JP7570075B2 - Anti-human haptoglobin antibody and method for determining inflammatory bowel disease using the same - Google Patents

Description

Inflammatory bowel disease (IBD), broadly classified into ulcerative colitis and Crohn's disease, is an intractable chronic inflammatory disease that causes repeated inflammation of the intestines, and the number of patients has been increasing in recent years. IBD is diagnosed by colonoscopy, gastrointestinal contrast examination, pathological tissue examination, etc. Patients with IBD have a higher risk of developing cancer than non-affected patients, so they need to continue undergoing examinations over the long term. However, colonoscopy is highly invasive and places a large physical burden on the patient being examined.

Therefore, diseases are usually diagnosed comprehensively, taking into account blood test items such as serum C-reactive protein (CRP) and white blood cell count, which reflect inflammatory responses, as well as the disease activity index, which is calculated based on the severity of the condition as determined by the doctor.

However, blood test results do not necessarily reflect the pathology of inflammatory bowel disease. In addition, the disease activity index has the problem of lacking objectivity in diagnosis.

For this reason, diagnosis using biomarkers that can diagnose inflammatory bowel disease non-invasively is also becoming more common.

For example, fecal calprotectin is known as a non-invasive marker that reflects the degree of intestinal inflammation. Compared to serum biomarkers, fecal calprotectin levels are thought to more directly reflect local intestinal inflammation. However, patients suspected of having inflammatory bowel disease often have loose or watery stools. Measurements using samples in such a condition are prone to errors in the measurement.

In addition, human haptoglobin is an acute phase reaction protein synthesized in the liver, and it is known that the blood concentration varies in patients with ulcerative colitis and Crohn's disease (Patent Document 1, Patent Document 2). Serum human haptoglobin concentrations have also been measured in patients with ulcerative colitis and Crohn's disease (Patent Document 3).

Patent Publication No. 2009-526233 Patent Publication No. 2012-529508 Patent Publication No. 2012-507723

There is still no known biomarker that is highly specific for diagnosing inflammatory bowel disease and that appropriately reflects disease activity and treatment effects of inflammatory bowel disease. In this situation, there is a demand for the development of a method for diagnosing inflammatory bowel disease that is minimally invasive, reflects the pathology of inflammatory bowel disease, and allows for objective diagnosis.
The present invention has been made in view of the above-mentioned circumstances, and an objective of the present invention is to provide a method for determining inflammatory bowel disease which is simple to measure and accurately reflects clinical symptoms.

The present inventors conducted extensive research to find an excellent biomarker that can accurately diagnose inflammatory bowel disease, and as a result, established an antibody that specifically binds to a region of the amino acid sequence of the α-chain of human haptoglobin, represented by SEQ ID NO: 1. They then found that inflammatory bowel disease can be diagnosed with high accuracy by measuring human haptoglobin using this antibody, and thus completed the present invention.

The present invention comprises the following configurations.
"[1] An anti-human haptoglobin antibody that specifically binds to the region of the amino acid sequence represented by SEQ ID NO:1 of the α chain of human haptoglobin.
[2] A method for diagnosing inflammatory bowel disease, comprising the following steps:
(1) measuring the amount of human haptoglobin in a sample derived from a subject using Antibody 1, which is an anti-human haptoglobin antibody that specifically binds to a region of the amino acid sequence represented by SEQ ID NO:1 of the α-chain of human haptoglobin;
(2) A step of diagnosing inflammatory bowel disease based on the results of (1) above.
[3] The method for diagnosing inflammatory bowel disease according to the above-mentioned [2], wherein the step (1) comprises the following steps:
(1-i) contacting a sample derived from a subject with the antibody 1 to form a complex 1 between human haptoglobin and the antibody 1;
(1-ii) measuring the amount of complex 1.
[4] A method for diagnosing inflammatory bowel disease described in [2] or [3] above, characterized in that step (1) further uses in combination with antibody 2 which recognizes human haptoglobin and which recognizes an epitope different from that of antibody 1.
[5] The method for assessing inflammatory bowel disease according to any one of the above-mentioned [2] to [4], wherein the step (1) comprises the following steps:
(1-i) contacting a sample derived from a subject with the antibody 1 and with antibody 2, which is an antibody that recognizes human haptoglobin and recognizes a different epitope from that of antibody 1, to form a complex 2 between human haptoglobin, the antibody 1, and the antibody 2;
(1-ii) Measuring the amount of complex 2.
[6] The method for determining inflammatory bowel disease described in [4] or [5] above, wherein either the antibody 1 or the antibody 2 is immobilized on an insoluble carrier, and the other is labeled with a labeling substance.
[7] The method for determining inflammatory bowel disease described in any one of [2] to [6] above, wherein the subject-derived sample is serum, plasma, or whole blood.
[8] A method for obtaining data for diagnosing inflammatory bowel disease, comprising measuring the amount of human haptoglobin in a sample derived from a subject using an anti-human haptoglobin antibody that specifically binds to a region of the amino acid sequence represented by sequence number 1 of the alpha chain of human haptoglobin.
[9] A kit for measuring the amount of human haptoglobin to determine inflammatory bowel disease, comprising an anti-human haptoglobin antibody that specifically binds to a region of the amino acid sequence represented by SEQ ID NO: 1 of the α chain of human haptoglobin.
[10] The kit according to the above [9], further comprising an antibody that binds to human haptoglobin and recognizes an epitope different from that of the anti-human haptoglobin antibody.

