepared as a solid phase, and a latex agglutination (latex agglutination: LA) using latex particles among PAs. .
In the competition method, for example, an antibody is bound to a solid phase, a test sample and a certain amount of labeled antigen are reacted simultaneously, and the amount of antigen in the sample can be measured from the amount of bound label.
The antibody used for preceptin measurement is preferably used in the above-described measurement method.
　プレセプシン測定に用いられる検体は、対象から採取したものである。
　ここで、対象は、例えば、ヒト、非ヒト哺乳動物（例えば、ウサギ、ヤギ、馬、羊、ブタ、ラット、およびマウス）が挙げられる。いくつかの態様では対象は、ヒトである。
　検体は、特に限定されないが、水性の検体が好ましく、例えば、血液（全血、血漿、血清等）、尿、組織液、リンパ液、関節液、乳汁、脳脊髄液、膿、唾液、涙液、粘液、鼻水、痰、腹水、用水、***などの体液、また、鼻腔、気管支、肺、皮膚、腹腔、各種臓器、関節、骨などを洗浄した後の洗浄液、あるいは、細胞培養上清、またはカラム溶出液等が挙げられる。これらの試料は、そのまま、あるいは各種緩衝液等で希釈あるいは抽出後濃縮され、測定に用いられる。プレセプシン測定に用いられる検体は、好ましくは、血液検体であり、より好ましくはヒト血液検体である。 The specimen used for preceptin measurement is collected from a subject.
Here, examples of the subject include humans and non-human mammals (eg, rabbits, goats, horses, sheep, pigs, rats, and mice). In some embodiments, the subject is a human.
The sample is not particularly limited, but an aqueous sample is preferable. For example, blood (whole blood, plasma, serum, etc.), urine, tissue fluid, lymph fluid, joint fluid, milk, cerebrospinal fluid, pus, saliva, tear fluid, mucus Body fluids such as runny nose, sputum, ascites, irrigation fluid, semen, and washing fluid after washing nasal cavity, bronchi, lung, skin, abdominal cavity, various organs, joints, bones, etc., cell culture supernatant, or column elution Liquid and the like. These samples are used as they are or after dilution or extraction with various buffers and the like, and are concentrated. The specimen used for preceptin measurement is preferably a blood specimen, more preferably a human blood specimen.
　また、プレセプシン測定に用いる検体として、全血検体を用いる場合には、全血検体を採取してから７２時間、４８時間以内、２４時間以内、１２時間以内、６時間以内、又は４時間以内に分析を実施されてもよい。また、ＥＤＴＡ採血管又はヘパリン採血管により全血検体を採取されてもよい。好ましくは、ＥＤＴＡ採血管に全血検体を採取して６時間以内に分析すること、又は、ヘパリン採血管により全血検体を採取して４時間以内に分析することが挙げられる。 In addition, when a whole blood sample is used as a sample for preceptin measurement, the whole blood sample is collected within 72 hours, 48 hours, 24 hours, 12 hours, 6 hours, or 4 hours. An analysis may be performed. A whole blood sample may be collected by an EDTA blood collection tube or a heparin blood collection tube. Preferably, a whole blood sample is collected in an EDTA blood collection tube and analyzed within 6 hours, or a whole blood sample is collected in a heparin blood collection tube and analyzed within 4 hours.
４．プレセプシン測定キット
　また、ここでは、抗ＣＤ１４抗体、クロマトグラフィー用カラム、および膜からなる群から選択される少なくとも１つを含む、プレセプシン測定用キット（「測定キット」ともいう）が提供される。
　測定キットは、好ましくは、検体中のプレセプシン値を安定化させる方法、検体中のｓＣＤ１４由来の偽プレセプシン産生を抑制させる方法、検体中のｓＣＤ１４の変性抑制方法、検体中の真のプレセプシン量の定量方法、真のプレセプシンのみを定量する方法、偽プレセプシン値のみを定量する方法などに用いる、プレセプシン測定用キットである。測定キットは、好ましくは、抗ＣＤ１４抗体、クロマトグラフィー用カラム、または膜を含む。
　「抗ＣＤ１４抗体」、「クロマトグラフィー用カラム」、または「膜」は、具体的には、前述の通りである。 4). Presepsin Measurement Kit Also provided herein is a presepsin measurement kit (also referred to as “measurement kit”) comprising at least one selected from the group consisting of an anti-CD14 antibody, a chromatography column, and a membrane.
The measurement kit is preferably a method for stabilizing the preceptin value in the sample, a method for suppressing the production of pseudopreceptin derived from sCD14 in the sample, a method for suppressing the denaturation of sCD14 in the sample, and the determination of the amount of true preceptin in the sample. It is a kit for measuring presepsin used for a method, a method for quantifying only true preceptin, a method for quantifying only pseudo-preceptin value, and the like. The measurement kit preferably includes an anti-CD14 antibody, a chromatography column, or a membrane.
The “anti-CD14 antibody”, “chromatography column”, or “membrane” is specifically as described above.
　測定キットは、より好ましくは、プレセプシン測定のための補助試薬を含む。補助試薬としては、例えば、一次抗体、二次抗体、標識抗体、標識酵素、金コロイド等の標識物質、発色基質、蛍光基質（ＡｍｐｌｅｘＴＭ　Ｒｅｄ，ＡｔｔｏＰｈｏｓＴＭ，４－ＭＵＰ等）、化学発光基質（ルミノール、ＣＤＰ－ＳｔａｒＴＭ，ＡＭＰＰＤＴＭ，ＣＳＰＤＴＭ等）、ビオチン－ストレプトアビジン等の特異結合物質、不溶性担体、ブロッキング剤、希釈液、洗浄液、標準物質等が挙げられるが、これらに限定されない。 The measurement kit more preferably contains an auxiliary reagent for preceptin measurement. Examples of auxiliary reagents include primary antibodies, secondary antibodies, labeled antibodies, labeled enzymes, labeling substances such as gold colloids, chromogenic substrates, fluorescent substrates (Amplex ™ Red, AttoPhos ™, 4-MUP, etc.), chemiluminescent substrates (luminol, CDP-StarTM, AMPPDTM, CSPDTM, etc.), specific binding substances such as biotin-streptavidin, insoluble carriers, blocking agents, diluents, washings, standard substances and the like, but are not limited thereto.
