JP2021063020A - Antibodies that bind to decomposition product of hmgb1, methods and reagents for measuring hmgb1 decomposition product - Google Patents

Abstract

To provide antibodies of which affinity to the HMGB1 decomposition product is higher than to HMGB1, as well as methods and reagents for quantitative, precise and exclusive determination of HMGB1 decomposition product.SOLUTION: Disclosed are antibodies that specifically bind to a HMGB1 decomposition product of a specific amino acid sequence produced by thrombin or thrombin-thrombomodulin complex, where the antibodies are prepared by using a conjugate of a synthetic peptide and a carrier as an immunogen. Methods and a reagents for measuring the HMGB1 decomposition product by using the antibody are also disclosed.SELECTED DRAWING: None

Description

The present invention relates to an antibody that specifically binds to a degradation product of HMGB1 whose cytotoxicity is reduced as compared with HMGB1, and a method and reagent for measuring the degradation product of HMGB1.
The present invention is useful in the fields of life science such as clinical examination, clinical pathology, immunology and medicine, and the field of chemistry such as analytical chemistry.

HMGB (High Mobility Group Box Protein; High Mobility Group Box Protein) was formerly called HMGB (High Mobility Group Protein; High Mobility Group Protein), but it is a large amount of non-histone protein contained in the chromatin structure. Discovered in 1964, it is a protein ubiquitously contained in all higher animals and plants, and its primary structure is extremely conserved among races.
Moreover, it is known that it is abundant not only in the nucleus but also in the cytoplasm.

Although the physiological action is not clearly known, HMGB loosens the double helix structure when it binds to DNA, so it is said that the higher-order structure of DNA is changed to the optimum structure during the transcription reaction to enhance transcriptional activity. It is believed to function as a very wide range of transcriptional promoters and nucleosome relaxation factors.

There are several types of HMGB.
For example, HMGB1 (High Mobility Group Box Protein 1), HMGB2 (High Mobility Group Box Protein 2), HMGB3 (High Mobility Group Box Protein 3), HMGB8 (High Mobility Group Box Protein 8), HMGB17 (High Mobility Group Box Protein 17) ), HMGB I (High Mobility Group Box Protein I), HMGB Y (High Mobility Group Box Protein Y), HMGB I (Y) (High Mobility Group Box Protein I (Y)), HMGB IC (High Mobility Group Box) Protein IC) and the like can be mentioned.

The amino acid sequence homology between human HMGB1 and porcine HMGB1 is 99.1%, and the amino acid sequence homology between human HMGB2 and porcine HMGB2 is also 99.1%.

In 1999, Wang et al. Quantitatively measured HMGB1 in serum (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 could be a marker for sepsis.
Then, it was shown that it is possible to distinguish between a surviving patient and a dying patient in a septic patient by accurately measuring HMGB1 in blood (see Non-Patent Document 1).

It was previously shown that the antibody used for the measurement of HMGB1, that is, the antibody that binds to HMGB1, can be prepared by Parkkinen et al. And Lepp et al. (Non-Patent Documents 2 and Non). See Patent Document 3).

Using this antibody, Rep et al. State that solid-phase enzyme-linked immunosorbent assay (Solid-phase Immunoassay) is possible for HMGB1.

In addition, Cabart et al. Have shown a method for preparing human HMGB1 and human HMGB2 (see Non-Patent Document 4).

In addition, literature has been published that HMGB1 is also induced by inflammation, which is considered to be the cause of mass secretion of various cytokines (see Non-Patent Document 5, Non-Patent Document 6 and Non-Patent Document 7). ..

In addition, the present inventors can obtain an accurate measurement value including an error without measuring an antibody that binds to human HMGB1 but does not bind to human HMGB2, and HMGB2. We have developed an immunological measurement method and an immunological measurement reagent (see Patent Document 1).

In addition, the present inventors have previously included a bird-derived anti-human HMGB1 antibody, which is a high-titer antibody having a high binding ability to human HMGB1 and which can obtain the antibody with high probability, in a sample. We have developed an immunological measurement method and immunological measurement reagent for human HMGB1 in a highly sensitive sample capable of accurately measuring even a very small amount of human HMGB1 (see Patent Document 2).

By the way, the present inventors cleave the 10th arginine (R10) -11th glycine (G11) of human HMGB1 by thrombin or the thrombin-thrombomodulin complex, and 10 of the N-terminal "MGKGDPKKPR" of this HMGB1. It was found that the peptide consisting of the amino acid residues of HMGB1 was separated to produce a degradation product of HMGB1 having a newly exposed N-terminal "GKMSSYA ...". (The present inventors have named this degradation product of human HMGB1 "des-HMGB1".)
As a result of the studies by the present inventors, it was found that the degradation product of HMGB1 has lower cytotoxicity than HMGB1.

Therefore, in order to reduce the cytotoxicity of HMGB1, HMGB1 is degraded (cleaved) as described above using thrombin or a thrombin / thrombomodulin complex to convert it into a degradation product of HMGB1 having less cytotoxicity. Was conceived.

Then, in order to understand the effect of decomposition of HMGB1 by this thrombin or thrombin / thrombomodulin complex, quantitative measurement of the degradation product of HMGB1 was required, and a measurement method was examined. However, the conventional method is the decomposition of HMGB1. HMGB1 and the like were also measured together with the product, and it was not possible to specifically measure only the degradation product of HMGB1 (see Non-Patent Document 8).

In addition, we have previously found that the affinity of HMGB1 for thrombin or the degradation product of the thrombin-thrombomodulin complex is at least 1.5 times greater than the affinity for HMGB1 for thrombin or thrombin for HMGB1. -An antibody that binds to a degradation product of a thrombin modulus complex, and an immunological measurement method and an immunological measurement reagent for an HMGB1 degradation product using the antibody have been developed, but they are not always sufficient (see Patent Document 3). ).

Japanese Unexamined Patent Publication No. 2003-96099 International Publication No. 2008/07578 Pamphlet International Publication No. 2014/147873 Pamphlet

H. Wang et al., SCIENCE, Vol. 285, No. 9, pp. 248-251, 1999 J. Parkkinen et al., The Journal of Biological Chemistry, Vol. 268, No. 26, pp. 19726-19738, 1993. W. A. Lepp et al., Journal of Immunoassay, Vol. 10, No. 4, pp. 449-465, 1989 P. Cabart et al., Cell Biochemistry and Function 13, 125-133, 1995 Andersson, U et al., J. Mol. Exp. Med. , 192, 565-570, 2000 Scaffidl et al., Nature, 418, pp. 191-195, 2002 Park et al., The Journal of Biological Chemistry, Vol. 279, No. 27, 2004 Ito et al., Arteriosclerosis, Thrombosis, and Vascular Biology, Vol. 28, pp. 1825-1830, 2008

As described above, it has been required to accurately quantitatively measure the degradation products of HMGB1 by thrombin or thrombin-thrombomodulin complex.
However, conventional antibodies have not been highly specific for the degradation products of HMGB1.
The conventional measuring method and measuring reagent also do not have high specificity for the HMGB1 degradation product, and not only measure the HMGB1 degradation product, but also measure HMGB1, that is, the measured value is It contained a positive error due to HMGB1.
Moreover, since the entire amino acid sequence of the degradation product of HMGB1 is present in HMGB1, it was expected that it would be difficult to obtain an antibody having an affinity for the degradation product higher than that for HMGB1.
However, as a result of repeated studies aimed at solving the above-mentioned problems, the present inventors have succeeded in obtaining the target antibody, and by using the antibody, accurately quantitatively measure only the HMGB1 degradation product. We have found that we can do this, and have completed the present invention.

The gist of the present invention is as follows.
(1) An antibody that specifically binds to a thrombin of HMGB1 or a degradation product of a thrombin / thrombomodulin complex, which is characterized by specifically recognizing the amino acid sequence represented by SEQ ID NO: 1.
(2) A method for measuring a degradation product of HMGB1 by thrombin or a thrombin / thrombomodulin complex, which comprises using the antibody according to (1) above.
(3) A reagent for measuring a degradation product of HMGB1 by thrombin or a thrombin / thrombomodulin complex, which comprises the antibody according to (1) above.

The antibody of the present invention is an antibody having a high affinity for a thrombin or thrombomodulin complex degradation product of HMGB1, that is, an antibody having a high specificity for the HMGB1 degradation product.
In addition, the measuring method and measuring reagent of the present invention have high specificity for the degradation product of thrombin or thrombin / thrombomodulin complex of HMGB1, and the measurement of HMGB1 is suppressed, that is, a positive error derived from HMGB1. Is suppressed, and only the HMGB1 degradation product can be accurately quantitatively measured.

FIG. 1 is a photograph showing the results of SDS-polyacrylamide gel electrophoresis of human HMGB1 and human HMGB2 with and without thrombin contact treatment, respectively. It is a photograph of the result of Western blotting showing the reactivity of the anti-human des-HMGB1 monoclonal antibody (47C1) to each of human HMGB1, human des-HMGB1 and human des-HMGB2. It is a graph of the calibration curve when the pig des-HMGB1 is measured by the ELISA method using the anti-human des-HMGB1 monoclonal antibody (47C1).

[I] An antibody that specifically binds to a thrombin of HMGB1 or a degradation product of a thrombin / thrombomodulin complex, which is characterized by specifically recognizing the amino acid sequence shown in SEQ ID NO: 1.

(1) Anti-HMGB1 Degradation Product Antibody Specific binding to a thrombin or thrombin-thrombomodulin complex degradation product of HMGB1, which is characterized by specifically recognizing the amino acid sequence shown in SEQ ID NO: 1 of the present invention. The "antibody to be used" is not particularly limited as long as it is an antibody capable of specifically recognizing the amino acid sequence shown in SEQ ID NO: 1. (In addition, the antibody of the present invention may be hereinafter referred to as "the present anti-HMGB1 degradation product antibody".)

In this anti-HMGB1 degradation product antibody, the 10th arginine (R10) -11th glycine (G11) of human HMGB1 is cleaved by thrombin or the thrombin-thrombomodulin complex, and the N-terminal "MGKGDPKKPR" of this HMGB1 is cleaved. A peptide consisting of 10 amino acid residues is separated to specifically recognize the newly exposed N-terminal "GKMSSYA" amino acid sequence.

As the anti-HMGB1 degradation product antibody, a monoclonal antibody is preferable.

The anti-HMGB1 degradation product antibody is a polyclonal antibody, an anti-serum containing a polyclonal antibody, a monoclonal antibody, a fragment of these antibodies (Fab, F (ab') 2 or Fab', etc.), or a single chain antibody (scFv). ) Etc. may be used.

(2) HMGB1 degradation product 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. In the present invention, in particular, thrombin or a thrombin-thrombomodulin complex cleaves the 10th arginine (R10) -11th glycine (G11) of human HMGB1 and 10 of the N-terminal "MGKGDPKKPR" of this HMGB1. It refers to a degradation product of HMGB1 having a newly exposed N-terminal "GKMSSYA ...", which is produced by the separation of a peptide consisting of amino acid residues.

In the present invention, as the degradation product of human HMGB1 by thrombin or thrombin / thrombomodulin complex, deletion, substitution, insertion, or addition of one or several amino acid residues to the amino acid sequence of des-HMGB1. Alternatively, it may be a protein consisting of an amino acid sequence obtained by subjecting the modification, or a protein to which a saccharide, a lipid, or the like is bound.

The number of amino acid residues in the deletion, substitution, insertion, addition or modification of the amino acid residues is usually 1 to 10, preferably 1 to 8, more preferably 1 to 6, and even more preferably 1. The number is ~ 4, more preferably 1-2, and particularly preferably 1.

Further, in the present invention, the thrombin or thrombin / thrombomodulin complex is not only a thrombin or thrombin / thrombomodulin complex existing in the human body, but also an artificially prepared one such as a genetically recombinant or the like. It also includes those that have been artificially isolated.
(Note that the above-mentioned "degradation product of HMGB1 by thrombin or thrombin / thrombomodulin complex" may be hereinafter referred to as "the degradation product of HMGB1").

(3) Immunogen The immunogen for obtaining an anti-HMGB1 degradation product antibody will be described below.
Examples of the immunogen for obtaining the anti-HMGB1 degradation product antibody include human HMGB1, HMGB1 of an animal having high homology to the amino acid sequence of human HMGB1 (for example, bovine or pig), or the HMGB1 degradation product. All or part of it can be mentioned.

As a part of the HMGB1 degradation product, for example, a peptide consisting of 7 amino acid residues of "GKMSSYA" at the N-terminal of the HMGB1 degradation product can be mentioned.