The method for determining inflammatory bowel disease of the present invention can be used as a method to assist doctors and others in making diagnoses.
Furthermore, all of the above methods are carried out in vitro.

The method of the present invention using the antibody of the present invention has the excellent effect of enabling inflammatory bowel disease to be diagnosed in a minimally invasive and highly accurate manner.
In addition, the result of the determination method of the present invention can be one of the indicators for determining whether or not a patient needs to undergo a colonoscopy. Therefore, by carrying out the determination method of the present invention, it is possible to avoid overtreatment, such as performing a colonoscopy on patients who do not actually need the examination.

FIG. 1 is a schematic diagram of human haptoglobin. FIG. 2 is an electrophoretic diagram showing the results of examining the reactivity of 10-7G2A antibody against Hpt1-1 and Hpt2-2 obtained in Example 1. FIG. 3 is an electrophoretic diagram showing the results obtained in Example 2 in which the reactivity of 10-7G2A antibody to human haptoglobin α-chain fragments was examined. FIG. 4 is a list of peptide fragments of the human haptoglobin α chain used in the secondary screening of the epitope of the 10-7G2A antibody in Example 2. Fig. 5 shows the results of an inhibition experiment of the 10-7G2A antibody obtained in Example 2. In Fig. 5, (a) shows the results of an inhibition assay using, as a sample, a culture supernatant obtained by culturing an established line in which a human haptoglobin gene was introduced into the colon cancer cell line HCT116 and stably overexpressing the gene in an L-fucose-added culture medium. Fig. 5(b) shows the results of an inhibition assay using, as a sample, the serum of a pancreatic cancer patient. In Figs. 5(a) and 5(b), (1) shows the measurement results using a control peptide solution, (2) shows the measurement results using synthetic peptide 1 (broken line), and (3) shows the measurement results using synthetic peptide 3 (solid line). FIG. 6 shows a distribution diagram of human haptoglobin amounts (relative units) obtained by measurement using the 10-7G2A antibody and the 3-1 antibody in the sera of healthy subjects, ulcerative colitis patients, and Crohn's disease patients obtained in Example 3. FIG. 7 shows a distribution diagram of human haptoglobin amounts (relative units) obtained in Example 4 by measurement using 10-7G2A antibody and 3-1 antibody in the sera of healthy subjects and healthy subjects with high CRP levels. FIG. 8 is a correlation diagram between serum CRP values (logarithm) and serum human haptoglobin amounts (logarithm) of healthy subjects, obtained in Example 4. FIG. 9 is a correlation diagram obtained in Example 5 between the Clinical Activity Index (CAI) and the serum human haptoglobin level in patients with ulcerative colitis.

The present invention relates to a novel anti-human haptoglobin antibody, a method for diagnosing inflammatory bowel disease using the same, and a kit used in the same.

<1. Inflammatory bowel disease>
The inflammatory bowel disease according to the present invention includes ulcerative colitis and Crohn's disease, which are generally classified as inflammatory bowel disease, and is preferably ulcerative colitis and Crohn's disease.