　プレセプシン測定のための補助試薬は、プレセプシン測定の方法に合わせて、適宜組み合わせて用いられる。 Auxiliary reagents for presepsin measurement are used in appropriate combination according to the method of presepsin measurement.
　一次抗体は、好ましくは、プレセプシンに結合する抗体であり、より好ましくは、抗体と異なるエピトープを認識する抗体が好ましい。例えば、ＷＯ２００４／０４４００５号公報の実施例３に記載のＦ１１０６－１３－３抗体やＦ１０３１－８－３抗体などが挙げられる。ＷＯ２００５／１０８４２９号公報も同様に参照できる。 The primary antibody is preferably an antibody that binds to preceptin, and more preferably an antibody that recognizes an epitope different from the antibody. Examples thereof include the F1106-13-3 antibody and the F1031-8-3 antibody described in Example 3 of WO2004 / 044005. Reference can also be made to WO 2005/108429.
　プレセプシン測定に使用する抗体と一次抗体のいずれを標識抗体としてもよい。プレセプシン測定に使用する抗体と一次抗体のいずれも標識されていない場合は、標識された二次抗体を用いてもよい。 Either the antibody used for preceptin measurement or the primary antibody may be used as the labeled antibody. When neither the antibody used for preceptin measurement nor the primary antibody is labeled, a labeled secondary antibody may be used.
　不溶性担体としては、例えば、磁性粒子、ビーズ、ガラス、セルロース、ニトロセルロース、多孔性合成ポリマー、グラスファイバー、ポリアクリルアミド、ナイロン、ポリスチレン、ポリビニルクロライド、ポリプロピレン、プラスチックプレート、ラテックス粒子、不織布、濾紙等が挙げられる。 Examples of insoluble carriers include magnetic particles, beads, glass, cellulose, nitrocellulose, porous synthetic polymers, glass fibers, polyacrylamide, nylon, polystyrene, polyvinyl chloride, polypropylene, plastic plates, latex particles, non-woven fabric, filter paper, and the like. Can be mentioned.
　プレセプシン測定に使用する抗体の標識は、ペルオキシダーゼ（ＨＲＰ）、アルカリフォスファターゼ（ＡＬＰ）、β－ガラクトシダーゼ等の酵素、金コロイド等、好ましく用いられるが、これらに限定されない。 The label of the antibody used for preceptin measurement is preferably used, but is not limited to enzymes such as peroxidase (HRP), alkaline phosphatase (ALP), β-galactosidase, colloidal gold, and the like.
　例えば、ＨＲＰを用いる場合は、発色基質として、３，３’，５，５’－テトラメチルベンジジン（ＴＭＢ）、ｏ－フェニレンジアミン（ＯＰＤ）等が挙げられる。ＡＬＰを用いる場合は、発色基質としてｐ－ニトロフェニルフォスフェート（ｐＮＰＰ）等が挙げられる。β－ガラクトシダーゼを用いる場合の発色基質としては、ｏ－ニトロフェニル－β－Ｄ－ガラクトピラノシド（ｏ－Ｎｉｔｒｏｐｈｅｎｙｌ－β－Ｄ－Ｇａｌａｃｔｏｐｙｒａｎｏｓｉｄｅ：ＯＮＰＤ）等が例示される。 For example, when HRP is used, examples of the chromogenic substrate include 3,3 ', 5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (OPD), and the like. When ALP is used, p-nitrophenyl phosphate (pNPP) and the like can be mentioned as a color developing substrate. Examples of the chromogenic substrate in the case of using β-galactosidase include o-nitrophenyl-β-D-galactopyranoside (o-Nitrophenyl-β-D-Galactopyranoside: ONPD).
　例えば、サンドイッチＥＬＩＳＡ法用の測定キットは、プレセプシン測定に使用する抗体及び一次抗体（いずれかの抗体が酵素標識されていてもよい）、発色基質、希釈液、標準物質等を含有しうる。抗体及び一次抗体が標識されていない場合、標識された二次抗体を含有してもよい。 For example, the measurement kit for the sandwich ELISA method may contain an antibody and a primary antibody (any antibody may be enzyme-labeled) used for preceptin measurement, a chromogenic substrate, a diluent, a standard substance, and the like. When the antibody and the primary antibody are not labeled, a labeled secondary antibody may be contained.
　化学発光酵素免疫法（ＣＬＥＩＡ）用の測定キットは、例えば、磁性粒子等に固相化した抗体、酵素標識抗体、化学発光基質、希釈液、洗浄液等を含有しうる。 A measurement kit for chemiluminescent enzyme immunoassay (CLEIA) can contain, for example, an antibody immobilized on magnetic particles, an enzyme-labeled antibody, a chemiluminescent substrate, a diluent, a washing solution, and the like.
　蛍光酵素免疫測定法（ＦＥＩＡ）の測定キットは、例えば、磁性粒子等に固相化した抗体、酵素標識抗体、蛍光基質、希釈液、洗浄液等を含有しうる。 A fluorescent enzyme immunoassay (FEIA) measurement kit can contain, for example, an antibody immobilized on magnetic particles, an enzyme-labeled antibody, a fluorescent substrate, a diluent, a washing solution, and the like.
　電気化学発光免疫測定法（ＥＣＬＩＡ）の測定キットは、例えば、ビオチン化抗体、Ｒｕ（ｂｐｙ）３標識抗体、ストレプトアビジンコーティング磁性粒子、トリプロピルアミン等を含有しうる。 The measurement kit for electrochemiluminescence immunoassay (ECLIA) can contain, for example, biotinylated antibody, Ru (bpy) 3-labeled antibody, streptavidin-coated magnetic particles, tripropylamine and the like.
　イムノクロマト法による測定キットは、試料添加部、試薬部、検出部及び吸収部を、試験添加部に添加される液性検体が上記の順に移動するように設けた試験ストリップである。例えば、試薬部に標識した第二の抗体を含浸させ、検出部に第一の抗体が結合した不溶性担体を設置することができる。 The measurement kit by immunochromatography is a test strip provided with a sample addition part, a reagent part, a detection part, and an absorption part so that the liquid sample added to the test addition part moves in the above order. For example, an insoluble carrier in which the second antibody labeled is impregnated in the reagent part and the first antibody is bound to the detection part can be installed.