Then, a peptide consisting of 7 amino acid residues of the N-terminal "GKMSSYA" of the HMGB1 degradation product or a conjugate of the peptide and a carrier (carrier) serves as an immunogen for obtaining the anti-HMGB1 degradation product antibody. preferable.

In addition, as an immunogen for obtaining this anti-HMGB1 degradation product antibody, deletion, substitution, insertion, addition or modification of one or several amino acid residues with respect to the above-mentioned protein or peptide as an immunogen. It may be a protein or peptide obtained by subjecting to.

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, and particularly preferably 1. It is an individual.

Further, as a substance that immunizes an animal or the like in order to obtain the present anti-HMGB1 degradation product antibody, a carrier may be bound to the protein or peptide as an immunogen described above.

In particular, when the protein or peptide as the immunogen is of a low molecular weight, the antibody-producing ability is improved by binding to the carrier, so that the one to which the carrier is bound is preferably used as the immunogen.

Examples of this carrier include hemocyanin (KLH), bovine serum albumin (BSA), human serum albumin (HSA), chicken serum albumin, poly-L-lysine, polyalanyl lysine, and dipalmityl lysine. Known carriers such as tetanus toxoid and polysaccharides can be used.

(4) Method for Obtaining Immunogen, etc. of the Anti-HMGB1 Degradation Product Antibody The method for obtaining an immunogen, etc. that immunizes an animal or the like in order to obtain the present anti-HMGB1 degradation product antibody will be described below.
Although it is an immunogen for obtaining the anti-HMGB1 degradation product antibody, the human HMGB1 described in (3) above or the animal HMGB1 having high homology with the amino acid sequence of human HMGB1 is, for example, human or 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 to the amino acid sequence of HMGB1 by a known method or the like. Etc., it can be obtained.

Further, the HMGB1 hydrolyzate is obtained by contacting HMGB1 of an animal having high homology with the amino acid sequence of human HMGB1 or human HMGB1 (for example, bovine or pig) with the thrombin or thrombin / thrombomodulin complex. It can be hydrolyzed and obtained by extracting, purifying or the like by a known method or the like.

In the present invention, the peptide consisting of 7 amino acid residues of "GKMSSYA" at the N-terminal of the HMGB1 degradation product or a conjugate of this peptide and a carrier (carrier) obtains the anti-HMGB1 degradation product antibody. It is preferable as an immunogen of HMGB1.

Further, 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" edited by the Japan Biochemistry Society, Tokyo Chemistry Dojin, 1975; Izumiya et al. "Basics and Experiments of Peptide Synthesis", Maruzen, 1985; or "Sequel Biochemistry" edited by the Japan Biochemistry Society. It can be synthesized according to the method described in "Experimental Course 2 Under Protein Chemistry", Tokyo Chemistry Dojin, 1987, etc.

Furthermore, the immunogen may be prepared from DNA or RNA having the corresponding nucleic acid base sequence using genetic engineering technology, "Sequel Biochemistry Experiment Course 1 Gene Research Method I" edited by the Japan Biochemistry Society, Tokyo Kagaku. Dojin, 1986; Japan Biochemistry Society ed., "Sequel Biochemistry Experiment Course 1 Gene Research Method II", Tokyo Kagaku Dojin, 1986; or Japan Biochemistry Society ed., "Sequel Biochemistry Experiment Course 1 Gene Research Method III", Tokyo Kagaku Dojin , 1987, etc.

For example, the amino acid sequence of human HMGB1, the amino acid sequence of the HMGB1 degradation product, or a gene corresponding to a part of these amino acid sequences is cloned, and the obtained gene is incorporated into an expression vector such as a plasmid.
Next, by introducing this expression vector into a host cell such as Escherichia coli and culturing the obtained transformant, a protein or peptide consisting of the above amino acid sequence or a part thereof can be expressed.

Examples of the method for cloning the base sequence of a gene include a PCR method, a recombinant PCR method, a ligation method, and a linker ligation method.

By the way, when the immunogen is a low molecular weight 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 for this. Since there is a report that a specific antibody was produced (Kiyama et al., "Abstracts of the 112th Annual Meeting of the Japanese Society of Pharmaceutical Sciences," p. 122, published in 1992), it is not essential to use a carrier.

When an animal or the like is immunized with a carrier bound to the above-mentioned protein or peptide, the carrier includes hemosianin (KLH), bovine serum albumin (BSA), human serum albumin (HSA), and chicken serum albumin. , Poly-L-lysine, polyalanyllysine, dipalmityllysine, tetanus toxoid, polysaccharides and the like known as carriers can be used.

The method for binding the protein or peptide to the carrier is a glutaraldehyde method, a 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide method, a maleimide benzoyl-N-hydroxysuccinimid ester method, a bisdiazated benzidine method, or a method. A known binding method such as N-succimidyl-3- (2-pyridyldithio) propionic acid method can be used.

Further, a carrier having the above-mentioned protein or peptide adsorbed on a carrier such as nitrocellulose particles, polyvinylpyrrolidone or liposomes can also be used for immunization of the above-mentioned animals or the like in order to obtain the anti-HMGB1 degradation product antibody.

(6) Method for Obtaining This Anti-HMGB1 Degradation Product Antibody (polyclonal Antibody) In this anti-HMGB1 degradation product antibody, the polyclonal antibody or antiserum can be obtained by the following operation.
(I) First, the immunogen or a combination of the immunogen and a carrier is immunized against a mammal (mouse, rabbit, rat, sheep, goat, horse, etc.) or a bird (chicken, etc.).

The immunogenicity of the immunogen or the conjugate of the immunogen and the carrier is determined by the type of immunogen, the site of immunoinjection, and the like. For example, in the case of rabbits, one per rabbit. 10 μg to several tens of mg of the immunogen or a combination of the immunogen and the carrier is immunoinjected at a time.

It is preferable that the immunogen or a conjugate of the immunogen and a carrier is added and mixed with an adjuvant and immunoinjected.
As the adjuvant, known adjuvants such as Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide adjuvant, and Bordetella pertussis adjuvant can be used.

The immunoinjection may be performed subcutaneously, intravenously, intraperitoneally, or on the back.
After the initial immunization, the above-mentioned immunogen or a combination of the above-mentioned immunogen and a carrier is additionally immunized at a site such as subcutaneous, intravenous, intraperitoneal or back at intervals of 2 to 3 weeks. Also in this case, it is preferable that the immunogen or the conjugate of the immunogen and the carrier is additionally immunoinjected by adding and mixing an adjuvant.

After the initial immunization, the antibody titer in the serum of the immunized animal is repeatedly measured by the ELISA method or the like, and when the antibody titer reaches the plateau, the whole blood is collected, the serum is separated, and the antiserum containing the anti-HMGB1 degradation product antibody is contained. To get.

This antiserum is purified by a method such as salting out with ammonium sulfate, sodium sulfate or the like, ion exchange chromatography, gel filtration or affinity chromatography, or a combination of these methods to obtain a polyclonal antibody.

(Ii) The polyclonal antibody obtained here is further contacted by passing it through an affinity chromatography column immobilized on a solid phase using HMGB1 as a ligand.

The anti-HMGB1 antibody binds to the solid phase via the ligand of this column (HMGB1) and is collected.
On the other hand, this anti-HMGB1 degradation product antibody is difficult to bind to the ligand (HMGB1) of this column, and many of them pass through this column. Therefore, by obtaining a bypassed fraction, this anti-HMGB1 degradation product antibody Can be obtained.

This is brought into contact with an affinity chromatography column immobilized on a solid phase using the HMGB1 degradation product as a ligand, and the anti-HMGB1 degradation product antibody is solidified via a ligand (the HMGB1 degradation product) of this column. And other antibodies etc. are separated by passing through this column, and then the anti-HMGB1 degradation product antibody bound to this solid phase is separated from the ligand (the HMGB1 degradation product) from this column. By obtaining the outflowing fraction, the present anti-HMGB1 degradation product antibody having a higher purity can be obtained.

(Iii) When an animal or the like is immunized using a conjugate of an immunogen and a carrier, an antibody against this carrier is present in the obtained antiserum or polyclonal antibody, and thus an antibody against such a carrier. It is preferable to carry out the removal treatment of.
Examples of this removal treatment method include removing aggregates formed by adding the carrier to the obtained polyclonal antibody or antiserum solution, or immobilizing the carrier on an insolubilized solid phase and removing it by affinity chromatography. Can be used.

(7) Method for obtaining the present anti-HMGB1 degradation product antibody (monoclonal antibody) In the present anti-HMGB1 degradation product antibody, the monoclonal antibody can be obtained by the following operation.
Monoclonal antibodies are hybridomas produced by Keller et al.'S cell fusion method (G. Koehler et al., Nature, Vol. 256, pp. 495-497, published in 1975), or antibody-producing cells such as tumorigenic cells caused by viruses such as Epstan-Bar virus. Can be obtained by

The monoclonal antibody can be prepared by the cell fusion method by the following operation.
First, the immunogen or a combination of the immunogen and a carrier is immunized against a mammal (mouse, nude mouse, rat, etc., for example, BALB / c of an inbred mouse) or a bird (chicken, etc.). ..

The immunogenicity of the immunogen or the conjugate of the immunogen and the carrier is appropriately determined depending on the type of immunogen, the site of immunoinjection, and the like. For example, in the case of mice, one per animal. It is preferable to immunoinject 0.1 μg to 5 mg of the immunogen or a combination of the immunogen and the carrier at a time.

It is preferable that the immunogen or a conjugate of the immunogen and a carrier is immunoinjected by adding and mixing an adjuvant.
As the adjuvant, known adjuvants such as Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide adjuvant, and Bordetella pertussis adjuvant can be used.

The immunoinjection may be performed subcutaneously, intravenously, intraperitoneally, or at a site such as the back.
After the initial immunization, the immunogen or a combination of the immunogen and the carrier is additionally immunized at intervals of 1 to 2 weeks under the skin, intravenously, intraperitoneally, or on the back.
The number of booster injections is generally 2 to 6 times.
In this case as well, it is preferable that the immunogen or the conjugate of the immunogen and the carrier is additionally immunoinjected by adding and mixing an adjuvant.

After the initial immunization, the antibody titer in the serum of the immunized animal is repeatedly measured by the ELISA method or the like, and when the antibody titer reaches a plateau, the above-mentioned immunogen or a combination of the above-mentioned immunogen and the carrier is subjected to physiological saline. A solution dissolved in (0.9% sodium chloride aqueous solution) is injected intravenously or intraperitoneally for final immunization.

Three to five days after this final immunization, cells capable of producing antibodies, such as splenocytes, lymph node cells, and peripheral lymphocytes of immune animals, are obtained.

The cells having the antibody-producing ability obtained from this immune animal are fused with the myeloma cells (myeloma cells) of mammals (mouse, nude mouse, rat, etc.). Cell lines lacking enzymes such as guanine phosphoribosyl transferase (HGPRT) or thymidine kinase (TK) are preferred, for example, P3-X63-Ag8 line (ATCC), which is an HGPRT-deficient cell line derived from BALB / c mice. TIB9), P3-X63-Ag8-U1 strain (Cancer Research Research Source Bank (JCRB) 9085), P3-NS1-1-Ag4-1 strain (JCRB 0009), P3-X63-Ag8.653 strain (JCRB 0028) Alternatively, SP2 / O-Ag-14 strain (JCRB 0029) or the like can be used.

Cell fusion can be carried out using a fusion accelerator having various molecular weights such as polyethylene glycol (PEG), liposomes or Sendai virus (HVJ), or by an electric fusion method.

When the myeloma cells are of an HGPRT-deficient strain or a TK-deficient strain, a selection medium (HAT medium) containing hypoxanthine, aminopterin, and thymidine is used to fuse the cells having an antibody-producing ability and the myeloma cells. Only cells (hybridoma) can be selectively cultured and proliferated.

The hybridoma culture supernatant thus obtained is measured by an immunological measurement method such as an ELISA method or a Western blotting method using the HMGB1 degradation product or the like to produce the anti-HMGB1 degradation product antibody. Hybridomas can be selected.

Further, by measuring the culture supernatant of the hybridoma by an immunological measurement method such as ELISA method or Western blotting using HMGB1 or the like, a hybridoma that produces an antibody that does not bind to HMGB1 or the like is selected. Can be done.

By combining these two types of hybridoma selection methods and known cloning methods such as the limiting dilution method, the present anti-HMGB1 degradation product antibody (monoclonal antibody), that is, "the amino acid sequence represented by SEQ ID NO: 1 is specific. An antibody producing a "monoclonal antibody" (monoclonal antibody) that specifically binds to a thrombin or a degradation product of a thrombin-thrombomodulin complex of HMGB1 can be isolated and obtained.

