WO2019167935A1 - Utilisation d'un anticorps anti-cd14 utile pour la mesure de la présepsine - Google Patents

Utilisation d'un anticorps anti-cd14 utile pour la mesure de la présepsine Download PDF

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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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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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    • 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.

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Abstract

La présente invention concerne un procédé de stabilisation de mesures de présepsine dans des échantillons, le procédé comprenant une étape de mise en contact d'un échantillon avec un anticorps anti-CD14 immédiatement après la collecte de l'échantillon. Ce procédé permet d'obtenir des niveaux de présepsine réels dans des échantillons sans augmenter les mesures de présepsine même lorsque des échantillons à utiliser sont soumis à des stimuli physiques tels qu'une agitation intensive pendant la mesure de l'échantillon.
PCT/JP2019/007291 2018-02-27 2019-02-26 Utilisation d'un anticorps anti-cd14 utile pour la mesure de la présepsine WO2019167935A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005108429A1 (fr) * 2004-05-11 2005-11-17 Mochida Pharmaceutical Co., Ltd. Nouvel antigène cd14 soluble
WO2011093459A1 (fr) * 2010-01-29 2011-08-04 三菱化学メディエンス株式会社 PROCÉDÉ DE DOSAGE DU sCD14-ST HUMAIN
WO2017008894A1 (fr) * 2015-07-13 2017-01-19 University Of Geneva Panels de biomarqueurs pour complications de lésions cérébrales
WO2017033281A1 (fr) * 2015-08-25 2017-03-02 持田製薬株式会社 Anticorps anti-présepsine spécifiquement purifiés

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005108429A1 (fr) * 2004-05-11 2005-11-17 Mochida Pharmaceutical Co., Ltd. Nouvel antigène cd14 soluble
WO2011093459A1 (fr) * 2010-01-29 2011-08-04 三菱化学メディエンス株式会社 PROCÉDÉ DE DOSAGE DU sCD14-ST HUMAIN
WO2017008894A1 (fr) * 2015-07-13 2017-01-19 University Of Geneva Panels de biomarqueurs pour complications de lésions cérébrales
WO2017033281A1 (fr) * 2015-08-25 2017-03-02 持田製薬株式会社 Anticorps anti-présepsine spécifiquement purifiés

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May 2015 (2015-05-01), Retrieved from the Internet <URL:https://www.jamt.or.jp/congress/j64/pdf/general/0563.pdf> [retrieved on 20190426] *
vol. 64, May 2015 (2015-05-01), Retrieved from the Internet <URL:https://www.jamt.or.jp/congress/j64/pdf/general/0576.pdf> [retrieved on 20190426] *

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