　試験ストリップは、多孔性担体等を用いることが例示される。多孔性担体は、例えば、ニトロセルロース、セルロース、セルロース誘導体、ナイロン、ナイロン繊維、ガラス線維、多孔性合成ポリマー等が挙げられる。吸収部は、水吸収性材料のスポンジ等の吸収ポリマー、セルロース濾紙、濾紙等が挙げられる。 The test strip is exemplified by using a porous carrier or the like. Examples of the porous carrier include nitrocellulose, cellulose, cellulose derivatives, nylon, nylon fibers, glass fibers, and porous synthetic polymers. Examples of the absorption part include an absorption polymer such as a water-absorbing material sponge, cellulose filter paper, filter paper and the like.
　敗血症患者では、特徴的にプレセプシン血中濃度が増加することが報告されているため、プレセプシン測定用キットは、敗血症の検出用キット、又は、敗血症の検出若しくは診断を補助するためのキットであってもよい。 It has been reported that presepsin blood levels are characteristically increased in septic patients, so the presepsin measurement kit is a kit for detecting sepsis or a kit for assisting in the detection or diagnosis of sepsis. Also good.
　また、測定キットは、敗血症の診断薬、あるいは、敗血症診断の補助薬として用いることができる。プレセプシン測定用キットは、このような敗血症の検出等を目的とするとき、抗体を用いて測定した、被験者の検体中のプレセプシン濃度が、カットオフ値より高値であるときに、被験者を敗血症の可能性があると判定し、検出又は診断を補助することができる。このとき、カットオフ値は、３１４～６００ｐｇ／ｍＬであり、好ましくは４００～５８０ｐｇ／ｍＬ、より好ましくは４５０～５５０ｐｇ／ｍＬ、さらに好ましくは、５００ｐｇ／ｍＬである。 In addition, the measurement kit can be used as a sepsis diagnostic agent or as an auxiliary agent for sepsis diagnosis. When the preceptin measurement kit is used for the detection of such sepsis, etc., the subject can be septic when the preceptin concentration in the subject's sample measured using an antibody is higher than the cutoff value. It can be determined that there is sex, and detection or diagnosis can be assisted. At this time, the cut-off value is 314 to 600 pg / mL, preferably 400 to 580 pg / mL, more preferably 450 to 550 pg / mL, and still more preferably 500 pg / mL.
　また、いくつかの態様では、プレセプシン測定用キットは、例えば、敗血症と全身性炎症反応症候群（ｓｙｓｔｅｍｉｃ　ｉｎｆｌａｍｍａｔｏｒｙ　ｒｅｓｐｏｎｓｅ　ｓｙｎｄｒｏｍｅ、ＳＩＲＳ）との判別；敗血症の重症化のリスク評価；敗血症の予後予測（死亡率予測）；敗血症の重症度評価；術後感染症の検出；感染性血管内凝固症候群（ｄｉｓｓｅｍｉｎａｔｅｄ　ｉｎｔｒａｖａｓｃｕｌａｒ　ｃｏａｇｕｌａｔｉｏｎ、ＤＩＣ）の検出；感染性ＤＩＣの検出；心疾患の検出；細菌感染を伴う呼吸器感染症、炎症性腸疾患（クローン病、潰瘍性大腸炎）、発熱性好中球減少症（ｆｅｂｒｉｌｅ　ｎｅｕｔｒｏｐｅｎｉａ、ＦＮ）、又は血球貪食症候群（ｈｅｍｏｐｈａｇｏｃｙｔｉｃ　ｓｙｎｄｒｏｍｅ、ＨＰＳ）の検出から選ばれる少なくとも１つの疾患の検出又評価のために使用することができる。プレセプシン測定用キットは、上記の少なくとも１つの疾患の検出又は評価のためのキットであってもよい。 Further, in some embodiments, the kit for measuring presepsin, for example, distinguishes between sepsis and systemic inflammatory response syndrome (SIRS); risk assessment of severe sepsis; prognosis prediction of septicemia (mortality rate) Prediction); severity assessment of sepsis; detection of postoperative infection; detection of infectious intravascular coagulation (DIC); detection of infectious DIC; detection of heart disease; respiratory infection with bacterial infection Detection of inflammation, inflammatory bowel disease (Crohn's disease, ulcerative colitis), febrile neutropenia (FN), or hemophagocytic syndrome (HPS) Chosen can be used for detection The evaluation of at least one disease. The kit for measuring presepsin may be a kit for detecting or evaluating at least one disease.
５．偽プレセプシンと真のプレセプシンの分別定量方法
　検体中の偽プレセプシンと真のプレセプシンの分別定量方法は、ゲル濾過法を用いることを含む。
　「偽プレセプシンと真のプレセプシンの分別定量」とは、具体的には、実施例４のように、ｓＣＤ１４由来の偽プレセプシンとプレセプシンとを分離することを意味する
。
　例えば、実施例４に準じる方法に従って、ゲル濾過法を用いて行うことができ、各々の分画中のプレセプシン値を測定する。より具体的には、プレセプシン測定前の検体中を、例えば、ゲル濾過クロマトグラフィーで分離し、真のプレセプシンを含む分画中を得て、プレセプシン値を測定することで、真のプレセプシン値を定量することが可能である。定量にあたっては、前記のプレセプシン測定方法を用いるが、例えば、実施例４で言えば、標準濃度のプレセプシンによって作成した検量線と各プレセプシンを含む分画のプレセプシン量を比較することで、真のプレセプシン値を得ることができる。
　あるいは、真のプレセプシンを含む分画の代わりに、偽プレセプシンを含む分画を得て、偽プレセプシン値を測定することで、偽プレセプシン値を得ることができる。偽プレセプシン値が得られれば、検体のプレプシン値を測定し、真のプレセプシン値と偽プレセプシン値の合計値（総プレセプシン量）から真のプレセプシンの測定値を得ることも可能である。 5. Fractional presepsin and true preceptin differential quantification method The pseudo preceptin and true preceptin differential quantification method in a sample includes using a gel filtration method.
Specifically, “separate quantification of pseudo-presepsin and true pre-sepsin” means that, as in Example 4, sCD14-derived pseudo-preceptin and preceptin are separated.