This monoclonal antibody-producing cell line can be cultured in an appropriate medium, and the present anti-HMGB1 degradation product antibody (monochrome antibody) can be obtained from the culture supernatant. As the medium, a serum-free medium, a low-concentration serum medium, or the like can be used. It may be used, and in this case, it is preferable in that the purification of the antibody is facilitated, and a medium such as DMEM medium, RPMI1640 medium or ASF medium 103 can be used.

In addition, a monoclonal antibody-producing cell line is injected into the abdominal cavity of a mammal that is compatible with this and has been pre-stimulated with pristan or the like, and after a certain period of time, this anti-HMGB1 degradation product antibody (monoclonal antibody) is injected from the ascites accumulated in the abdominal cavity. ) Can also be obtained.

The monoclonal antibody thus obtained was purified by a method such as 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. The present anti-HMGB1 degradation product antibody (monoclonal antibody) can be obtained.

[II] Method for measuring degradation product of HMGB1 thrombin or thrombin / thrombomodulin complex (1) General remarks Method for measuring degradation product of HMGB1 thrombin or thrombin / thrombomodulin complex of the present invention (hereinafter, "measurement method of the present invention") ) Uses an antibody that specifically binds to the thrombin of HMGB1 or the degradation product of the thrombin-thrombomodulin complex, which is characterized by specifically recognizing the amino acid sequence shown in SEQ ID NO: 1. It is not particularly limited as long as it is characterized by.

In the measuring method of the present invention, the measurement may be carried out using only the present anti-HMGB1 degradation product antibody as the antibody, or another anti-HMGB1 antibody or another anti-HMGB1 degradation product antibody may be added to the present anti-HMGB1 degradation product antibody. Measurements may be performed in combination.

In the measurement method of the present invention, the present anti-HMGB1 degradation product antibody is a polyclonal antibody, an anti-serum containing a polyclonal antibody, a monoclonal antibody, and fragments of these antibodies (Fab, F (ab') 2 or Fab', etc.). , Or a single chain antibody (scFv) or the like.

Then, in the measuring method of the present invention, the anti-HMGB1 degradation product antibody is preferably a monoclonal antibody.

Further, in the measuring method of the present invention, not only one type of the present anti-HMGB1 degradation product antibody but also a plurality of types may be used at the same time.

Further, in the measuring method of the present invention, the other anti-HMGB1 antibody or other anti-HMGB1 degradation product antibody is a polyclonal antibody, an anti-serum containing a polyclonal antibody, a monoclonal antibody, and fragments of these antibodies (Fab, F (Fab, F). It may be either ab') 2 or Fab'), or a single chain antibody (scFv) or the like.

Further, in the measuring method of the present invention, not only one type of the other anti-HMGB1 antibody or the other anti-HMGB1 degradation product antibody may be used at the same time, but a plurality of types may be used at the same time.

Further, the measuring method of the present invention uses a immobilized antibody and a labeled antibody, and is any of the present anti-HMGB1 degradation product antibody and the above-mentioned other anti-HMGB1 antibody or other anti-HMGB1 degradation product antibody. One antibody may be used as the immobilized antibody and the other antibody may be used as the labeled antibody.

By using the anti-HMGB1 degradation product antibody in the measuring method of the present invention, the specificity for the HMGB1 degradation product is high, and only the HMGB1 degradation product can be accurately quantitatively measured.

(2) This anti-HMGB1 degradation product antibody For details of this anti-HMGB1 degradation product antibody, thrombin of HMGB1 or HMGB1 thrombin, which is characterized by specifically recognizing the amino acid sequence shown in "[I] SEQ ID NO: 1" described above. As described in the section "Antibodies that specifically bind to the degradation products of the thrombin-thrombomodulin complex".

(3) The other anti-HMGB1 antibody or other anti-HMGB1 degradation product antibody The other anti-HMGB1 antibody or other anti-HMGB1 degradation product antibody is an antibody capable of specifically binding to HMGB1 or HMGB1 degradation. Any antibody that can specifically bind to the product is sufficient, and there is no other limitation.
For example, anti-HMGB1 antibody derived from birds (preferably chicken), anti-HMGB1 antibody derived from mouse, anti-HMGB1 degradation product antibody derived from mouse, and the like can be mentioned.

(4) Measurement method The measurement method of the present invention specifically binds to a thrombin of HMGB1 or a degradation product of a thrombin / thrombomodulin complex, which is characterized by specifically recognizing the amino acid sequence shown in SEQ ID NO: 1. As long as an antibody is used, the measurement principle is not particularly limited, and the desired effect is achieved.

Examples of this measuring method include enzyme-linked immunosorbent assay (ELISA, EIA), fluorescence immunoassay (FIA), radioimmunoassay (RIA), luminescence immunoassay (LIA), enzyme antibody method, and fluorescent antibody method. Immunological measurement methods such as immunochromatography method, immunoturbidimetric method, latex agglutination method, latex agglutination reaction measurement method, hemagglutination reaction method, and particle agglutination reaction method can be mentioned.

Then, in the above-mentioned measuring method, the measuring 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).

Further, the measurement in the measuring method of the present invention may be carried out by a manual method, or may be carried out by using an apparatus such as an analyzer.

(5) Samples The samples in the measurement method of the present invention include body fluids such as human blood, serum, plasma, urine, spinal fluid, saliva, sweat, tears, ascites or sheep water; stool; organs such as blood vessels or liver; tissues. Any sample such as a biological sample that may contain the HMGB1 degradation product, such as cells; or an extract of stool, organs, tissues, cells, etc., is a target.

(6) Immunological measurement method using a labeled antibody The measurement method of the present invention uses an enzyme immunoassay using a labeled antibody in which a labeling substance is bound to an antibody and an immobilized antibody in which the antibody is immobilized on a solid phase carrier. When it is carried out by an immunological measurement method such as a measurement method, a fluorescence immunoassay method, a radiation immunoassay method or a luminescence immunoassay method, it can be carried out by a sandwich method or a competitive method, but it should be carried out by the sandwich method. Is preferable.

When the measurement method of the present invention is carried out by the sandwich method, the antibody of the present anti-HMGB1 degradation product and one of the other anti-HMGB1 antibody or the other anti-HMGB1 degradation product antibody is solid phase. It may be used as a chemical antibody and the other antibody may be used as a labeled antibody.

Examples of the solid phase carrier used for the solid phase antibody used in the above measurement method include polystyrene, polycarbonate, polyvinyltoluene, polypropylene, polyethylene, polyvinyl chloride, nylon, polymethacrylate, polyacrylamide, latex, liposomes, gelatin, agarose, and the like. Solid-state carriers in the form of microcapsules, beads, microplates (microtiter plates), test tubes, sticks or test pieces made of materials such as cellulose, sepharose, glass, ceramics, metal or magnetic material can be used. it can.

The immobilized antibody is an antibody such as the present anti-HMGB1 degradation product antibody, or the above-mentioned other anti-HMGB1 antibody or other anti-HMGB1 degradation product antibody, and the solid phase carrier by a physical adsorption method, a chemical binding method, or a method thereof. It can be prepared by adsorbing and binding by a known method such as combined use.

In the case of the physical adsorption method, the antibody and the solid phase carrier are mixed and contacted in a solution such as a buffer solution according to a known method, or the antibody dissolved in the buffer solution and the solid phase carrier are brought into contact with each other. It can be carried out.

In addition, when using the chemical binding method, "Clinical Pathology Extraordinary Special Issue No. 53: Immunoassays for Clinical Examinations-Technology and Applications-" edited by the Japanese Society of Clinical Pathology, Clinical Pathology Publishing Society, published in 1983; Japan Society for Biochemistry A divalent cross-linking reagent such as glutaraldehyde, carbodiimide, imide ester, or maleimide, which uses a known method described in "New Chemistry Experiment Course 1 Protein IV", Tokyo Kagaku Dojin, published in 1991, etc. It can be carried out by mixing and contacting with, and reacting with each of the amino group, carboxyl group, thiol group, aldehyde group, hydroxyl group and the like of the antibody and the solid phase carrier.

Further, if it is necessary to further perform a treatment to suppress a non-specific reaction or natural aggregation of the solid phase carrier, for example, bovine serum albumin is placed on the surface or inner wall surface of the solid phase carrier on which the antibody is immobilized. (BSA), human serum albumin (HSA), ovalbumin, casein, proteins such as gelatin or salts thereof, surfactants, defatted milk powder, etc. are treated by a known method such as contacting and coating, and the solid phase carrier. Blocking process (masking process) may be performed.

As the labeling substance, in the case of enzyme immunoassay, for example, peroxidase (POD), alkaline phosphatase (ALP), β-galactosidase, urease, catalase, glucose oxidase, lactate dehydrogenase, amylase and the like can be used. it can.

In the case of fluorescence immunoassay, for example, fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, substituted rhodamine isothiocyanate, dichlorotriazine isothiocyanate and the like can be used.

Then, in the case of radioimmunoassay, for example, tritium, iodine-125, iodine-131 or the like can be used.

Further, in the luminescence immunoassay method, for example, substances related to the reaction system such as NADH-FMNH2-luciferase reaction system, luminol-hydrogen peroxide-POD reaction system, acridinium ester reaction system, or dioxetane compound reaction system are used. Can be used.

The method of binding this anti-HMGB1 degradation product antibody, or the antibody such as the other anti-HMGB1 antibody or other anti-HMGB1 degradation product antibody, and a labeling substance such as an enzyme is described in "Clinical Pathology Extraordinary Special Issue No." edited by the Japanese Society of Clinical Pathology. No. 53 Immunoassay for Clinical Examination-Technology and Applications- ", Clinical Pathology Publishing Association, published in 1983; Japan Biochemical Society ed.," Neochemical Experiment Course 1 Protein IV ", Tokyo Kagaku Dojin, published in 1991, etc. The antibody and the labeling substance are mixed and contacted with a divalent cross-linking reagent such as glutaaldehyde, carbodiimide, imide ester or maleimide according to the above method, and the amino group, carboxyl group, thiol group and aldehyde group of the antibody and the labeling substance are respectively mixed and contacted. Alternatively, the bond can be formed by reacting with a hydroxyl group or the like.

Known methods are known as methods for measuring immunoassays such as the above-mentioned enzyme immunoassay, radioimmunoassay, radioimmunoassay, and luminescence immunoassay. No. 53 Immunoassay for Clinical Examination-Technology and Applications- ", Clinical Pathology Publishing Society, 1983; Eiji Ishikawa et al.," Enzyme Immunoassay ", 3rd Edition, Medical School, 1987; Tsunehiro Kitagawa et al. It can be performed by ed., "Protein Nucleic Acid Enzyme Supplement No. 31 Enzyme Immunoassay", Kyoritsu Shuppan, 1987).

For example, by reacting a sample with an immobilized antibody (“solid phase carrier-antibody”) and simultaneously reacting with a labeled antibody (“antibody-labeled substance”), or by reacting the labeled antibody after washing, “ A complex of "solid phase carrier-antibody" = "the HMGB1 degradation product" = "antibody-labeled substance" is formed.

Then, the unbound labeled antibody is washed and separated, and the amount of the labeled antibody indirectly bound to the solid phase carrier by binding "solid phase carrier-antibody" = "the HMGB1 degradation product" = "antibody-labeled substance". Alternatively, the amount (concentration) of the HMGB1 degradation product contained in the sample can be measured by measuring the amount of the unbound labeled antibody.

Specifically, in the case of the enzyme immunoassay method, the enzyme labeled with the antibody is reacted with the substrate under the optimum conditions or the like, and the amount of the enzyme reaction product is measured by an optical method or the like.

Moreover, in the case of the fluorescence immunoassay method, the fluorescence intensity by the fluorescent substance label is measured.

Then, in the case of the radioimmunoassay method, the radiation amount by the radioactive substance label is measured.

Further, in the case of the luminescence immunoassay method, the amount of luminescence by the luminescence reaction system is measured.

(7) Measurement method by agglutination reaction method The measurement method of the present invention is included in a sample by measuring the formation of immune complex aggregates by an optical method or by visually measuring the transmitted light or scattered light. It is also carried out by an immunological measurement method such as an immunoturbidimetry method, a latex agglutination method, a latex agglutination reaction method, an hemagglutination reaction method or a particle agglutination reaction method, which measures the amount (concentration) of the HMGB1 degradation product. can do.