2. Human Haptoglobin
Haptoglobin is a liver-derived glycoprotein present in the blood of mammals, and is generally known to bind to hemoglobin released during hemolysis, thereby decreasing the blood concentration during hemolysis. The haptoglobin of the present invention refers to human haptoglobin derived from humans.
Human haptoglobin is composed of two subunits, the α-chain and the β-chain. The precursor prohaptoglobin, which is a combination of the α-chain and the β-chain, is cleaved into the α-chain and the β-chain by the serine protease C1RLP, and the two chains are linked via disulfide bonds to form mature haptoglobin.
A schematic diagram of human haptoglobin (αβ) is shown in FIG.

There are two types of α chains in human haptoglobin, α1 chain and α2 chain, with the α1 chain being approximately 10 kDa and the α2 chain being approximately 18 kDa. The β chain is approximately 39 kDa. Thus, human haptoglobin is classified into three types: Hpt1-1 type ((α1β) ₂ ), Hpt2-1 type ((α1β) _m (α2β) _n ), and Hpt2-2 type ((α2β) _n ). m and n are integers of 1 or more and may be the same or different.
The β chains of the three types are the same. Furthermore, since the α2 chain has two S-type bonds in the chain, Hpt2-1 and Hpt2-2 types exist as multimers of α and β chains.

Examples of the amino acid sequences of the α chain of the present invention include the amino acid sequence of the α1 chain represented by SEQ ID NO: 2 and the amino acid sequence of the α2 chain represented by SEQ ID NO: 3.

The amino acid sequence of the α1 chain represented by SEQ ID NO:2 has the amino acid sequence represented by SEQ ID NO:1 (QCKNYY) at positions 51 to 56 from the N-terminus.
The amino acid sequence of the α2 chain represented by SEQ ID NO:3 has the amino acid sequence represented by SEQ ID NO:1 at two positions from the N-terminus, 51st to 56th and 110th to 115th.

Specific examples of the amino acid sequence of the α chain according to the present invention include, in addition to the amino acid sequence represented by SEQ ID NO: 2, an amino acid sequence in which one or more amino acids of the amino acid sequence represented by SEQ ID NO: 2 have been substituted, deleted, inserted or added, and which has the amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence which has a sequence homology of 70% or more, preferably 80% or more, more preferably 95% or more, and even more preferably 97% or more with the amino acid sequence represented by SEQ ID NO: 2, and which has the amino acid sequence represented by SEQ ID NO: 1.

Further, other specific examples of the amino acid sequence of the α chain according to the present invention include, in addition to the amino acid sequence represented by SEQ ID NO: 3, an amino acid sequence in which one or more amino acids of the amino acid sequence represented by SEQ ID NO: 3 have been substituted, deleted, inserted or added, and which has the amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence which has a sequence homology of 70% or more, preferably 80% or more, more preferably 95% or more, and even more preferably 97% or more with the amino acid sequence represented by SEQ ID NO: 3, and which has the amino acid sequence represented by SEQ ID NO: 1.

3. Antibody of the present invention (Antibody 1)
The antibody of the present invention is an "anti-haptoglobin antibody that specifically binds to a region of the amino acid sequence of the α chain of human haptoglobin represented by SEQ ID NO: 1". Hereinafter, this antibody may be abbreviated as "antibody 1 of the present invention" or simply "antibody 1".

As described above, the amino acid sequence of the α1 chain of human haptoglobin contains one amino acid sequence represented by SEQ ID NO: 1. The amino acid sequence of the α2 chain contains two amino acid sequences represented by SEQ ID NO: 1. Antibody 1 of the present invention is an antibody that specifically binds to the region of the amino acid sequence represented by SEQ ID NO: 1 contained in the α1 chain and the α2 chain.
In other words, antibody 1 of the present invention is an antibody that recognizes the region of the amino acid sequence represented by sequence number 1 of the α1 chain and the region of the amino acid sequence represented by sequence number 1 of the α2 chain (at least one of the two), and binds to Hpt1-1, Hpt2-1, and Hpt2-2.

Antibody 1 of the present invention includes "an antibody that specifically recognizes the primary structure of the amino acid sequence represented by SEQ ID NO: 1 of the α chain of human haptoglobin" or "an antibody that specifically recognizes the three-dimensional structure within the region of the amino acid sequence represented by SEQ ID NO: 1 of the α chain of human haptoglobin."