For example, the gel filtration method can be used according to the method according to Example 4, and the preceptin value in each fraction is measured. More specifically, the sample before presepsin measurement is separated by, for example, gel filtration chromatography, the fraction containing true presepsin is obtained, and the presepsin value is measured to quantify the true preceptin value. Is possible. For quantification, the above-described preceptin measurement method is used. For example, in Example 4, a true preceptin is obtained by comparing the standard curve prepared with standard concentration of preceptin and the amount of preceptin in the fraction containing each preceptin. A value can be obtained.
Alternatively, a pseudo preceptin value can be obtained by obtaining a fraction containing a pseudo preceptin instead of a fraction containing a true preceptin and measuring the pseudo preceptin value. If a pseudo preceptin value is obtained, the prepsin value of the specimen can be measured, and a true preceptin measurement value can be obtained from the total value (total preceptin amount) of the true preceptin value and the pseudo preceptin value.
６．プレセプシン測定システム
　また、ここでは、検体中のプレセプシン値を安定化させる方法、検体中のｓＣＤ１４由来の偽プレセプシン産生を抑制させる方法、検体中のｓＣＤ１４の変性抑制方法、検体中の真のプレセプシン量の定量方法、真のプレセプシンのみを定量する方法、偽プレセプシン値のみを定量する方法などをシステム中に組み込んだ全自動装置等の装置が提供される。
　また、当該分離と定量を行うシステムを構築することで、検体を直接アプライするだけで真のプレセプシン値を得るシステムを構築することも可能となる。
　当該システムの各構成は、例えば、次の通りである。
１）検体カートリッジ部：例えば、測定に用いる採取した検体をマイクロチューブに入れ、そのマイクロチューブを保持する部分等が該当する；
２）検体処理部：検体サンプリングを行う部分であり、例えば、直接サンプリングするノズル、またはディスポーザブルチップを装着できる部分を備え、サンプルを採取する部分が該当する；
３）試薬カートリッジ部：プレセプシン測定に必要な試薬等を含む部分であり、例えば、前記のプレセプシン測定、又はプレセプシン測定キットの項目に記載のプレセプシン測定に必要な試薬等が該当する；
４）プレセプシン測定部：検体中のプレセプシン測定を行う部分であり、前記プレセプシン測定、またはプレセプシン測定キットの項目に記載に記載のプレセプシン測定を行う部分が該当する；
５）計算部：４）の測定部での測定に基づきプレセプシン値を計算する部分が該当する；
６）表示部：５）の計算部で計算したプレセプシン値を表示する部分が該当する；
等が挙げられ、上記１）から６）からなる群から選択される少なくとも１つを含む、プレセプシン測定システムが挙げられる。上記の１）～６）の構成は、基本的には、当該システム内において、その順番、すなわち、１）→２）→３）→４）→５）→６）の順で処理が進むこととなる。 6). Presepsin measurement system In addition, here, a method for stabilizing the preceptin value in the sample, a method for suppressing the production of pseudopreceptin derived from sCD14 in the sample, a method for suppressing degeneration of sCD14 in the sample, the amount of true preceptin in the sample An apparatus such as a fully automatic apparatus in which a quantification method, a method of quantifying only true preceptin, a method of quantifying only a pseudopreceptin value, and the like are incorporated in the system is provided.
In addition, by constructing a system that performs the separation and quantification, it is possible to construct a system that obtains a true preceptin value simply by directly applying a specimen.
Each configuration of the system is, for example, as follows.
1) Sample cartridge unit: For example, a sample sample used for measurement is put in a microtube, and a portion for holding the microtube corresponds.
2) Specimen processing unit: a part that performs specimen sampling, and includes, for example, a part that can be directly mounted with a nozzle for sampling or a disposable chip, and corresponds to a part for collecting a sample;
3) Reagent cartridge part: a part containing a reagent necessary for presepsin measurement, for example, a reagent necessary for presepsin measurement described in the above-mentioned presepsin measurement or presepsin measurement kit item;
4) Presepsin measurement unit: a part for measuring presepsin in a sample, which corresponds to a part for measuring presepsin according to the above-mentioned preceptin measurement or preceptin measurement kit item;
5) Calculation part: The part which calculates a preceptin value based on the measurement in the measurement part of 4) corresponds;
6) Display part: The part which displays the preceptin value calculated by the calculation part of 5) corresponds;
And a presepsin measurement system including at least one selected from the group consisting of 1) to 6) above. The above configurations 1) to 6) basically proceed in the order of the system, that is, 1) → 2) → 3) → 4) → 5) → 6). It becomes.
　尚、本明細書において引用した全ての文献、及び公開公報、特許公報、その他の特許文献は、参照として本明細書に組み込むものとする。
　以下の実施例により本発明を更に詳述するが、本発明はこれら実施例に限定して理解されるべきものではない。 It should be noted that all documents cited in the present specification, as well as published publications, patent gazettes, and other patent documents are incorporated herein by reference.
The present invention will be described in more detail with reference to the following examples, but the present invention should not be understood to be limited to these examples.
実施例１：プレセプシン測定値への検体振とうの影響
　ＴＥＮＮＥＳＳＥＥ Ｂｌｏｏｄ　Ｓｅｒｖｉｃｅｓ社の正常ヒト血漿（ＥＤＴＡ）２例（＃Ｒ２５５２９２、＃Ｒ２５５２８７）を検体として、プレセプシンＥＬＡＩＳＡキット（持田製薬（株）、Ｌｏｔ：２Ｓ０１５）を用いて、振とうした各検体中の各々のプレセプシン値を測定した。
　検体のプレセプシン値の測定は、振とう前（０分）及び振とう後１０分及び６０分で行った。
　尚、振とうは、検体２ｍＬをマイクロチューブに分注し、ＴＡＩＴＥＣ社　Ｖｏｒｔｅｘ　Ｍｉｘｅｒで６０秒まで、それ以降６０分まではＥＹＥＬＡ　ＣＵＴＥ　ＣＭ－１０００（１８００ｒｐｍ）を用いた。各測定検体は各々の検体２ｍＬより５０μＬずつサンプリングして使用した。
　各プレセプシンの測定値を表１に示した。 Example 1: Influence of specimen shaking on preceptin measured value Two specimens of normal human plasma (EDTA) (# R255292, # R255287) from TENNESSEE Blood Services were used as specimens for preceptin ELISA kit (Mochida Pharmaceutical Co., Ltd., Lot: 2S015) was used to measure each preceptin value in each shaken sample.
The presepsin value of the specimen was measured before shaking (0 minutes) and 10 minutes and 60 minutes after shaking.