The present anti-HMGB1 degradation product antibody, and in some cases additionally, the above-mentioned other anti-HMGB1 antibody or other anti-HMGB1 degradation product antibody (hereinafter, may be referred to as “antipolymer such as the present anti-HMGB1 degradation product antibody”) is solid-phase. When used by immobilizing on a carrier, examples of the solid phase carrier include polystyrene, styrene-styrene sulfonate copolymer, acrylonitrile-butadiene-styrene copolymer, and vinyl chloride-acrylic acid ester copolymer. , Vinyl acetate-acrylic acid copolymer, polyacrolein, styrene-methacrylic acid copolymer, styrene-glycidyl (meth) acrylic acid copolymer, styrene-butadiene copolymer, methacrylic acid polymer, acrylic acid polymer, Particles made of materials such as latex, gelatin, liposomes, microcapsules, erythrocytes, silica, alumina, carbon black, metal compounds, ceramics, metals and magnetic materials can be used.

As a method for immobilizing an antibody such as the anti-HMGB1 degradation product antibody on a solid phase carrier, a known method such as a physical adsorption method, a chemical binding method, or a combination thereof can be used.

In the case of the physical adsorption method, the antibody and the solid phase carrier are mixed and contacted in a solution such as a buffer solution according to a known method, or the antibody dissolved in the buffer solution or the like is brought into contact with the solid phase carrier. It can be carried out.

In addition, when using the chemical binding method, "Clinical Pathology Extraordinary Special Issue No. 53: Immunoassays for Clinical Examinations-Technology and Applications-" edited by the Japanese Society of Clinical Pathology, Clinical Pathology Publishing Society, published in 1983; Japan Society for Biochemistry A divalent cross-linking reagent such as glutaraldehyde, carbodiimide, imide ester, or maleimide, which uses a known method described in "New Chemistry Experiment Course 1 Protein IV", Tokyo Kagaku Dojin, published in 1991, etc. It can be carried out by mixing and contacting with, and reacting with each of the amino group, carboxyl group, thiol group, aldehyde group, hydroxyl group and the like of the antibody and the solid phase carrier.

Further, if it is necessary to carry out a treatment in order to further suppress a non-specific reaction or natural aggregation of the solid phase carrier, the surface or inside of the solid phase carrier on which an antibody such as the anti-HMGB1 degradation product antibody is immobilized. Treatment by a known method such as contacting and coating a wall surface with a protein such as bovine serum albumin (BSA), human serum albumin (HSA), egg white albumin, casein, gelatin or a salt thereof, a surfactant, or defatted milk powder. Then, the solid phase carrier may be blocked (masked).

When the latex turbidimetric method is used as the measurement principle, the particle size of the latex particles used as the solid phase carrier is not particularly limited, but the latex particles are antibodies such as the anti-HMGB1 degradation product antibody and the substance to be measured (the above). The average particle size of the latex particles is 0.04 to the average particle size for reasons such as the degree of binding via HMGB1 decomposition products) to generate agglomerates and the ease of measurement of the agglomerates formed. It is preferably 1 μm.

When the latex turbidimetry method is used as the measurement principle, the concentration of the latex particles on which the antibody such as the anti-HMGB1 degradation product antibody is immobilized is the concentration of the HMGB1 degradation product contained in the sample. The optimum concentration varies depending on various conditions such as the distribution density of an antibody such as an anti-HMGB1 degradation product antibody on the surface of the latex particle, the particle size of the latex particle, and the mixing ratio of the sample and the measurement reagent, and therefore cannot be unequivocally stated.

However, usually, an antigen-antibody reaction between an antibody such as this anti-HMGB1 degradation product antibody immobilized on latex particles by mixing a sample and a measurement reagent with the "HMGB1 degradation product" contained in the sample occurs. At the time of the measurement reaction to be carried out, the concentration of the latex particles on which the antibody such as the anti-HMGB1 degradation product antibody is immobilized should be 0.005 to 1% (w / v) in the reaction mixture. In this case, the measurement reagent contains "latex particles on which an antibody such as the anti-HMGB1 degradation product antibody is immobilized" having such a concentration in the reaction mixture.

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, the particle size of the particles used as the solid phase carrier is not particularly limited, but the average particles thereof. The diameter is preferably in the range of 0.01 to 100 μm, more preferably in the range of 0.5 to 10 μm.

The specific gravity of these particles is preferably in the range of 1 to 10, and more preferably in the range of 1 to 2.

Examples of the container used for the measurement when the indirect agglutination reaction method such as the latex agglutination reaction method, the erythrocyte agglutination reaction method or the particle agglutination reaction method is used as the measurement principle include glass, polystyrene, polyvinyl chloride or polymethacrylate. Examples thereof include a test tube, a microplate (microtiter plate), a tray, and the like.

The bottom surface of the solution accommodating portion (microplate well, etc.) of these containers is preferably U-shaped, V-shaped, UV-shaped, or the like having an inclination from the center to the periphery of the bottom surface.

When the measurement method of the present invention is carried out by an immunological measurement method such as an immunoturbidimetric method, a latex agglutination method, a latex agglutination reaction method, an agglutination reaction method or a particle agglutination reaction method, phosphate is used as a solvent. A buffer solution, a glycine buffer solution, a Tris buffer solution, a Good buffer solution, or the like can be used, and a reaction accelerator such as polyethylene glycol or a non-specific reaction inhibitor may be further included.

The operation method of the measurement in the immunological measurement method such as the immunoturbidimetry method, the latex turbidimetry method, the latex agglutination reaction method, the hemagglutination reaction method or the particle agglutination reaction method can be performed by a known method or the like. For example, in the case of measurement by an optical method, the sample and the above-mentioned "antibody such as the anti-HMGB1 degradation product antibody", or the sample and the solid phase carrier immobilized on the solid phase carrier "the present anti-HMGB1 degradation product antibody or the like". "Antibody" is reacted, and transmitted light and scattered light are measured by the endpoint method or the rate method.

In the case of visual measurement, the sample and the "antibody such as the anti-HMGB1 degradation product antibody" immobilized on the solid-phase carrier are reacted in the container such as a plate or a microplate to cause agglutination. Visually determine the condition.

Instead of this visual measurement, a device such as a microplate reader may be used for the measurement.

[III] Reagent for Measuring Degradation Product of Thrombin or Thrombomodulin Complex of HMGB1 (1) General Reagent for Measuring Degradation Product of Thrombin or Thrombomodulin Complex of HMGB1 of the Present Invention (hereinafter, "Measuring Reagent of the Present Invention") ) Includes an antibody that specifically binds to the thrombin of HMGB1 or the degradation product of the thrombin-thrombomodulin complex, which is characterized by specifically recognizing the amino acid sequence set forth in SEQ ID NO: 1. It is not particularly limited as long as it is characterized by.

In the measurement reagent of the present invention, the antibody may contain only the present anti-HMGB1 degradation product antibody, or the present anti-HMGB1 degradation product antibody may be combined with another anti-HMGB1 antibody or another anti-HMGB1 degradation product antibody. May be included.

In the measurement reagent of the present invention, the present anti-HMGB1 degradation product antibody is a polyclonal antibody, an anti-serum containing a polyclonal antibody, a monoclonal antibody, and fragments of these antibodies (Fab, F (ab') 2 or Fab', etc.). , Or a single chain antibody (scFv) or the like.

In the measuring reagent of the present invention, the anti-HMGB1 degradation product antibody is preferably a monoclonal antibody.

Further, in the measuring reagent of the present invention, the anti-HMGB1 degradation product antibody may contain not only one type but also a plurality of types.

Further, in the measuring reagent of the present invention, the other anti-HMGB1 antibody or other anti-HMGB1 degradation product antibody is a polyclonal antibody, an anti-serum containing a polyclonal antibody, a monoclonal antibody, and fragments of these antibodies (Fab, F (Fab, F). It may be either ab') 2 or Fab'), or a single chain antibody (scFv) or the like.

Further, in the measuring reagent of the present invention, the other anti-HMGB1 antibody or the other anti-HMGB1 degradation product antibody may contain not only one type but also a plurality of types at the same time.

Further, the measuring reagent of the present invention uses an immobilized antibody and a labeled antibody, and is any of the present anti-HMGB1 degradation product antibody and the above-mentioned other anti-HMGB1 antibody or other anti-HMGB1 degradation product antibody. One antibody may be used as the immobilized antibody and the other antibody may be used as the labeled antibody.

By including the anti-HMGB1 degradation product antibody, the measuring reagent of the present invention has high specificity for the HMGB1 degradation product, and only the HMGB1 degradation product can be accurately quantitatively measured.

(2) This anti-HMGB1 degradation product antibody For details of this anti-HMGB1 degradation product antibody, thrombin of HMGB1 or HMGB1 thrombin, which is characterized by specifically recognizing the amino acid sequence shown in "[I] SEQ ID NO: 1" described above. As described in the section "Antibodies that specifically bind to the degradation products of the thrombin-thrombomodulin complex".

(3) The other anti-HMGB1 antibody or the other anti-HMGB1 degradation product antibody For details of the other anti-HMGB1 antibody or the other anti-HMGB1 degradation product antibody, refer to the above-mentioned "[II] HMGB1 thrombin or thrombin. As described in the section "Method for measuring degradation products by thrombin modulin complex".

(4) Measuring reagent In the measuring reagent of the present invention, an immunological measuring method (sandwich) using a labeling substance such as an enzyme immunoassay, a fluorescent immunoassay, a radioimmunoassay or a luminescent immunoassay is used as a measuring principle. (Method or competition method, etc.), or immunological measurement to measure the formation of immune complex aggregates such as immunoturbidimetric method, latex agglutination method, latex agglutination reaction method, hemagglutination reaction method or particle agglutination reaction method. It can be applied without any particular limitation, such as that of a method.

For example, in immunoassay reagents based on the sandwich method in enzyme immunoassay (ELISA method), fluorescence immunoassay, luminescence immunoassay, etc., this anti-HMGB1 degradation product antibody and the other anti-antibodies described above. Of the HMGB1 antibody or the other anti-HMGB1 degradation product antibody, one of the antibodies may be used as the immobilized antibody and the other antibody may be used as the labeled antibody.

Further, for example, in an immunological measurement reagent based on a turbidimetry method, a latex agglutination reaction method, an hemagglutination reaction method, a particle agglutination reaction method, or the like, an antibody that immobilizes on a solid phase carrier such as latex particles. Is the present anti-HMGB1 degradation product antibody, and the present anti-HMGB1 degradation product antibody may be used as the antibody in the measurement reagent based on the immunoturbidimetry method.

In addition, this anti-HMGB1 degradation product antibody may be used in combination with the above-mentioned other anti-HMGB1 antibody or other anti-HMGB1 degradation product antibody.

(5) Other Reagent Components In the measuring reagent of the present invention, various aqueous solvents can be used as the solvent.
Examples of this aqueous solvent include purified water, physiological saline, and various buffer solutions such as Tris buffer solution, phosphate buffer solution, and phosphate buffered saline solution.

The pH of this buffer solution may be appropriately selected and used, and although there is no particular limitation, it is common to select and use a pH in the range of pH 3 to 12.

In addition, the measurement reagent of the present invention includes a "immobilized antibody" in which the anti-HMGB1 degradation product antibody or the like is immobilized on a solid phase carrier, and / or a labeling substance such as the present anti-HMGB1 degradation product antibody or the like and an enzyme. In addition to the reagent components such as bound "labeled antibody", proteins such as bovine serum albumin (BSA), human serum albumin (HSA), egg white albumin, casein, gelatin or salts thereof; various salts; various sugars; defatted milk powder Various animal sera such as normal rabbit serum; various preservatives such as sodium azide or antibody; activator; reaction promoter; sensitivity increasing substance such as polyethylene glycol; non-specific reaction inhibitor; or nonionic One or two or more kinds of various surfactants such as a surfactant, an amphoteric surfactant, a cationic surfactant, and an anionic surfactant may be appropriately contained.

The concentration when these are contained in the measurement reagent is not particularly limited, but is preferably 0.001 to 10% (w / v), and particularly preferably 0.01 to 5% (w / v). ..

Examples of the surfactant include sorbitan fatty acid ester, glycerin fatty acid ester, decaglycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene glycerin fatty acid ester, polyethylene glycol fatty acid ester, and polyoxyethylene alkyl ether. Nonionic surfactants such as polyoxyethylene phytosterol, phytostanol, polyoxyethylene polyoxypropylene alkyl ether, polyoxyethylene alkyl phenyl ether, polyoxyethylene castor oil, hardened castor oil or polyoxyethylene lanolin; betaine acetate, etc. Amphoteric surfactants; cationic surfactants such as amine salts or quaternary ammonium salts; or anionic surfactants such as polyoxyethylene alkyl ether sulfates or polyoxyethylene alkyl ether acetates. Can be mentioned.