Furthermore, the 6 amino acid region of the α chain of human haptoglobin, represented by SEQ ID NO: 1, recognized by antibody 1 of the present invention is a region of human haptoglobin to which no glycan is bound. Therefore, antibody 1 of the present invention binds not only to human haptoglobin but also to pro-human haptoglobin.

Antibody 1 of the present invention may be any anti-human haptoglobin antibody having the above-mentioned characteristics, and may be a monoclonal or polyclonal antibody. Monoclonal antibodies are more preferable. In addition, it may be a commercially available product or one appropriately prepared by a conventional method. Furthermore, in the measurement of human haptoglobin using antibody 1 of the present invention described below, these may be used alone or in appropriate combination.

Antibody 1 of the present invention may be an antigen-binding fragment of antibody 1. An antigen-binding fragment means a fragment of an antibody that has an antigen-binding site. Specifically, for example, Fab, Fab', F(ab')2, Fv, Fd, single-chain Fv (scFv), disulfide-bonded Fv (sdFv), VL, VH, diabody ((VL-VH)2 or (VH-VL)2), triabody (trivalent antibody), tetrabody (tetravalent antibody), minibody ((scFV-CH3)2), IgG-delta-CH2, scFv-Fc, (scFv)2-Fc fragments of antibody 1, which specifically bind to the region of the amino acid sequence represented by SEQ ID NO: 1 of the α chain of human haptoglobin, may be mentioned.

Examples of immunogens (antigens) that can be used to obtain antibody 1 of the present invention include (a) a polypeptide having the amino acid sequence of the α chain of human haptoglobin of the present invention (e.g., the amino acid sequence represented by SEQ ID NO:2 or SEQ ID NO:3), (b) a polypeptide that is a partial sequence of the amino acid sequence of the α chain of human haptoglobin of the present invention (e.g., the amino acid sequence represented by SEQ ID NO:2 or SEQ ID NO:3) and contains the amino acid sequence represented by SEQ ID NO:1, or (c) human haptoglobin (full length).

(a) A polypeptide having the amino acid sequence of the α chain of human haptoglobin according to the present invention (e.g., the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3) used as an immunogen to obtain antibody 1 of the present invention, or (b) a polypeptide which is a partial sequence of the amino acid sequence of the α chain of human haptoglobin according to the present invention (e.g., the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3) and contains the amino acid sequence represented by SEQ ID NO: 1, can be produced by a general chemical method according to the amino acid sequence. For example, the polypeptide can be obtained by a general chemical synthesis method such as the fluorenylmethyloxycarbonyl method (Fmoc method) or the t-butyloxycarbonyl method (tBoc method). It can also be chemically synthesized using a commercially available peptide synthesizer. It may also be a product manufactured by outsourcing to a company that manufactures peptide synthesis products.

(c) Human haptoglobin (full length) used as an immunogen to obtain antibody 1 of the present invention can be obtained by extraction and purification, for example, by a method using an anti-human haptoglobin antibody column, from a culture medium or culture supernatant of a cancer cell line, such as the culture supernatant of cells in which a human haptoglobin gene has been introduced into the human colon cancer cell line HCT116 (ATCC) and stably overexpressed. Alternatively, a commercially available purified product of human haptoglobin can also be used.

The above-mentioned antigen protein serving as an immunogen can be purified by a method known per se, for example, by combining several chromatographic techniques such as affinity chromatography using Sepharose beads coated with human haptoglobin antibody or anti-human haptoglobin α-chain antibody.

The method for obtaining polyclonal antibody 1 of the present invention is to immunize animals such as horses, cows, sheep, rabbits, goats, guinea pigs, rats, and mice with the immunogen obtained by the method described above in a conventional manner [such as the method described in Introduction to Immunological Experiments, 2nd Edition, Matsuhashi Nao et al., Academic Press Center, 1981, etc.], and then select an antibody that specifically binds to the region of the amino acid sequence represented by SEQ ID NO: 1 of the α chain of human haptoglobin in a conventional manner.