For shaking, 2 mL of the sample was dispensed into a microtube, and EYELA CUTE CM-1000 (1800 rpm) was used for 60 seconds with a TAITEC Vortex Mixer until 60 minutes thereafter. Each measurement sample was used by sampling 50 μL from 2 mL of each sample.
The measured values of each preceptin are shown in Table 1.
　表１：振とうによる経時的なプレセプシン濃度変化
Figure JPOXMLDOC01-appb-T000001
 Table 1: Changes in preceptin concentration over time due to shaking
Figure JPOXMLDOC01-appb-T000001
　以上の結果より、検体の激しい振とうにより検体中のプレセプシン値が経時的に増加する現象が認められた。なお、この増加した値は、敗血症診断のカットオフ値；５００ｐｇ／ｍＬより大きな値であった。なお、本実験で加えた物理的刺激（振とう）モデルは、検討の為の極端な条件であり、血液検体の通常の取扱又は輸送状態等とは異なり、極めて異常で激しいものであることを補足説明しておく。 From the above results, a phenomenon was observed in which the preceptin value in the sample increased over time due to vigorous shaking of the sample. The increased value was a cutoff value for diagnosis of sepsis; a value larger than 500 pg / mL. Note that the physical stimulus (shaking) model added in this experiment is an extreme condition for examination, and it is extremely abnormal and intense, unlike the normal handling or transportation state of blood samples. Supplementary explanation.
実施例２：プレセプシン測定値の増加原因の検討
　Ａｃｃｅｓｓ Ｂｉｏｌｏｇｉｃａｌｓ社の正常ヒト血清（＃ＳＴＬ１３０１４５）、及び抗ＣＤ１４抗体で前処理したヒト血清（以下、ＣＤ１４吸収ヒト血清という。）を用い、各々の検体とも１．５ｍＬのマイクロチューブ当り１００μＬずつ分注し、１０分間振とうした（振とう条件は実施例１と同じ）。尚、ＣＤ１４吸収ヒト血清の調製は、Ｆ１０２４－１－３抗体（ＷＯ２００１／７２９９３の公報の実施例１及び２に記載の方法で調製した。）をクロマトグラフィー用の樹脂（Ｓｅｐｈａｒｏｓｅ　４ＦＦ）に結合させたアフィニティークロマトグラフィーを用いて調整した。正常ヒト血清を当該アフィニティーカラムに通して、ＣＤ１４を樹脂に吸着させ、その非吸着画分（素通り画分）を使用した。
　正常ヒト血清およびＣＤ１４吸収ヒト血清の各々の振とう前、及び１０分間振とう後の検体について、パスファーストＰｒｅｓｅｐｓｉｎ、ＰＡＴＨＦＡＳＴ（商品名）（（株）ＬＳＩメディエンス）を用いて検体中のプレセプシン濃度（ｐｇ／ｍＬ）を測定した。また、ＣＤ１４ＥＬＩＳＡキット（Ｒ＆Ｄ ｓｙｓｔｅｍｓ社）を用いて各検体中のｓＣＤ１４濃度（ｎｇ／ｍＬ）を測定した。
　各検体中のプレセプシン濃度及びｓＣＤ１４濃度を表２に示した。 Example 2: Examination of cause of increase in preceptin measurement value Using normal human serum (# STL130145) from Access Biologicals and human serum pretreated with anti-CD14 antibody (hereinafter referred to as CD14-absorbing human serum), each sample was used. In both cases, 100 μL was dispensed per 1.5 mL microtube and shaken for 10 minutes (the shaking conditions were the same as in Example 1). The CD14-absorbed human serum was prepared by binding F1024-1-3 antibody (prepared by the method described in Examples 1 and 2 of WO2001 / 72993) to a chromatography resin (Sepharose 4FF). Were prepared using affinity chromatography. Normal human serum was passed through the affinity column, CD14 was adsorbed to the resin, and the non-adsorbed fraction (through fraction) was used.
For samples of normal human serum and CD14-absorbed human serum before shaking and after shaking for 10 minutes, the preceptin concentration ( pg / mL). Further, the sCD14 concentration (ng / mL) in each sample was measured using a CD14 ELISA kit (R & D systems).
Table 2 shows the presepsin concentration and sCD14 concentration in each specimen.
　表２：各検体中のプレセプシン濃度（ｐｇ／ｍＬ）及びｓＣＤ１４濃度（ｎｇ／ｍＬ）
Figure JPOXMLDOC01-appb-T000002
 Table 2: Preceptin concentration (pg / mL) and sCD14 concentration (ng / mL) in each specimen
Figure JPOXMLDOC01-appb-T000002
　以上の結果より、特別な処理を行っていない正常ヒト血清では、１０分の振とうで著しいプレセプシン濃度の上昇が認められたが、ＣＤ１４吸収ヒト血清では、振とう後でも、プレセプシンの測定値の顕著な増加は認められなかった。
　ｓＣＤ１４濃度は、正常ヒト血清では、１０分の振とうで半分以下に減少したが、ＣＤ１４吸収ヒト血清ではほとんど変化しなかった。
　なお、アフィニティークロマトグラフィーによるＣＤ１４吸収処理により、正常ヒト血清中のｓＣＤ１４含量は２ケタ減らすことができていることが確認された。（表２）
　これらの結果から、振とうによるプレセプシン値の増加は、血清中のｓＣＤ１４に由来する可能性が示唆された。 From the above results, in normal human serum not subjected to special treatment, a significant increase in preceptin concentration was observed after shaking for 10 minutes, but in the case of CD14-absorbed human serum, the measured value of preceptin was not improved even after shaking. There was no significant increase.
The sCD14 concentration decreased to less than half in the normal human serum after 10 minutes of shaking, but hardly changed in the CD14-absorbed human serum.
It was confirmed that the sCD14 content in normal human serum could be reduced by two digits by CD14 absorption treatment by affinity chromatography. (Table 2)
From these results, it was suggested that the increase in the preceptin level due to shaking may be derived from sCD14 in serum.