(6) Structure of Measuring Reagent The measuring reagent of the present invention can be used alone for measuring the HMGB1 degradation product contained in a sample. And it can be sold by itself.

In addition, the measuring reagent of the present invention can be used in combination with other reagents for measuring the HMGB1 degradation product contained in the sample. Then, it can be sold in combination with other reagents.

The other reagents mentioned above include, for example, a buffer solution, a sample diluent, a reagent diluent, a reagent containing a labeling substance, a reagent containing a substance that generates a signal such as color development, and a reagent involved in the generation of a signal such as color development. Examples thereof include reagents containing substances, reagents containing substances for performing calibration, reagents containing substances for performing quality control, and the like.

Then, the other reagent described above may be used as the first reagent and the measuring reagent of the present invention may be used as the second reagent, or the measuring reagent of the present invention may be used as the first reagent and the other reagent may be used as the second reagent. , Can be used and sold in various combinations as appropriate.

Further, the measuring reagent of the present invention may be a measuring reagent kit composed of a plurality of constituent reagents such as the first reagent and the second reagent, or the other reagents described above.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

[Example 1] (Preparation of human HMGB1, human HMGB2, pig HMGB1, pig HMGB2, human des-HMGB1, human des-HMGB2, pig des-HMGB1 and pig des-HMGB2)

[1] Preparation of human HMGB1 and human HMGB2 Human HMGB1 and human HMGB2 were purified from HL60 cells as follows according to the literature (P. Cabart et al. Cell Biochemistry and Function 13; 125-133: 1995).

(1) HL60 cells were cultured in 300 mL of RPMI1640 medium (Gibco) containing 10% immobilized FCS (fetal bovine serum; Gibco) for about one week.

(2) The cultured HL60 cells were collected, washed with RPMI1640 medium, and then cultured with 3 L of PFHM-II medium (Invitrogen) for about 2 weeks.

(3) Next, this culture supernatant was passed through a heparin-cepharose column (Sigma) equilibrated with PBS.

(4) After washing well with PBS, elution was performed with PBS containing 0.5 M sodium chloride.

This elution was monitored by absorption at 280 nm and the absorptive portion was pooled.

The pooled solution was well dialyzed against 5 mM borate buffer (pH 9.0) containing 200 mM sodium chloride.

This dialyzed pool solution was added to a CM-Sephadex C25 column (GE Healthcare) equilibrated with 7.5 mM borate buffer (pH 9.0).

Then, it was eluted with 7.5 mM sodium borate buffer (pH 9.0) containing 200 mM sodium chloride.

As a result, human HMGB1 and human HMGB2 were prepared, respectively.

[2] Preparation of porcine HMGB1 and porcine HMGB2 Next, porcine HMGB1 and porcine HMGB2 were prepared.
(1) First, 500 g of porcine thymus was crushed with 600 mL of a solution containing 140 mM sodium chloride and 0.5 mM PMSF.

(2) Next, the crushed material was centrifuged with a centrifuge to remove the supernatant.

(3) A solution containing 140 mM sodium chloride and 0.5 mM PMSF was added thereto, and the mixture was stirred and then centrifuged with a centrifuge to remove the supernatant.
This cleaning operation was repeated twice.

(4) Next, 300 mL of 0.75 M perchloric acid was added to the obtained precipitate.
Then, after centrifuging with a centrifuge, the supernatant liquid was separated.
To the remaining precipitate was added 400 mL of 0.75 M perchloric acid.
Again, after centrifuging with a centrifuge, the supernatant was separated.
This supernatant was combined with the previously separated supernatant.
The precipitate was discarded.

(5) 0.75 M perchloric acid was added to the combined supernatant to bring the total volume to 1,000 mL.
Next, after centrifuging with a centrifuge, the supernatant was filtered through a glass filter (grade 4).

(6) A mixed solution of 3,500 mL of acetone and 21 mL of concentrated hydrochloric acid was added to the above-mentioned filtered filtrate.
Since turbidity was generated, the supernatant was separated by centrifuging with a centrifuge.

To this supernatant was added 2,500 mL of acetone.
Then, since turbidity was generated again, this was centrifuged by a centrifuge to separate the supernatant liquid, and the remaining precipitate was collected.

(7) The collected precipitate was air-dried at room temperature.
By the above operation, a protein fraction containing porcine HMGB1 and porcine HMGB2 was obtained in an amount of about 450 mg.

(8) The protein fraction containing porcine HMGB1 and porcine HMGB2 is dissolved in 10 mL of 7.5 mM sodium borate buffer (pH 9.0) containing 200 mM sodium chloride, and then containing this 200 mM sodium chloride. Sufficient dialysis was performed with 5 mM sodium borate buffer (pH 9.0).

(9) After this dialysis, it was added to a column of CM-Sephadex C25 equilibrated with 7.5 mM sodium borate buffer (pH 9.0).
Then, it was eluted with 7.5 mM sodium borate buffer (pH 9.0) containing 200 mM sodium chloride, and cation exchange chromatography was performed.

(10) Each fraction eluted here was subjected to SDS-polyacrylamide gel electrophoresis, and the fraction containing porcine HMGB1 and the fraction containing porcine HMGB2 were identified from their mobility.
By the above operation, pig HMGB1 and pig HMGB2 were separated.
The amino acid sequence homology between human HMGB1 and porcine HMGB1 is 99.1%, and the amino acid sequence homology between human HMGB2 and porcine HMGB2 is also 99.1%.

[3] Preparation of human des-HMGB1, human des-HMGB2, pig des-HMGB1 and pig des-HMGB2 Next, human des-HMGB1, human HMGB2, pig HMGB1 and pig HMGB2 are treated with thrombin to treat human des-HMGB1, human des. -HMGB2, pig des-HMGB1 and pig des-HMGB2 were prepared.

(1) First, human HMGB1, human HMGB2, porcine HMGB1 and porcine HMGB2 were dissolved in 50 mM phosphate buffer (pH 7.0) at 200 μg / mL, respectively.

(2) Next, thrombin 20u (unit) / mL (Mochida Pharmaceutical Co., Ltd.) was added to each of the human HMGB1, human HMGB2, porcine HMGB1 and porcine HMGB2 at 37 ° C. for 0 hours, 1 hour and 2 respectively. Processed for hours and 3 hours.

(3) With this, thrombin degradation products of human HMGB1, human HMGB2, porcine HMGB1 and porcine HMGB2, that is, human des-HMGB1, human des-HMGB2, porcine des-HMGB1 and porcine des-HMGB2 were obtained.

(4) As a result, it was clarified for the first time that human HMGB2 and porcine HMGB2 are substrates for thrombin.

In FIG. 1, 200 μg / mL human HMGB1 and 200 μg / mL human HMGB1 were contact-treated with 20 u (unit) / mL thrombin for 0 hours, 1 hour, 2 hours, or 3 hours, respectively. The migration image is shown.

Specifically, lane 1 shows a human HMGB1 of 200 μg / mL that has been contact-treated with 20u (unit) / mL of thrombin for 0 hours (that is, that has not been contact-treated with thrombin), and lane 2 has been treated. Indicates that 200 μg / mL human HMGB1 was contacted with 20u (unit) / mL thrombin for 1 hour, and lane 3 was contacted with 200 μg / mL human HMGB1 with 20u (unit) / mL thrombin for 2 hours. The treated product is shown, and lane 4 shows a human HMGB1 of 200 μg / mL treated with 20 u (unit) / mL thrombin for 3 hours.

In addition, lane 5 shows 200 μg / mL human HMGB2 contacted with 20 u (unit) / mL thrombin for 0 hours (that is, no contact treatment with thrombin), and lane 6 shows 200 μg / mL. The mL human HMGB2 was contacted with 20u (unit) / mL thrombin for 1 hour, and lane 7 was contacted with 200 μg / mL human HMGB2 with 20u (unit) / mL thrombin for 2 hours. And lane 5 shows 200 μg / mL human HMGB2 contacted with 20 u (unit) / mL thrombin for 3 hours.

From this electrophoretic image, as compared with lane 1 and lane 5 which were not subjected to the contact treatment with thrombin, lane 2, lane 3 and lane 4, and lane 6, lane 7 and lane 8 had bands on the lower molecular weight side. It was observed, and the formation of des-HMGB1 and des-HMGB2 was confirmed.

[Example 2] (Preparation of peptide of amino acid sequence of SEQ ID NO: 2)
A peptide having the amino acid sequence of SEQ ID NO: 2 in which cysteine for binding to a carrier was attached to the N-terminal of the amino acid sequence of SEQ ID NO: 1 was synthesized.

First, the peptide of the amino acid sequence of SEQ ID NO: 2 was synthesized by the t-butoxycarbonyl amino acid solid phase method according to the instruction manual using the model 430A peptide synthesizer (Model 430A peptide synthesizer) of Applied Biosystems. went.

Peptides synthesized from the resin by the hydrogen fluoride method were desorbed in the presence of dimethylsulfide, p-thiocresol, m-cresol and anisole as scavengers to suppress side reactions.

Then, the scavenger was extracted with dimethyl ether, and the peptide synthesized with 2N acetic acid was extracted.

Then, anion exchange column chromatography is performed and purified by Dawex 1-X2 (DOWNEX 1-X2), which is an anion exchange resin, and high performance liquid chromatography (HPLC) on an octadecyl (ODS) column is used for main. The peak pattern was confirmed.

Then, it was freeze-dried by an evaporator to concentrate, and then purified by HPLC and separated.

As the equipment and conditions for this HPLC purification, a reverse phase ODS column YMC-D-ODS-5 (20 mm × 300 mm) manufactured by Yamamura Chemical Laboratory Co., Ltd. was used, and the TWINCLE pump manufactured by JASCO Corporation and JASCO Corporation. A GP-A40 type gradienter was used to gradient 0% to 70% of acetonitrile in 0.1% trifluoroacetic acid (TFA) at a flow rate of 7.0 mL / min, and a UVIDEC-100V type detector (210 nm) manufactured by JASCO Corporation. , 1.28AUFS).

The synthetic peptide purified and separated here was freeze-dried with an evaporator and concentrated.

Next, the purity of the obtained synthetic peptide was analyzed by HPLC.
The equipment and conditions used for this analysis were the reverse phase ODS column YMC-R-ODS-5 (4.9 mm x 300 mm) manufactured by Yamamura Chemical Laboratory Co., Ltd., and the Twincle pump manufactured by JASCO Corporation and JASCO Corporation. UVIDEC-100V type detection by JASCO Corporation by performing 0% to 70% gradient of acetonitrile in 0.1% trifluoroacetic acid (TFA) with GP-A40 type gradienter at a flow rate of 1.0 mL / min for 25 minutes. Detection was performed with a vessel (210 nm, 1.28 AUFS).

As a result of this analysis, it was found that the purity of the obtained synthetic peptide was almost 100%.

[Example 3] (Preparation of a conjugate of a peptide consisting of a synthesized amino acid sequence and a carrier)

Two types of carriers (carriers) were bound to the peptide having the amino acid sequence of SEQ ID NO: 2 synthesized in Example 2, respectively, to prepare a conjugate (two types) of the peptide and the carrier.

(1) Preparation of a conjugate with a synthetic peptide using bovine serum albumin as a carrier 10 mg of bovine serum albumin (BSA) (Biochemical Industry Co., Ltd.) as a carrier (carrier) is 10 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate. Dissolve in potassium buffer (pH 7.0), and add 150 μL of 2.5% maleimide benzoyl-N-hydroxysuccinimid ester (MBS) (Pierce) solution dissolved in N, N-dimethylformamide. The reaction was carried out for 30 minutes with stirring at room temperature.

A Sephadex G-25 column (Sephadex G-25), which is a gel filtration column in which this is equilibrated with 10 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH 7.0) at 4 ° C. The MBS-carrier binding component was collected by subjecting to GE Healthcare) and monitoring by the absorbance at 280 nm.

The MBS-carrier binding component was adjusted to pH 7.0 with trisodium phosphate, and the peptide having the amino acid sequence of SEQ ID NO: 2 synthesized in Example 2 was added and mixed, and the mixture was reacted for 150 minutes.

After the reaction, the mixture was dialyzed against water three times and then freeze-dried to obtain a conjugate of the peptide having the amino acid sequence of SEQ ID NO: 2 and the carrier BSA, which consisted of a carrier bound to the peptide.

(2) Preparation of a solution with a synthetic peptide using hemocyanin as a carrier 10 mg of hemocyanin (KLH) (Kalbiochem) as a carrier is 10 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer. Add 150 μL of a 2.5% maleimide benzoyl-N-hydroxysuccinimid ester (MBS) (Pierce) solution dissolved in N, N-dimethylformamide to the solution (pH 7.0) at room temperature. The reaction was carried out for 30 minutes with stirring.