The method for obtaining the monoclonal antibody 1 of the present invention may, for example, be the following method. That is, immune-sensitized cells, such as spleen cells or lymphocytes, of an animal, such as a rat or a mouse, immunized with the immunogen obtained by the above-mentioned method are fused with cells having the property of permanently growing, such as myeloma cells, by a cell fusion technique developed by Keller and Milstein et al. (Nature, 256, 495, 1975), which is known per se, to prepare hybridomas, and a hybridoma that produces a monoclonal antibody that specifically binds to the region of the amino acid sequence of the α chain of human haptoglobin represented by SEQ ID NO: 1 is selected. The selected hybridoma is cultured in a medium or administered into the peritoneal cavity of an animal to produce an antibody in the ascites, and the desired monoclonal antibody is collected from the culture or ascites. Alternatively, cells that produce antibodies having the above-mentioned properties can be prepared by a method known per se (Eur. J. Immunol., 6 , 511, 1976) that utilizes gene recombination technology, etc., and the desired monoclonal antibody 1 of the present invention can be obtained by culturing these cells.

An example of a method for producing antibody 1 of the present invention is described below using a method for producing a monoclonal antibody as an example.

That is, an immunogen [(a) a polypeptide having the amino acid sequence of the α-chain of human haptoglobin according to the present invention (e.g., the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3), (b) a polypeptide which is a partial sequence of the amino acid sequence of the α-chain of human haptoglobin according to the present invention (e.g., the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3) and contains the amino acid sequence shown in SEQ ID NO: 1, or (c) human haptoglobin (full length)] is mixed with an adjuvant such as complete (or incomplete) Freund's adjuvant to prepare a suspension. This suspension is administered subcutaneously, intravenously, or intraperitoneally to a suitable animal as described above, usually in an amount of 1 ng to 10 mg of the antigen, usually every 1 to 5 weeks, preferably every 2 to 5 weeks, usually 2 to 10 times, preferably 3 to 8 times, for immunization.

Three to four days after the final immunization, the spleen and lymph nodes are aseptically removed from the immunized animal. The most preferred organ to be removed is the spleen. Splenocytes are prepared from the removed spleen by standard methods. The resulting splenocytes are then fused with myeloma cells, such as NS-1, Sp2, Sp2/0, and X63, by standard methods. Examples of cell fusion methods include a method using polyethylene glycol and cell electric fusion, with the method using polyethylene glycol being simple and preferred.

Hybridomas are then selected using HAT medium according to standard methods.
The selected hybridomas are cultured, and the culture supernatant is collected. The culture supernatant is subjected to a conventional ELISA method, an indirect immunofluorescence method, or a Western blot immunostaining method using a polyvinylidene difluoride (PVDF) membrane after SDS-polyacrylamide gel electrophoresis, to select cells (hybridomas) that produce an antibody that specifically recognizes human haptoglobin and specifically binds to the amino acid sequence represented by SEQ ID NO:1.
Next, cloning is carried out several times by limiting dilution method, and those that are found to stably produce high-titer antibodies are selected as hybridoma strains producing monoclonal antibody 1 of the present invention.
By the above method, the desired hybridoma producing the monoclonal antibody 1 of the present invention can be obtained.

To obtain monoclonal antibody 1 of the present invention from the obtained hybridoma, the hybridoma obtained by the above method is cultured, and monoclonal antibody 1 is purified from the obtained culture supernatant by conventional methods.

Methods for culturing hybridomas include conventional methods such as the ascites formation method, in which the hybridoma is administered into the peritoneal cavity of an animal, and the cell culture method, in which the hybridoma is cultured.

Purification of monoclonal antibodies from culture supernatants or mouse ascites can be carried out by appropriately selecting and combining known methods such as ammonium sulfate salt separation, affinity chromatography, ion exchange chromatography, and molecular sieve chromatography.

The inventors have established a novel antibody 1 that specifically binds to the region of the amino acid sequence represented by sequence number 1 of the α chain of human haptoglobin, and named it "10-7G2A antibody."

<4. Method for determining inflammatory bowel disease>
The method for determining inflammatory bowel disease of the present invention comprises the steps of:
"A method for determining inflammatory bowel disease, comprising the steps of:
(1) measuring the amount of human haptoglobin in a sample derived from a subject using Antibody 1, which is an anti-human haptoglobin antibody that specifically binds to a region of the amino acid sequence represented by SEQ ID NO:1 of the α-chain of human haptoglobin;
(2) A step of diagnosing inflammatory bowel disease based on the results of (1) above."
It is.

The method of the present invention also includes a method for obtaining data for diagnosing inflammatory bowel disease, characterized by measuring the amount of human haptoglobin in a sample derived from a subject using antibody 1 of the present invention.