実施例３：抗ＣＤ１４抗体によるプレセプシン濃度変化へ影響の検討
　Ｆ１０２４－１－３抗体（抗ＣＤ１４抗体）を約３０μｇ、１ｍLの正常ヒト血漿（ＥＤＴＡ）に加えた後、１０分間振とう後、各検体のプレセプシン濃度をプレセプシンキット（パスファーストＰｒｅｓｅｐｓｉｎ、ＰＡＴＨＦＡＳＴ（商品名））を用いて測定し、抗ＣＤ１４抗体の振とうによるプレセプシン濃度変化への影響を検討した。
　結果を図１に示した。正常人の血漿を用い、Ｆ１０２４－１－３抗体（抗ＣＤ１４抗体）の存在下・非存在下でプレセプシンを測定したところ、振とうによって生じるプレセプシン値の増加をＦ１０２４－１－３抗体が抑制することが示された。 Example 3: Examination of the effect of anti-CD14 antibody on changes in preceptin concentration F1024-1-3 antibody (anti-CD14 antibody) was added to about 30 μg of 1 mL of normal human plasma (EDTA), shaken for 10 minutes, The presepsin concentration of the specimen was measured using a preceptin kit (Path First Presepsin, PATHHFAST (trade name)), and the influence of shaking of the anti-CD14 antibody on the change of the preceptin concentration was examined.
The results are shown in FIG. When presepsin was measured using normal human plasma in the presence or absence of F1024-1-3 antibody (anti-CD14 antibody), F1024-1-3 antibody suppressed the increase in preceptin level caused by shaking. It was shown that.
　また、敗血症患者の血漿を用いた場合でも、抗ＣＤ１４抗体の添加により、振とうによるプレセプシン濃度の変化が抑えられることが確認できた。
　一方、血漿検体に、抗ＣＤ１４抗体以外の抗体、例えば、抗アラキドン酸抗体、抗ｈＣＧ抗体等の非抗ＣＤ１４抗体を添加した場合には、振とうによるプレセプシン濃度変化抑制効果は認められなかった。 Moreover, even when using the plasma of a septic patient, it was confirmed that the addition of the anti-CD14 antibody can suppress changes in the preceptin concentration due to shaking.
On the other hand, when a non-anti-CD14 antibody such as an anti-arachidonic acid antibody or anti-hCG antibody was added to the plasma specimen, for example, anti-CD14 antibody, the effect of suppressing changes in preceptin concentration due to shaking was not observed.
　以上の結果より、抗ＣＤ１４抗体の添加により、振とう、すなわち激しい物理的刺激によるプレセプシン濃度の増加を抑制できることが明らかとなった。 From the above results, it was clarified that the addition of anti-CD14 antibody can suppress the increase in preceptin concentration due to shaking, that is, intense physical stimulation.
実施例４：振とう処理したヒト血漿のゲル濾過分析
　正常ヒト血漿（＃ＳＴＬ１３０１４５）を０．５ｍＬずつ３本のチューブに分注し、１本は、コントロールとしてそのまま静置した後、ゲル濾過分析に用いた。残り２本は、
１０分間の振とう処理した後に、それぞれゲル濾過分析を行った。このとき、１本にはＦ１０２４－１－３抗体を約２００μｇ添加し室温で１０分間静置後に振とう処理を行った。
　・Ｃｏｌｕｍｎ　Ｓｕｐｅｒｄｅｘ　７５　１０／３００　ＧＬ（１ｃｍφ×３０ｃｍ）
　・フラクション　０．５ｍＬ／チューブ
　・カラムへの検体添加量　１００μＬ Example 4: Gel filtration analysis of shaken human plasma Normal human plasma (# STL130145) was dispensed in 0.5 mL aliquots into three tubes, one was allowed to stand as a control, and then gel filtration analysis Used for. The remaining two are
After 10 minutes of shaking treatment, gel filtration analysis was performed. At this time, about 200 μg of F1024-1-3 antibody was added to one, and the mixture was allowed to stand at room temperature for 10 minutes and then shaken.
・ Column Superdex 75 10/300 GL (1cmφ × 30cm)
・ Fraction 0.5mL / tube ・ Sample addition amount to the column 100μL
（１）ゲル濾過の各フラクションをプレセプシンＥＬＩＳＡキットで測定し、当該測定液の４５０－６５０ｎｍの吸光度を図２に示した。 (1) Each fraction of gel filtration was measured with a preceptin ELISA kit, and the absorbance at 450 to 650 nm of the measured solution is shown in FIG.
（２）ゲル濾過の各フラクションをＣＤ１４ＥＬＩＳＡキット（Ｒ＆Ｄ ｓｙｓｔｅｍｓ社）で測定し、当該測定液の４５０－６５０ｎｍの吸光度を図３に示した。 (2) Each fraction of gel filtration was measured with a CD14 ELISA kit (R & D Systems), and the absorbance at 450-650 nm of the measurement solution is shown in FIG.
　図２に示した通り、コントロールとした正常血漿は、フラクション１９付近に単一ピークで溶出された。この溶出位置は、分子量１３ＫＤとされる真のプレセプシンの溶出位置である。
　一方、振とう処理したヒト血漿検体のゲル濾過分析結果は、フラクション１３付近と１９付近の２つのピークが認められた。フラクション１３は、ゲル濾過カラムのボイドの位置に相当し、高分子量の画分である。
　抗ＣＤ１４抗体を添加して振とう処理した検体では、コントロールと同様の溶出パターンを示した。
　このことから、検体の振とうにより高分子量のプレセプシン様物質（真のプレセプシンとは異なるが、抗プレセプシン抗体に反応する物質：以後「偽プレセプシン」と呼ぶことがある）が生成し、偽プレセプシンの生成に伴い、プレセプシン測定値の増加（見掛け上の濃度の上昇）が起きていることが分かった。また、偽プレセプシンは、抗ＣＤ１４抗体を共存させることでその生成を効果的に抑制されることが明らかとなった。 As shown in FIG. 2, normal plasma as a control was eluted with a single peak in the vicinity of fraction 19. This elution position is an elution position of true preceptin having a molecular weight of 13 KD.
On the other hand, the gel filtration analysis result of the shake-treated human plasma specimen showed two peaks near the fraction 13 and around 19. Fraction 13 corresponds to the void position of the gel filtration column and is a high molecular weight fraction.
The sample treated with shaking after adding the anti-CD14 antibody showed the same elution pattern as the control.
From this, the shake of the specimen produces a high molecular weight preceptin-like substance (which is different from true preceptin but reacts with an anti-preceptin antibody: hereinafter referred to as “pseudopreceptin”). It was found that an increase in preceptin measurement (apparent increase in concentration) occurred with the generation. In addition, it has been clarified that pseudopreceptin is effectively suppressed in the presence of anti-CD14 antibody.
