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WO2014147873A1 - Anticorps se liant de manière spécifique à un produit de dégradation de hmgb1, et procédé de mesure ainsi que réactif de mesure de produit de dégradation de hmgb1 - Google Patents

Anticorps se liant de manière spécifique à un produit de dégradation de hmgb1, et procédé de mesure ainsi que réactif de mesure de produit de dégradation de hmgb1 Download PDF

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Publication number
WO2014147873A1
WO2014147873A1 PCT/JP2013/076177 JP2013076177W WO2014147873A1 WO 2014147873 A1 WO2014147873 A1 WO 2014147873A1 JP 2013076177 W JP2013076177 W JP 2013076177W WO 2014147873 A1 WO2014147873 A1 WO 2014147873A1
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Prior art keywords
antibody
hmgb1
degradation product
thrombin
sample
Prior art date
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PCT/JP2013/076177
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English (en)
Japanese (ja)
Inventor
東　義則
山田　晋吾
幸恵 小野
丸山　征郎
伊藤　隆史
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Kagoshima University NUC
Shino Test Corp
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Kagoshima University NUC
Shino Test Corp
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Priority to JP2015506540A priority Critical patent/JPWO2014147873A1/ja
Publication of WO2014147873A1 publication Critical patent/WO2014147873A1/fr
Anticipated expiration legal-status Critical
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6875—Nucleoproteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons

Definitions

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

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Abstract

L'invention fournit un anticorps de spécificité élevée par rapport à un produit de dégradation de HMGB1, et un procédé de mesure ainsi qu'un réactif de mesure permettant de mesurer avec exactitude une quantité fixe uniquement du produit de dégradation de HMGB1. Plus précisément, l'invention concerne un anticorps dont la compatibilité par rapport à un produit de dégradation provenant d'un complexe thrombine/thrombomoduline ou d'une thrombine de HMGB1, est d'au moins 1,5 fois en comparaison à sa compatibilité par rapport à un HMGB1, et qui est lié au produit de dégradation provenant d'un complexe thrombine/thrombomoduline ou d'une thrombine de HMGB1. Enfin, l'invention concerne un procédé de mesure immunologique et un réactif de mesure immunologique dudit anticorps, ainsi que du produit de dégradation de HMGB1 qui met en œuvre cet anticorps lié au produit de dégradation provenant d'un complexe thrombine/thrombomoduline ou d'une thrombine de HMGB1, et dont la compatibilité par rapport à un produit de dégradation provenant d'un complexe thrombine/thrombomoduline ou d'une thrombine de HMGB1, est élevée.
PCT/JP2013/076177 2013-03-19 2013-09-19 Anticorps se liant de manière spécifique à un produit de dégradation de hmgb1, et procédé de mesure ainsi que réactif de mesure de produit de dégradation de hmgb1 Ceased WO2014147873A1 (fr)

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WO2018079393A1 (fr) * 2016-10-26 2018-05-03 扶桑薬品工業株式会社 Anticorps spécifique de hmgb1 de type disulfure, procédé de mesure de hmgb1 de type disulfure et kit pour ladite mesure, ainsi qu'un procédé de mesure capable de quantifier toutes les molécules de hmgb1 comprenant une hmgb1 réduite, une hmgb1 de type disulfure et une hmgb1 pouvant être clivée par la thrombine et kit pour ladite mesure
JP2020148557A (ja) * 2019-03-12 2020-09-17 株式会社シノテスト 試料中のｈｍｇｂ１の測定方法及び測定試薬
JP2021063707A (ja) * 2019-10-11 2021-04-22 株式会社シノテスト 安定化されたｈｍｇｂ１含有溶液

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Publication number Priority date Publication date Assignee Title
WO2018079393A1 (fr) * 2016-10-26 2018-05-03 扶桑薬品工業株式会社 Anticorps spécifique de hmgb1 de type disulfure, procédé de mesure de hmgb1 de type disulfure et kit pour ladite mesure, ainsi qu'un procédé de mesure capable de quantifier toutes les molécules de hmgb1 comprenant une hmgb1 réduite, une hmgb1 de type disulfure et une hmgb1 pouvant être clivée par la thrombine et kit pour ladite mesure
JPWO2018079393A1 (ja) * 2016-10-26 2019-09-12 扶桑薬品工業株式会社 ジスルフィド型ｈｍｇｂ１特異的抗体、ジスルフィド型ｈｍｇｂ１の測定方法および測定用キット、ならびに、還元型ｈｍｇｂ１、ジスルフィド型ｈｍｇｂ１、トロンビン分解ｈｍｇｂ１等の全てのｈｍｇｂ１を定量することが可能な測定方法および測定用キット
US11174310B2 (en) 2016-10-26 2021-11-16 Fuso Pharmaceutical Industries, Ltd. Disulfide-type HMGB1-specific antibody, method for measuring disulfide-type HMGB1 and kit for said measurement, and measurement method capable of quantitating all of HMGB1 molecules including reduced HMGB1, disulfide-type HMGB1 and thrombin-cleavable HMGB1 and kit for said measurement
JP2020148557A (ja) * 2019-03-12 2020-09-17 株式会社シノテスト 試料中のｈｍｇｂ１の測定方法及び測定試薬
JP7313659B2 (ja) 2019-03-12 2023-07-25 株式会社シノテスト 試料中のｈｍｇｂ１の測定方法及び測定試薬
JP2021063707A (ja) * 2019-10-11 2021-04-22 株式会社シノテスト 安定化されたｈｍｇｂ１含有溶液
JP7425459B2 (ja) 2019-10-11 2024-01-31 株式会社シノテスト 安定化されたｈｍｇｂ１含有溶液

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