A Sephadex G-25 column (Sephadex G-25), which is a gel filtration column in which this is equilibrated with 10 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH 7.0) at 4 ° C. The MBS-carrier binding component was collected by subjecting to GE Healthcare) and monitoring by the absorbance at 280 nm.

The MBS-carrier binding component was adjusted to pH 7.0 with trisodium phosphate, and the peptide having the amino acid sequence of SEQ ID NO: 2 synthesized in Example 2 was added and mixed, and the mixture was reacted for 150 minutes.

After the reaction, the mixture was dialyzed against water three times and then freeze-dried to obtain a conjugate of the peptide having the amino acid sequence of SEQ ID NO: 2 and the carrier KLH, which consisted of a carrier bound to the peptide.

[Example 4] (Preparation of anti-human des-HMGB1 monoclonal antibody derived from a mouse using a conjugate of a synthesized peptide and a carrier as an immunogen)
(1) A mouse is immunized with a conjugate of BSA as a carrier prepared in Example 3 and a peptide having the amino acid sequence of SEQ ID NO: 2 as an immunogen, and a mouse anti-human des-HMGB1 monoclonal antibody is used using this mouse spleen. (47C1) was acquired.

For 1 volume of the conjugate of the peptide of the amino acid sequence of SEQ ID NO: 2 and BSA as a carrier prepared in Example 3 as an immunogen, 1 FREUND adjuvant (DIFCO LABORATORIES) as a chemically synthesized adjuvant was used. Mixed in volume proportions.

(2) Next, as an immunogen in the abdominal cavity of a mouse (BALB / c), a peptide having the amino acid sequence of SEQ ID NO: 2 prepared in Example 3 of 300 to 500 μg / animal / dose and BSA as a carrier were added. A mixture of the conjugate and the FREUND complete adjuvant was injected, and after 2 and 4 weeks, the peptide of the amino acid sequence of SEQ ID NO: 2 described above, the conjugate of the carrier BSA, and the FREUND incomplete adjuvant were injected intraperitoneally into the mouse. The mixture with was injected.

(3) Four weeks after the final immunization, the undiluted solution of the conjugate of the peptide of the amino acid sequence of SEQ ID NO: 2 prepared in Example 3 and the carrier BSA was boosted with 300 μg / animal, and the next day, immunization was performed. Mieloma cells (P3U1) were mixed at a ratio of 1: 1 to 10: 1 and polyethylene glycol (PEG1500; Roche) was added by a general method for cell fusion and grown hybridomas. Colonies were sorted.

Specifically, cell fusion was performed as follows.
The mixed spleen cells and myeloma cells (P3U1) were centrifuged to remove the supernatant, suspended in 1 mL of polyethylene glycol (PEG1500; Roche) at room temperature for 1 minute, and then stirred at 37 ° C. for 1 minute.

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.

After washing the cells several times, they were suspended in a medium containing hypoxanthine, aminopterin, and thymidine, dispensed into wells of a 96-well microtiter plate, and cultured at _{37 ° C. in the presence of 5% CO 2.}

As a method for selecting the grown monoclonal antibody-producing cell line (fusion cell line), 7 to 14 days after the cell fusion, the peptide of the amino acid sequence of SEQ ID NO: 2 was immobilized, and the fusion cell culture supernatant was used as the primary antibody. It was performed by the system of the ELISA method.

Specifically, this ELISA method was carried out as follows (i) to (viii).
(I) A peptide of the amino acid sequence of SEQ ID NO: 2 prepared in Example 2 and KLH bound thereto was prepared with phosphate buffered saline to a concentration of 1 μg / mL, or as a control. 100 μL of Phosphate Buffered Saline was injected into the wells of each 96-well microtiter plate [Thermo Fisher Scientific Inc.], and allowed to stand at 5 ° C. for 16 to 24 hours (or 37 ° C. for 2 hours). A peptide having the amino acid sequence of No. 2 bound to KLH was immobilized, and further allowed to stand at 5 ° C. for 16 to 24 hours (or 37 ° C. for 2 hours) in phosphate buffered saline containing 1% BSA. , Blocked.

(Ii) Next, each well of the microtiter plate of (i) was washed three times with a washing solution [phosphate buffered saline containing 0.05% Tween 20].

(Iii) Next, 100 μL of each of these fusion cell culture supernatant solutions was injected into the wells of the microtiter plate washed in (ii) as a sample, and allowed to stand at 37 ° C. for 2 hours. The peptide of the amino acid sequence of SEQ ID NO: 2 immobilized on the well of the titer plate was reacted with the antibody contained in each of the fusion cell culture supernatant solutions.

(Iv) Next, each well of the microtiter plate of (iii) was washed three times with the cleaning solution of (ii).

(V) Next, a POD-labeled anti-mouse IgG antibody (DakoCytomation) diluted 1000-fold with phosphate buffered saline containing 1% BSA was applied to each well of the microtiter plate washed in (iv). 100 μL was injected and allowed to stand at 37 ° C. for 2 hours to carry out the reaction.

(Vi) Next, each well of the microtiter plate of (v) was washed three times with the washing liquid.

(Vii) Next, a color-developing solution containing 0.045% 3,3', 5,5'-tetramethylbenzidine hydrochloride aqueous solution (pH 2.0) containing 0.2 mM EDTA.2 sodium and 5 mM hydrogen peroxide. 100 μL of a color-developing substrate solution prepared by mixing 1: 1 with a substrate solution consisting of a 60 mM disodium phosphate aqueous solution (pH 4.3) containing hydrogen peroxide, 41 mM citric acid, and 0.2 mM EDTA.2 sodium. , Was injected into each well of the microtiter plate washed in (vi) above, allowed to stand at room temperature for 30 minutes, reacted, and developed a color.

Then, 100 μL of a reaction terminator solution consisting of 0.7N sulfuric acid was dispensed into each well to stop the color development reaction.

(Vii) Next, for each well of the microtiter plate of (vii), the absorbance at a main wavelength of 450 nm and a sub-wavelength of 550 nm was measured using a spectrophotometer.

By the above measurement, a fusion cell line producing an antibody that binds to human des-HMGB1 was selected, and one clone was established from the grown fusion cell lines, which was named "18120 47C1 strain".

(4) IgG (immunoglobulin G) was purified from this selected monoclonal antibody-producing cell line as follows.

This monoclonal antibody-producing cell line was cultured in PFHM-II medium (GIBCO) at 37 ° C. in _{a CO 2 incubator.}

After culturing, IgG in the supernatant was bound to a protein A column (GE Healthcare).

The bound IgG was eluted with a 100 mM aqueous citric acid solution (pH 3.0).

To 1 volume of eluate, 1 volume of 0.5 M phosphate buffer (pH 7.5) buffer was added, and as purified IgG, an antibody that binds to human des-HMGB1 was used as a monoclonal antibody-producing cell line (18120 47C1 strain). ) Obtained from.

This mouse-derived anti-human des-HMGB1 monoclonal antibody was named anti-human des-HMGB1 monoclonal antibody (47C1).

The 18120 47C1 strain, which is a monoclonal antibody-producing cell line of "anti-des-HMGB1 monoclonal antibody (47C1)", is a patented microorganism deposit center [NPMD] of the National Institute of Technology and Evaluation [NPMD] (Kazusakamatari, Kisarazu City, Chiba Prefecture, Japan). It was received as "Receipt number: NITE AP-03024" on September 20, 2019 in 2-5-8).

[Example 5] (Preparation of peroxidase-labeled anti-human des-HMGB1 antibody)
A peroxidase-labeled anti-human des-HMGB1 monoclonal antibody was prepared.

(1) Introduction of maleimide group into peroxidase After dissolving 4 mg of peroxidase (POD) [derived from Western wasabi] in 0.3 mL of 0.1 M phosphate buffer (pH 7.0), N-succinimidyl-4- 1.0 mg of (N-maleimidemethyl) cyclohexane-1-carboxylic acid dissolved in 60 μL of N, N'-dimethylformamide was added, and the mixture was reacted at 30 ° C. for 60 minutes.

Then, dialysis was performed overnight with 0.1 M phosphate buffer (pH 6.0).
By the above operation, a maleimide group was introduced into the peroxidase.

(2) Introduction of thiol group into anti-human des-HMGB1 monoclonal antibody (47C1) 10 mg of the anti-human des-HMGB1 monoclonal antibody (47C1) prepared by the present inventors described in (4) of Example 4 above. 0.6 mg of S-acetylmercaptohydride succinic acid was dissolved in 10 μL of N, N'-dimethylformamide in 0.5 mL of 0.1 M phosphate buffer solution (pH 6.5) containing at a concentration of / mL. The product was added and reacted at room temperature for 30 minutes.

Then, 20 μL of 0.1 M EDTA, 0.1 mL of 0.1 M Tris-chloride buffer (pH 7.0), and 0.1 mL of 1 M hydroxylamine hydrochloride (pH 7.0) were added thereto. Then, it was left at 30 ° C. for 5 minutes.

It was then passed through a column of Sephadex G-25 equilibrated with 0.1 M phosphate buffer (pH 6.0) containing 5 mM EDTA, subjected to simple gel filtration chromatography and excess S-. Acetylmercapto succinic anhydride was removed and antibody fractions were collected.

By the above operation, a thiol group was introduced into the anti-human des-HMGB1 monoclonal antibody (47C1).

(3) Preparation of peroxidase-labeled anti-human des-HMGB1 monoclonal antibody Peroxidase introduced with the maleimide group prepared in (1) above and anti-human des-HMGB1 monoclonal antibody introduced with the thiol group prepared in (2) above. (47C1) was mixed in equal amounts and reacted at 30 ° C. for 20 hours to introduce (label) peroxidase into the antibody.

This was then passed through a column of Ultragel AcA34 equilibrated with 0.1 M phosphate buffer (pH 6.5) for gel filtration chromatography.

Each fraction of this gel filtration chromatography was confirmed by 10% polyacrylamide gel electrophoresis, and only the fraction of the antibody to which peroxidase was bound was collected so as not to be contaminated with unbound peroxidase.

The fraction of the antibody to which the peroxidase was bound was concentrated to obtain an anti-human des-HMGB1 monoclonal antibody (47C1) to which the peroxidase was bound, that is, a peroxidase-labeled anti-human des-HMGB1 monoclonal antibody.

Then, the protein concentration of the solution containing the peroxidase-labeled anti-human des-HMGB1 antibody was measured.

[Example 6] (Confirmation of reactivity of anti-human des-HMGB1 monoclonal antibody (47C1) with human HMGB1, human des-HMGB1 and human des-HMGB2)

The reactivity of the anti-human des-HMGB1 monoclonal antibody (47C1) prepared in Example 4 with respect to human HMGB1, human des-HMGB1 and human des-HMGB2 was confirmed by Western blotting.

(1) 4% (w / v) of human HMGB1 (0.3 mg / mL), human des-HMGB1 (0.3 mg / mL) and human des-HMGB2 (0.3 mg / mL) prepared in Example 1. Equal amounts were mixed with 125 mM tris (hydroxymethyl) aminomethane buffer (pH 6.8) containing sodium dodecyl sulfate, 20% (w / v) glycerin and an appropriate amount of bromphenol blue.

(2) Next, this was electrophoresed using a 15% SDS-polyacrylamide gel.
Then, using a barbital buffer (pH 8.8) as the electrophoresis buffer, electrophoresis was performed by energizing with a current of 17 mA for 180 minutes.

(3) The transcription after the electrophoresis of (2) above was carried out by a dry method using a Nova Blot Electrophoretic Transfer Kit (GE Healthcare) according to the instruction manual thereof.

First, the gel subjected to the electrophoresis in (2) above was placed on a transfer device.

Next, an 8 cm × 8 cm nitrocellulose membrane (Biorad) was overlaid on this gel, and 48 mM tris (hydroxymethyl) aminomethane, 39 mM glycine, 0.0357% (w / v) sodium dodecyl sulfate (SDS). ) And 20% (v / v) methanol were used for transfer at a current of 80 mA for 2 hours.

(4) The transferred nitrocellulose membrane was subjected to phosphate buffered saline (5.59 mM disodium hydrogen phosphate, 1.47 mM potassium dihydrogen phosphate, 137 mM sodium chloride and 2.68 mM) containing 1% BSA. Blocking was performed by immersing in 20 mL of an aqueous solution containing potassium chloride (pH 7.2)) at 4 ° C. overnight.

Next, this was washed with shaking in 20 mL of a washing solution (phosphate buffered saline containing 0.05% Tween 20) for 10 minutes.
This cleaning operation was performed three times.