　また、図３に示したＣＤ１４測定キットで検出される物質のクロマトグラフィー溶出パターンは、コントロール検体がフラクション１７付近に単一ピークを示したパターンから、血漿を振とう処理することにより、ボイド位置付近に肩を持つピークに変化した。このことから、振とうにより高分子量化したｓＣＤ１４が生じていることが示唆された。また、この肩の部分は、偽プレセプシンが検出された溶出位置と一致するため、偽プレセプシンは、ｓＣＤ１４分子が変性・凝集し、高分子量化してものであることが示唆された。 In addition, the chromatographic elution pattern of the substance detected by the CD14 measurement kit shown in FIG. 3 is a pattern in which the control sample shows a single peak in the vicinity of the fraction 17 and the plasma is shaken to obtain a vicinity of the void position. It changed to a peak with a shoulder. This suggested that sCD14 having a high molecular weight by shaking was generated. Further, this shoulder portion coincides with the elution position where the pseudopreceptin was detected, suggesting that the pseudopreceptin was obtained by denaturing and aggregating the sCD14 molecule to increase the molecular weight.
　以上の実験結果に基づき考察する。
　ｓＣＤ１４－ＳＴ（プレセプシン）とは、非還元条件下ＳＤＳ－ＰＡＧＥにおいて分子量１３±２ｋＤａに泳動されることを特徴とし、血漿検体のゲル濾過でも低分子量側に溶出された（図２）。一方、血中に存在するＣＤ１４は、可溶性ＣＤ１４（ｓＣＤ１４）とも言われ、分子量約５５ｋＤａあるいは約４９ｋＤａの分子種が知られており、これが、図３のコントロール正常ヒト血漿中のクロマトグラフィーパターンに認められたものと考えられる。そして、振とうにより生じるより高分子量の物質が、プレセプシン測定キットに反応し、偽プレセプシンとして、プレセプシン測定値の異常値の原因となっていることが示唆された。これは、プレセプシン測定キットが、免疫科学的にｓＣＤ１４分子の特異的配列（立体構造）を認識する分析定量法であるから、変性した高分子量のｓＣＤ１４分子であっても、プレセプシン測定キットに反応することあることは、一応想定しなければいけないことであり、今回の実験結果から、このような状況が起きている事が示唆された。 Consideration based on the above experimental results.
sCD14-ST (preceptin) is characterized in that it migrates to a molecular weight of 13 ± 2 kDa in SDS-PAGE under non-reducing conditions, and it was also eluted to the low molecular weight side by gel filtration of plasma samples (FIG. 2). On the other hand, CD14 present in blood is also referred to as soluble CD14 (sCD14), and a molecular weight of about 55 kDa or about 49 kDa is known. This is observed in the chromatographic pattern in control normal human plasma in FIG. It is thought that it was done. Then, it was suggested that a higher molecular weight substance generated by shaking reacts with the preceptin measurement kit and causes an abnormal value of the preceptin measurement value as pseudo-preceptin. This is an analytical quantification method in which the preceptin measurement kit immunologically recognizes a specific sequence (three-dimensional structure) of the sCD14 molecule, so even a denatured high molecular weight sCD14 molecule reacts with the preceptin measurement kit. This is something that must be assumed for the time being, and the results of this experiment suggest that this situation is occurring.
　以上に基づき、以下の発明が生まれる。物理的刺激等により偽プレセプシンを生じることを抑制するためには、検体中のｓＣＤ１４分子を安定化させればよく、例えば、検体採取直後に検体と抗ＣＤ１４抗体とを接触させることで達成することができる。すなわち、プレセプシン値測定における振とう等によるプレセプシン測定値の異常値発生を防ぐことが可能となる。また、検体採取直後に、ｓＣＤ１４分子を吸収等により除去してしまえば、偽プレセプシンを生じることはない。 Based on the above, the following inventions are born. In order to suppress the generation of pseudopreceptin due to physical stimulation or the like, it is only necessary to stabilize the sCD14 molecule in the specimen, for example, by bringing the specimen into contact with the anti-CD14 antibody immediately after the specimen is collected. Can do. That is, it is possible to prevent occurrence of an abnormal value of the preceptin measured value due to shaking or the like in the preceptin value measurement. Moreover, if the sCD14 molecule is removed by absorption or the like immediately after the sample is collected, pseudo-preceptin is not generated.
　さらに、既に偽プレセプシンを生じている検体の測定においても、偽プレセプシンを真のプレセプシンを区別することにより、真のプレセプシンの定量が可能となる。例えば、プレセプシン測定の際、（ｉ）３０ｋＤａの限外濾過膜を通すことにより偽プレセプシンを除去する、（ｉｉ）真のプレセプシンには反応せずに、偽プレセプシンの反応する抗体を利用することにより、偽プレセプシンを吸着除去・分離する等の前処理をすることで、正しいプレセプシン測定をすることが可能となる。 Furthermore, even in the measurement of a sample that has already produced pseudo-presepsin, the true pre-sepsin can be quantified by distinguishing the pseudo-preceptin from the true pre-ceptin. For example, when measuring presepsin, (i) removing pseudopreceptin by passing through a 30 kDa ultrafiltration membrane, (ii) using an antibody that reacts with pseudopreceptin without reacting with true preceptin. It is possible to perform correct preceptin measurement by performing pretreatment such as adsorption removal / separation of pseudopreceptin.
　さらにまた、実施例４のようにクロマトグラフィーグラフで偽プレセプシンと真のプレセプシンを分離分析が可能となる。真のプレセプシンを含む分画中のプレセプシン値を定量すれば良い。また、いずれかの手段で、偽プレセプシンが定量できれば、計算により真のプレセプシン値が求められる。例えば、クロマトグラフィーによって分離した「偽プレセプシン」を含む分画でプレセプシン測定を行うことによって、「偽プレセプシン値」を得ることができる。偽プレセプシン値が得られれば、検体のプレプシン値を測定し、真のプレセプシン値と偽プレセプシン値の合計値（総プレセプシン量）から偽プレセプシンの測定値を得ることも可能である。
　さらには、偽プレセプシンには反応せず、真のプレセプシンのみに反応する測定系をスクリーニングすることにより、特別な処理を要せずに、真のプレセプシン値のみの定量が可能となる。 Furthermore, as in Example 4, it is possible to separate and analyze pseudo-presepsin and true pre-sepsin using a chromatographic graph. What is necessary is just to quantify the presepsin value in the fraction containing true presepsin. Moreover, if pseudo preceptin can be quantified by any means, a true preceptin value can be obtained by calculation. For example, a “pseudopreceptin value” can be obtained by performing preceptin measurement on a fraction containing “pseudopreceptin” separated by chromatography. If a pseudo preceptin value is obtained, it is also possible to measure the prepsin value of the specimen and obtain a measured value of the pseudo preceptin from the total value (total preceptin amount) of the true preceptin value and the pseudo preceptin value.