1) Samples Examples of samples used in the method for determining inflammatory bowel disease of the present invention include body fluids such as serum, plasma, blood, pancreatic juice, saliva, lymphatic fluid, and cerebrospinal fluid, intestinal tissues such as the large intestine and small intestine, extracts of the tissues, tissue slices of the tissues, biopsy samples of the tissues, washings of the tissues, and samples prepared from these, all of which are derived from a human subject. Among these, serum, plasma, or blood is preferred. Serum is particularly preferred.

2) Method for measuring human haptoglobin In the method for diagnosing inflammatory bowel disease of the present invention, the method for measuring the amount of human haptoglobin in the step of measuring the amount of human haptoglobin may be a conventional method for measuring the amount of human haptoglobin, except that antibody 1 of the present invention described in the above section <3. Antibody of the present invention (Antibody 1)> is used.
For example, a method includes contacting a sample derived from a subject with antibody 1 of the present invention to form a complex between human haptoglobin and antibody 1 (hereinafter sometimes abbreviated as complex 1), and measuring the amount of complex 1.
Of these, it is preferable to use the 10-7G2A antibody established by the present inventors as the antibody 1 of the present invention.

The method for measuring human haptoglobin according to the present invention is preferably a method using a combination of antibody 1 of the present invention and an antibody that recognizes human haptoglobin and recognizes an epitope different from that recognized by antibody 1.
Hereinafter, the "antibody that recognizes human haptoglobin and recognizes a different epitope from that recognized by antibody 1 of the present invention" may be abbreviated as "antibody 2 of the present invention" or simply "antibody 2".

The antibody 2 according to the present invention may be a monoclonal antibody or a polyclonal antibody. A monoclonal antibody is more preferable. It may be a commercially available product or one appropriately prepared by a conventional method. In addition, these may be used alone or in appropriate combination.

Antibody 2 may also be an antigen-binding fragment of antibody 2, specifically, for example, Fab, Fab', F(ab')2, Fv, Fd, single-chain Fv (scFv), disulfide-linked Fv (sdFv), VL, VH, diabody ((VL-VH)2 or (VH-VL)2), triabody (trivalent antibody), tetrabody (tetravalent antibody), minibody ((scFV-CH3)2), IgG-delta-CH2, scFv-Fc, (scFv)2-Fc fragments of antibody 2, which specifically recognize human haptoglobin.

The origin of antibody 2 is not particularly limited, but examples include those derived from rabbits, rats, mice, sheep, goats, horses, etc. and having the above-mentioned properties. Commercially available products or those obtained by methods described in, for example, "Introduction to Immunology Experiments, 2nd Edition, Matsuhashi Nao et al., Academic Press Center Co., Ltd., 1981" and the like and having the above-mentioned properties may be used.

Antibody 2 can be obtained by immunizing an animal with human haptoglobin or a fragment thereof, and collecting, purifying, and screening the antibodies produced in the body according to a conventional method for producing polyclonal antibodies or a conventional method for producing monoclonal antibodies. The antigen, human haptoglobin, can be obtained by extraction from the culture medium or culture supernatant of a cancer cell line according to a conventional method, for example, a method using an anti-human haptoglobin antibody column, and commercially available products may also be used. Antibody 2 may also be commercially available products.

The antibody used as antibody 2 is an antibody that recognizes human haptoglobin and recognizes an epitope different from that of antibody 1 of the present invention, and is preferably an antibody that does not compete with antibody 1 of the present invention in binding to human haptoglobin. For example, an antibody that recognizes the β chain of human haptoglobin can be mentioned.
Examples of such antibodies include commercially available products such as Affinity Purified Rabbit anti-Human Haptoglobin (catalog No. RHPT-80ALY, polyclonal antibody) from Immunology Consultants laboratory, Inc.
Further examples include the 3-1 antibody and the 3-5 antibody established by the present inventors and disclosed in WO2017/204295. The 3-1 antibody and the 3-5 antibody recognize the β chain of human haptoglobin but do not recognize human haptoglobin in which the S—S bond has been cleaved.