(5) 20 mL of the peroxidase-labeled anti-human des-HMGB1 antibody prepared in Example 5 diluted 1000-fold with phosphate buffered saline containing 1% BSA was prepared.

The nitrocellulose membrane subjected to the operation (4) above was immersed in this solution at room temperature for 2 hours for reaction.

Next, this nitrocellulose membrane was washed by shaking in 20 mL of a washing solution for 10 minutes.
This cleaning operation was performed three times.

(6) Next, the nitrocellulose membrane of (5) above was added to 20 mL of phosphate buffered saline containing 0.025% 3,3'-diaminobenzidine tetrachloride and 0.01% hydrogen peroxide. It was immersed for 15 minutes at room temperature to develop color.

(7) By the above operation, the result of Western blotting of the anti-human des-HMGB1 antibody (47C1) prepared in Example 4 was obtained.
The results of this Western blotting are shown in Fig. 2.

In this figure, the rightmost lane shows a molecular weight marker, lane 1 shows HMGB1, lane 2 shows des-HMGB1, and lane 3 shows des-HMGB2.

From this figure, it was confirmed that it is an anti-human des-HMGB1 monoclonal antibody (47C1) that does not react with human HMGB1 but binds to both human des-HMGB1 and human des-HMGB2.

[Example 7] (Synthesis of peptide of selected amino acid sequence portion)
A peptide having the amino acid sequence of SEQ ID NO: 4 in which cysteine for binding to a carrier was attached to the N-terminal of the amino acid sequence of SEQ ID NO: 3 was synthesized.
The amino acid sequence of SEQ ID NO: 3 is the amino acid sequence from the 167th lysine to the 180th lysine of HMGB1 from the N-terminal.

First, the peptide of the amino acid sequence of SEQ ID NO: 4 was synthesized by the t-butoxycarbonyl amino acid solid phase method according to the instruction manual using the model 430A peptide synthesizer (Model 430A peptide synthesizer) of Applied Biosystems. went.

Peptides synthesized from the resin by the hydrogen fluoride method were desorbed in the presence of dimethylsulfide, p-thiocresol, m-cresol and anisole as scavengers to suppress side reactions.

Then, the scavenger was extracted with dimethyl ether, and the peptide synthesized with 2N acetic acid was extracted.

Anion exchange column chromatography is performed on an anion exchange resin, DOWEX 1-X2, to purify the product, and then high performance liquid chromatography (HPLC) on an octadecyl (ODS) column is performed to obtain the main peak. The pattern was confirmed.

Then, it was freeze-dried by an evaporator to concentrate, and then purified by HPLC and separated.

As the equipment and conditions for this HPLC purification, a reverse phase ODS column YMC-D-ODS-5 (20 mm × 300 mm) manufactured by Yamamura Chemical Laboratory Co., Ltd. was used, and the TWINCLE pump manufactured by JASCO Corporation and JASCO Corporation. A GP-A40 type gradienter was used to gradient 0% to 70% of acetonitrile in 0.1% trifluoroacetic acid (TFA) at a flow rate of 7.0 mL / min for 25 minutes, and a UVIDEC-100V type detector manufactured by JASCO Corporation. Detection was performed at (210 nm, 1.28 AUFS).

The synthetic peptide purified and separated here was freeze-dried with an evaporator and concentrated.

Next, the purity of the obtained synthetic peptide was analyzed by HPLC.
The equipment and conditions used for this analysis were the reverse phase ODS column YMC-R-ODS-5 (4.9 mm x 300 mm) manufactured by Yamamura Chemical Laboratory Co., Ltd., and the Twincle pump manufactured by JASCO Corporation and JASCO Corporation. UVIDEC-100V type detection by JASCO Corporation by performing 0% to 70% gradient of acetonitrile in 0.1% trifluoroacetic acid (TFA) with GP-A40 type gradienter at a flow rate of 1.0 mL / min for 25 minutes. Detection was performed with a vessel (210 nm, 1.28 AUFS).

As a result of this analysis, it was found that the purity of the obtained synthetic peptide was almost 100%.

[Example 8] (Preparation of a conjugate of a peptide consisting of a selected amino acid sequence and a carrier)
A peptide having the amino acid sequence of SEQ ID NO: 4 synthesized in Example 7 was bound to a carrier (bovine serum albumin) to prepare a peptide.

10 mg of bovine serum albumin (BSA) [Biochemical Industry Co., Ltd.] as a carrier was dissolved in 10 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH 7.0), and N, N- 150 μL of a 2.5% maleimide benzoyl-N-hydroxysuccinimid ester (MBS) (Pierce) solution dissolved in dimethylformamide was added, and the mixture was reacted at room temperature for 30 minutes with stirring.

A Sephadex G-25 column (Sephadex G-25), which is a gel filtration column in which this is equilibrated with 10 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH 7.0) at 4 ° C. The MBS-carrier binding component was collected by subjecting to GE Healthcare) and monitoring by the absorbance at 280 nm.

The MBS-carrier binding component was adjusted to pH 7.0 with trisodium phosphate, and the peptide having the amino acid sequence of SEQ ID NO: 4 synthesized in Example 7 was added and mixed, and the mixture was reacted for 150 minutes.

After the reaction, the mixture was dialyzed against water three times and then freeze-dried to obtain a conjugate of the peptide having the amino acid sequence of SEQ ID NO: 4 and BSA as a carrier.

[Example 9] (Preparation of anti-human HMGB1 antibody derived from birds using a conjugate of a synthesized peptide and a carrier as an immunogen)
An anti-human HMGB1 antibody derived from birds was prepared using the "combined product of the synthesized peptide and carrier" prepared in Example 8 as an immunogen.

An anti-human HMGB1 antibody was obtained from chickens, which are birds, using the "combined product of synthesized peptide and carrier" prepared in Example 8 as an immunogen.

(1) The conjugate of the peptide having the amino acid sequence of SEQ ID NO: 4 and the carrier BSA prepared in Example 8 was dissolved in physiological saline (0.9% sodium chloride aqueous solution) so as to be 100 μg / mL. did.

Next, this was mixed with Freund's complete adjuvant in equal amounts to form an emulsion, and then 0.5 mL was injected into each of the wing roots of 10 female chickens (Asahi Technograss) using this as an immunogen. I was immunized.

(2) Two weeks after this initial immunization, the immunogen was dissolved in physiological saline to a concentration of 100 μg / mL, and this was mixed with Freund's incomplete adjuvant in equal amounts to form an emulsion, 0.5 mL thereof. A booster immunoinjection was performed.
This booster injection was continued every two weeks.

(3) Then, with respect to the eggs laid by the immunized chicken, the titer of the antibody in the yolk was measured weekly from the 6th week after the initial immunization by the enzyme-linked immunosorbent assay (ELISA method).

The operation of this ELISA method was performed as described in (i) to (vi) below.

(I) The conjugate of the peptide of the amino acid sequence of SEQ ID NO: 4 prepared in Example 8 and BSA as a carrier was dissolved in physiological saline to a concentration of 1 μg / mL, and this was dissolved in a 96-well-microtiter. 100 μL per well was added to a plate (Nunk), and the mixture was allowed to stand at 37 ° C. for 2 hours to solidify the conjugate of the peptide of the amino acid sequence of SEQ ID NO: 4 with BSA, which is a carrier, into the wells of this microtiter plate. It became.

(Ii) Next, each well of this microtiter plate is washed with a washing solution (0.05% Tween 20) in phosphate buffered saline (5.59 mM disodium hydrogen phosphate, 1.47 mM dihydrogen phosphate). After washing with an aqueous solution containing potassium, 137 mM sodium chloride and 2.68 mM potassium chloride (pH 7.2))), a 10 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH 7.2) containing 1% BSA. Was added in an amount of 300 μL per well, and the mixture was allowed to stand at 37 ° C. for 2 hours for blocking, and then washed again with the above washing solution.

(Iii) Next, for chicken eggs whose production of antibodies against human HMGB1 should be tested, 100 μL of the egg yolk is dissolved in 900 μL of physiological saline, diluted 10 times, and further diluted 1,000 times with physiological saline, 10 times. Diluted 000-fold and 100,000-fold, respectively.

Each of these diluted egg yolk solutions was added to the wells of the microtiter plate of (ii) in an amount of 100 μL, allowed to stand at 37 ° C. for 2 hours for reaction, and then washed with the washing solution.

(Iv) As a control, 100 μL of phosphate buffered saline containing 1% BSA was added in place of the diluted egg yolk solution, and the mixture was allowed to stand at 37 ° C. for 2 hours and then washed with the washing solution.

(V) Peroxidase (POD) -labeled anti-chicken IgY antibody (Up-Date) was diluted 5,000-fold with phosphate buffered saline containing 3% BSA, which was then diluted (iii). And 100 μL per well was added to the microtiter plate of (iv), and the mixture was allowed to stand at 37 ° C. for 2 hours to carry out the reaction.

(Vi) Next, after washing each well of this microtiter plate with the washing solution, a peroxidase reaction solution (3 mM 2,2'-azino-bis (3-ethylbenzthiazolin-6-sulfonic acid) [ABTS]. ] To 1 mL of 50 mM disodium hydrogen phosphate-24 mM citrate buffer (prepared by adding 2 μL of 1.7% hydrogen peroxide immediately before use) was added at room temperature by adding 100 μL per well. It was reacted.

Then, after 15 minutes, 150 μL of 6N sulfuric acid was added per well to stop the reaction.

This was measured by an EIA plate reader (Bio-Rad Laboratories, Inc.) for the absorbance of each well at 415 nm.

(4) As a result of measuring the titer of the antibody in the yolk in (3) above, it was confirmed that the antibody titer reached the plateau 1.5 to 2 months after the initial immunization. More antibody (IgY) was obtained.

(5) To 10 mL of egg yolk, 40 mL of 10 mM Tris-hydrochloric acid buffer (pH 7.4) containing 140 mM sodium chloride and 0.01% sodium azide was added, stirred well, and centrifuged to obtain a supernatant.

(6) Next, 7.5 mL of the Tris-hydrochloric acid buffer of (5) containing 1 M calcium chloride and the Tris-hydrochloric acid buffer of (5) containing 10% (w / v) dextran sulfate in this supernatant. 3 mL of the solution was added, and the mixture was stirred for 30 minutes and then centrifuged to obtain a supernatant and a precipitate.

The supernatant was left as it was, and the precipitate was re-extracted with the Tris-hydrochloric acid buffer of (5) above and centrifuged.

The supernatant obtained by re-extraction and centrifugation from this precipitate was combined with the supernatant before precipitation re-extraction to make 100 mL with the Tris-hydrochloric acid buffer solution of (5) above.

(7) 20 g of anhydrous sodium sulfate was added thereto, and the mixture was stirred for 30 minutes, centrifuged, and the precipitate was separated from the supernatant by decantation. The precipitate was dissolved in 10 mL of phosphate buffered saline.

Next, this was dialyzed against phosphate buffered saline, and then concentrated to obtain a globulin fraction.

(8) Next, this globulin fraction was passed through a column on which the peptide synthesized in Example 7 was immobilized, and affinity chromatography was performed.

This affinity chromatography was performed as described in (i) to (iv) below.

(I) 2 g of CNBr-Sepharose (GE Healthcare) was reacted with 10 mg of the peptide having the amino acid sequence of SEQ ID NO: 4 synthesized in Example 7 according to the instruction manual to obtain the synthetic peptide. Immobilized columns for affinity chromatography were prepared.

(Ii) This column was equilibrated with phosphate buffered saline, and then the globulin fraction obtained in (7) above was passed through this column.

(Iii) Then, a phosphate buffered saline was sufficiently passed through this column for washing, and then a 0.1 M acetate buffer solution (pH 3.0) was passed through the column.

(Iv) The eluted fractions were collected, dialyzed against phosphate buffered saline, and then concentrated.

By the above affinity chromatography operation, an antibody (polyclonal antibody) that binds to the peptide of the amino acid sequence of SEQ ID NO: 4 of the immunized chicken was separated.

It has already been clarified that this chicken-derived anti-human HMGB1 polyclonal antibody binds to human HMGB1 but not to human HMGB2. (International Publication No. 2008/07578 Pamphlet)

[Example 10] (Preparation of chicken-derived peroxidase-labeled anti-human HMGB1 antibody)
A chicken-derived peroxidase-labeled anti-human HMGB1 antibody was prepared.

(1) Introduction of maleimide group into peroxidase After dissolving 4 mg of peroxidase (POD) [derived from Western wasabi] in 0.3 mL of 0.1 M phosphate buffer (pH 7.0), N-succinimidyl-4- 1.0 mg of (N-maleimidemethyl) cyclohexane-1-carboxylic acid dissolved in 60 μL of N, N'-dimethylformamide was added, and the mixture was reacted at 30 ° C. for 60 minutes.