Furthermore, by screening a measurement system that does not react with pseudo-preceptin but reacts with only true preceptin, only the true preceptin value can be quantified without requiring special treatment.
　配列番号１：Ｐ０３ペプチドのアミノ酸配列である。
　配列番号２：Ｓ６８ペプチドのアミノ酸配列である。
　配列番号３：ヒト全長可溶型ＣＤ１４のアミノ酸配列である。 SEQ ID NO: 1 is the amino acid sequence of the P03 peptide.
SEQ ID NO: 2 is the amino acid sequence of the S68 peptide.
SEQ ID NO: 3 is the amino acid sequence of human full-length soluble CD14.

Claims (14)

  1. 　検体採取直後に検体と抗ＣＤ１４抗体とを接触させる工程を含む、検体中のプレセプシン測定値を安定化させる方法。 A method of stabilizing the preceptin measurement value in a sample, comprising a step of bringing the sample into contact with an anti-CD14 antibody immediately after sample collection.
  2. 　検体採取直後に検体と抗ＣＤ１４抗体とを接触させる工程を含む、検体中の偽プレセプシン産生を抑制させる方法。 A method of suppressing pseudopreceptin production in a sample, comprising a step of contacting the sample with an anti-CD14 antibody immediately after the sample is collected.
  3. 　検体採取直後に検体と抗ＣＤ１４抗体とを接触させる工程を含む、検体中のｓＣＤ１４の変性抑制方法。 A method for inhibiting denaturation of sCD14 in a sample, comprising a step of bringing the sample into contact with an anti-CD14 antibody immediately after the sample is collected.
  4. 　検体と抗ＣＤ１４抗体とを接触させる工程により、検体中のｓＣＤ１４を吸着除去することを含む、請求項１ないし請求項３のいずれか１項に記載の方法。 The method according to any one of claims 1 to 3, comprising adsorbing and removing sCD14 in the specimen by a step of contacting the specimen with the anti-CD14 antibody.
  5. 　検体と抗ＣＤ１４抗体とを接触させる工程により、検体中のｓＣＤ１４と抗体ＣＤ１４抗体とが結合物をつくることを特徴とする、請求項１ないし請求項３のいずれか１項に記載の方法。 The method according to any one of claims 1 to 3, wherein the sCD14 in the specimen and the antibody CD14 antibody form a conjugate by contacting the specimen with the anti-CD14 antibody.
  6. 　プレセプシン測定前に、検体と抗ＣＤ１４抗体とを接触させる工程を含む、検体中の真のプレセプシン量の定量方法。 A method for quantifying the amount of true preceptin in a sample, comprising a step of bringing the sample into contact with an anti-CD14 antibody before measuring presepsin.
  7. 　検体と抗ＣＤ１４抗体とを接触させる工程により、検体中の偽プレセプシンと反応する抗ＣＤ１４抗体を用いて偽プレセプシンを吸着除去することを特徴とする、請求項６に記載の方法。 The method according to claim 6, wherein the pseudo-preceptin is adsorbed and removed using an anti-CD14 antibody that reacts with the pseudo-presepsin in the sample in the step of contacting the sample with the anti-CD14 antibody.
  8. 　プレセプシン測定前に、偽プレセプシンと真のプレセプシンとを分別する工程を含む、真のプレセプシンのみを定量する方法。 A method of quantifying only true preceptin, including a step of separating pseudo preceptin and true preceptin before measuring preceptin.
  9. 　偽プレセプシンと真のプレセプシンとの分別が、クロマトグラフィーによる分離であることを特徴とする、請求項８に記載の方法。 The method according to claim 8, wherein the fractionation between pseudo preceptin and true preceptin is a chromatographic separation.
  10. 　偽プレセプシンと真のプレセプシンとの分別が、膜による分離であることを特徴とする、請求項８に記載の方法。 9. The method according to claim 8, wherein the separation between pseudo-presepsin and true pre-sepsin is separation by membrane.
  11. 　プレセプシン測定前に、偽プレセプシンと真のプレセプシンとを分別する工程を含む、偽プレセプシンのみを定量する方法。 A method of quantifying only pseudo-preceptin, including a step of separating pseudo-preceptin and true pre-ceptin before measuring pre-ceptin.
  12. 　検体のプレセプシンを測定してプレセプシン値を算出し、算出したプレセプシン値から請求項１１に記載の方法で得た偽プレセプシン値を減じることを含む、真のプレセプシン値を定量する方法。 A method for quantifying a true preceptin value, comprising measuring a preceptin value of a specimen to calculate a preceptin value, and subtracting the pseudo-preceptin value obtained by the method according to claim 11 from the calculated preceptin value.
  13. 　請求項１ないし請求項１２のいずれか１項に記載の方法に用いるための、抗ＣＤ１４抗体、クロマトグラフィー用カラム、および膜からなる群から選択される少なくとも１つを含む、プレセプシン測定キット。 A preceptin measurement kit comprising at least one selected from the group consisting of an anti-CD14 antibody, a chromatography column, and a membrane for use in the method according to any one of claims 1 to 12.
  14. 　請求項１ないし請求項１２のいずれか１項に記載の方法に用いるための、検体カートリッジ部、検体処理部、試薬カートリッジ部、プレセプシン測定部、計算部、及び表示部からなる群から選択される少なくとも１つを含む、プレセプシン測定システム。
          A sample cartridge unit, a sample processing unit, a reagent cartridge unit, a preceptin measurement unit, a calculation unit, and a display unit for use in the method according to any one of claims 1 to 12 are selected. A presepsin measurement system comprising at least one.
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