Then, dialysis was performed overnight with 0.1 M phosphate buffer (pH 6.0).
By the above operation, a maleimide group was introduced into the peroxidase.

(2) Introduction of thiol group into chicken-derived anti-human HMGB1 antibody The chicken-derived anti-human HMGB1 antibody prepared by the present inventors described in (8) of Example 9 is contained at a concentration of 10 mg / mL. To 0.5 mL of a 1 M phosphate buffer solution (pH 6.5), 0.6 mg of S-acetylmercaptosuccinic anhydride dissolved in 10 μL of N, N'-dimethylformamide was added, and 30 at room temperature. Reacted for minutes.

Then, 20 μL of 0.1 M EDTA, 0.1 mL of 0.1 M Tris-chloride buffer (pH 7.0), and 0.1 mL of 1 M hydroxylamine hydrochloride (pH 7.0) were added thereto. Then, it was left at 30 ° C. for 5 minutes.

It was then passed through a column of Sephadex G-25 equilibrated with 0.1 M phosphate buffer (pH 6.0) containing 5 mM EDTA, subjected to simple gel filtration chromatography and excess S-. Acetylmercapto succinic anhydride was removed and antibody fractions were collected.
By the above operation, a thiol group was introduced into the chicken-derived anti-human HMGB1 antibody.

(3) Preparation of chicken-derived peroxidase-labeled anti-human HMGB1 antibody The peroxidase introduced with the maleimide group prepared in (1) above and the chicken-derived anti-human HMGB1 antibody introduced with the thiol group prepared in (2) above were prepared. Equal amounts were mixed and reacted at 30 ° C. for 20 hours to introduce (label) peroxidase into the antibody.

This was then passed through a column of Ultragel AcA34 equilibrated with 0.1 M phosphate buffer (pH 6.5) for gel filtration chromatography.

Each fraction of this gel filtration chromatography was confirmed by 10% polyacrylamide gel electrophoresis, and only the fraction of the antibody to which peroxidase was bound was collected so as not to be contaminated with unbound peroxidase.

The fraction of the antibody to which this peroxidase was bound was concentrated to obtain a chicken-derived anti-human HMGB1 antibody to which peroxidase was bound, that is, a peroxidase-labeled anti-human HMGB1 antibody.

Then, the protein concentration of the solution containing this peroxidase-labeled anti-human HMGB1 antibody was measured.

[Example 11] (Measurement of des-HMGB1 in a sample)
Pig des-HMGB1 in the sample was measured by the ELISA method. (In addition, as a substitute for human des-HMGB1, pig des-HMGB1 having a homology of 99.7% was used as a sample.)

1. 1. Reagent i) Anti-human des-HMGB1 monoclonal antibody-immobilized plate The anti-human des-HMGB1 monoclonal antibody (47C1) obtained in Example 4 was diluted with phosphate buffer (pH 7.4) to 1 to 5 μg / mL.

Next, each of the above dilutions was injected into a 96-well microtiter plate at a rate of 100 μL per well and allowed to stand at 37 ° C. for 2 hours to immobilize each monoclonal antibody in each well.

Next, the mixture in each well was removed, and 300 μL of Tris buffer (pH 7.5) containing 2% BSA was dispensed into each well for blocking treatment.

After that, each plate was sealed with a plate seal and refrigerated until use so as not to evaporate.
This was used as an anti-human des-HMGB1 antibody-immobilized plate.

ii) Peroxidase-labeled anti-human HMGB1 antibody The chicken-derived peroxidase-labeled anti-human HMGB1 antibody prepared in Example 10 was diluted 1000 to 40,000 times with a labeled antibody diluent.

iii) Washing solution A phosphate buffered saline containing 0.05% Tween 20 was used as the washing solution.

iv) Diluted solution A "sample diluted solution" of a commercially available HMGB1 measuring reagent "HMGB1 ELISA Kit II" (Sinotest Co., Ltd.) was used as a diluted solution.

v) Color-developing solution A 0.045% 3,3', 5,5'-tetramethylbenzidine hydrochloride aqueous solution (pH 2.0) containing 0.2 mM EDTA.2 sodium was used as the color-developing solution.

vi) Substrate solution A 60 mM disodium phosphate aqueous solution (pH 4.3) containing 5 mM hydrogen peroxide, 41 mM citric acid, and 0.2 mM EDTA.2 sodium was used as the substrate solution.

vi) Color-developing substrate The color-developing solution of v) and the substrate solution of vi) were returned to room temperature before use and then mixed in equal amounts at the time of use to prepare a color-developing substrate.

viii) Reaction stop solution 0.7N sulfuric acid was used as the reaction stop solution.

ix) Labeled antibody diluent A "labeled antibody lysate" of a commercially available HMGB1 measurement reagent "HMGB1 ELISA Kit II" (Sinotest) was used as the labeled antibody diluent.

2. Sample The pig des-HMGB1 prepared in Example 1 was diluted with the above 1 iv) diluent so as to be 12.5 ng / mL, 25 ng / mL, and 50 ng / mL, respectively, to prepare a sample.

The iv) diluted solution of 1 above was used as a sample having a porcine des-HMGB1 concentration of 0 ng / mL.

3. 3. Measurement (1) Each well of the anti-human des-HMGB1 antibody-immobilized plate of 1 i) was washed 3 times with 400 μL of the iii) wash solution of 1 above.

(2) Next, 100 μL of the iv) diluent of 1 above was added to each well, and 10 μL of each sample prepared in 2 was added thereto, and the mixture was mixed with a plate mixer.

This was allowed to stand at room temperature for 20 to 24 hours, and the antigen-antibody reaction between each anti-human des-HMGB1 monoclonal antibody immobilized in the well of the anti-human des-HMGB1 antibody-immobilized plate and the added pig des-HMGB1 was carried out. Let me do it.

(3) Next, each well was washed three times with the washing solution of iii) described above.

(4) Next, the ii) chicken-derived peroxidase-labeled anti-human HMGB1 antibody of 1 above was diluted 1000-fold with the ix) -labeled antibody diluent of 1 above, and 100 μL of this was dispensed into each well.
The antigen-antibody reaction between pig des-HMGB1 bound to the anti-human des-HMGB1 antibody-immobilized plate via the anti-human des-HMGB1 antibody and the chicken-derived peroxidase-labeled anti-human HMGB1 antibody was allowed to stand at room temperature for 2 hours. Let me do it.

(5) Next, each well was washed three times with the washing solution of iii) described above.

(6) Next, 100 μL of the above 1 vii) color-developing substrate is added to each well, and the mixture is reacted at room temperature for 30 minutes to carry out a color development reaction by POD, and then the above 1 viii) reaction terminator is applied to each well. The color reaction was stopped by adding 100 μL each.

(7) Next, the absorbance (450 nm) of the liquid in each well was measured, and the value of the absorbance was obtained.

4. Measurement results The results of the measurement in 3 above are shown in FIG. This is the calibration curve.

In this figure, the vertical axis shows the concentration (ng / mL) of porcine des-HMGB1 in the sample, and the horizontal axis shows each measured value [absorbance of the liquid in each well (450 nm)].

Each measured value (absorbance value) in this table is obtained by subtracting reagent blinding (reagent blank).

From FIG. 3, in the sample containing pig des-HMGB1, the obtained absorbance increased in proportion to the concentration of pig des-HMGB1 contained in the sample, and was proportional to the concentration of pig des-HMGB1 contained in the sample. It was confirmed that the measured value was obtained.
That is, the calibration curve could be created.

[Example 12] (Measurement method of des-HMGB1 and measurement of a sample containing serum / plasma with a measurement reagent)
Regarding the method and reagent for measuring des-HMGB1 of the present invention, pig des-HMGB1 contained in a sample containing serum / plasma was measured to confirm the accuracy of the sample containing serum / plasma at the time of measurement. (In addition, as a substitute for human des-HMGB1, pig des-HMGB1 having a homology of 99.7% was used as a sample.)

2. Sample (1) The solution containing pig des-HMGB1 prepared in Example 1 was sufficiently prepared with 50 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH 7.4) containing 0.01% sodium azide. I dialyzed.

The protein concentration of the solution containing the porcine des-HMGB1 after dialysis was determined by a protein assay (manufactured by Bio-Rad).

Then, the solution containing the pig des-HMGB1 is diluted with 50 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH 7.4) containing 0.01% sodium azide, and the pig des-HMGB1 is added. A solution having a concentration of 20 ng / mL was prepared.

(2) The solution prepared in (1) above was diluted 2-fold with physiological saline.
Then, a sample containing no serum / plasma having a porcine des-HMGB1 concentration of 10 ng / mL was prepared.

(3) Further, the solution prepared in (1) above was diluted 2-fold with human serum or plasma known to have a human des-HMGB1 concentration and a porcine des-HMGB1 concentration of 0 ng / mL.
Then, a sample containing serum or plasma having a porcine des-HMGB1 concentration of 10 ng / mL was prepared.

3. 3. Measurement by enzyme immunoassay (sandwich method) (1) 100 μL of each sample prepared in 2 above was placed in the well of the microtiter plate-immobilized anti-human des-HMGB1 monoclonal antibody of 1 (i) of Example 11. The antibody was added and allowed to stand at room temperature for 12 hours to carry out an antigen-antibody reaction between the antibody immobilized on a microtiter plate and the pig des-HMGB1 contained in the sample.

(2) Next, each well of the microtiter plate-immobilized anti-human des-HMGB1 monoclonal antibody of Example 11-1 (i) was washed with the washing solution of Example 11-1 (iii).

(3) The chicken-derived peroxidase-labeled anti-human HMGB1 antibody of Example 11-1 (ii) was diluted 1,000-fold with phosphate buffered saline containing 3% BSA.

Next, 100 μL of this was added to each well of the microtiter plate-immobilized anti-human HMGB1 antibody subjected to the washing operation of (2), and then the antibody was allowed to stand at 37 ° C. for 2 hours.

As a result, porcine des-HMGB1 bound to the antibody immobilized on the microtiter plate was subjected to a reaction in which the peroxidase-labeled anti-human HMGB1 antibody was bound.

(4) After that, each well of the microtiter plate-immobilized anti-human des-HMGB1 monoclonal antibody of (3) was washed with the washing solution of 1 (iii) of Example 11.

(5) Next, 100 μL of the substrate solution of 1 (vi) of Example 11 was added to each well of the microtiter plate-immobilized anti-human des-HMGB1 monoclonal antibody of (4).
Then, it was reacted at room temperature.

(6) 15 minutes after the addition of the substrate solution of (5), the reaction terminator of 1 (viii) of Example 11 was applied to each well of the microtiter plate-immobilized anti-human des-HMBG1 monoclonal antibody. The reaction of the labeled peroxidase was stopped by adding 100 μL each.

(7) Next, the absorbance of the solution of the microtiter plate-immobilized anti-human des-HMGB1 monoclonal antibody of (6) in each well at 450 nm was measured by a microplate reader (Biorad).

(8) In the measured values (absorbency) of each of the samples obtained by the above operation, the measured value (absorbency) [B] of the sample containing serum or plasma is the measured value (absorbency) of the sample not containing serum / plasma. ) The ratio [B / A] (%) divided by [A] is shown in Table 1.
The measured absorbance shall be obtained by subtracting the absorbance of the measured value measured using phosphate buffered saline (pH 7.2) containing 3% BSA as a blind value (blank).

From this, the concentration of des-HMGB1 contained in serum and plasma can be accurately and quantitatively determined by the method and reagent for measuring des-HMGB1 in the sample of the present invention without being affected by the matrix in serum and plasma. It was confirmed that it could be measured.

The 18120 47C1 strain, which is a monoclonal antibody-producing cell line of "anti-des-HMGB1 monoclonal antibody (47C1)", is a patented microorganism deposit center [NPMD] of the National Institute of Technology and Evaluation [NPMD] (Kazusakamatari, Kisarazu City, Chiba Prefecture, Japan). It was received as "Receipt number: NITE AP-03024" on September 20, 2019 in 2-5-8).

Claims (3)

An antibody that specifically binds to a thrombin of HMGB1 or a degradation product of a thrombin / thrombomodulin complex, which is characterized by specifically recognizing the amino acid sequence represented by SEQ ID NO: 1. A method for measuring a degradation product of HMGB1 by thrombin or a thrombin / thrombomodulin complex, which comprises using the antibody according to claim 1. A reagent for measuring a degradation product of HMGB1 by thrombin or a thrombin / thrombomodulin complex, which comprises the antibody according to claim 1.

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