Specific examples of the method for measuring the amount of human haptoglobin according to the present invention include enzyme immunoassay (EIA), radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), fluorescent immunoassay (FIA), simple immunochromatography, high performance liquid chromatography (HPLC), electrophoresis, capillary electrophoresis, capillary tip electrophoresis, mass spectrometry, immunonephelometry, immunonephelometry, and other immunoagglutination-based measurement methods, immunoblotting, etc. Among these, enzyme immunoassay (EIA), radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), fluorescent immunoassay (FIA), immunonephelometry, and immunonephelometry are preferred, and enzyme immunoassay (EIA) is more preferred.
The measurement principles include, for example, the sandwich method, the competitive method, the double antibody method, etc., with the sandwich method being preferred. In addition, it is possible to perform the measurement by a heterogeneous method using an insoluble carrier or the like and performing B/F separation, or a homogeneous method not performing B/F separation.

[Method of measuring human haptoglobin according to the present invention by sandwich method]
In the method for measuring human haptoglobin according to the present invention, an example of the sandwich method is a method in which a sample derived from a subject is contacted with antibody 1 and antibody 2 to form a complex between human haptoglobin and antibody 1 and antibody 2 (hereinafter sometimes abbreviated as complex 2), and the amount of complex 2 is measured.

Antibody 1 and/or antibody 2 used in the above-mentioned sandwich method are preferably labeled with a labeling substance or the like. For example, when antibody 1 of the present invention is antibody 1 labeled with a labeling substance (labeled antibody 1), complex 2 can be measured based on the amount of labeling substance in labeled antibody 1, and when antibody 2 is antibody 2 labeled with a labeling substance (labeled antibody 2), complex 2 can be measured based on the amount of labeling substance in labeled antibody 2.

Examples of labeling substances used to label antibody 1 or antibody 2 include enzymes such as peroxidase (POD), microperoxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, acetylcholinesterase, malate dehydrogenase, and luciferase, which are used in conventional immunoassays; radioisotopes such as 99mTc, 131I, 125I, 14C, 3H, 32P, and 35S, which are used in radioimmunoassays (RIA); and fluorescein, dansyl, fluorescamine, coumarin, naphthylamine, fluorescein isothiocyanate (FITC), rhodamine, and rhodamine X isothiocyanate, which are used in fluorescent immunoassays (FIA). fluorescent substances such as, for example, luciferin, isoluminol, luminol, bis(2,4,6-trifluorophenyl)oxalate; luminescent substances such as, for example, phenol, naphthol, anthracene, or derivatives thereof, which have absorption in the ultraviolet region; labeling substances such as, for example, 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl, 3-amino-2,2,5,5-tetramethylpyrrolidine-1-oxyl, 2,6-di-t-butyl-α-(3,5-di-t-butyl-4-oxo-2,5-cyclohexadiene-1-ylidene)-p-tolyloxyl, which have properties as spin labeling agents; HiLyte dyes such as Alexa Fluor Dye 350, Alexa Fluor Dye 430, Alexa Fluor Dye 488, Alexa Fluor Dye 532, Alexa Fluor Dye 546, Alexa Fluor Dye 555, Alexa Fluor Dye 568, Alexa Fluor Dye 594, Alexa Fluor Dye 633, Alexa Fluor Dye 647, Alexa Fluor Dye 660, Alexa Fluor Dye 680, Alexa Fluor Dye 700, Alexa Fluor Dye 750 (all available from Molecular Probes, Inc.); Probes (trade name); CyDye dyes such as Cy3, Cy3.5, Cy5, Cy5.5, and Cy7 (all trade names of Amersham Biosciences); dyes such as Coomassie Brilliant Blue R250 and methyl orange, and all of the labeling substances usually used in this field are exemplified. Among them, enzymes such as peroxidase, microperoxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, acetylcholinesterase, malate dehydrogenase, and luciferase are preferred, and peroxidase is more preferred.

In addition, in order to bind (label) the above-mentioned labeling substance to antibody 1 or antibody 2, a labeling method commonly used in immunoassays such as EIA, RIA, FIA, etc., which are well known per se, may be appropriately used.

For example, the method described in Enzyme Labeling Method, p. 62, Ishikawa Eiji, Academic Press Center, 1991; Medical Chemistry Experiment Lectures, Vol. 8, edited by Yamamura Yuichi, 1st edition, Nakayama Shoten, 1971; Illustrated Fluorescent Antibody, Kawao Akira, 1st edition, Soft Science Co., Ltd., 1983; Enzyme Immunoassay Method, Ishikawa Eiji, Kawai Tadashi, Muroi Kiyoshi, 2nd edition, Igaku Shoin, 1982, etc.; Molecular Cloning: A Laboratory Manual, Second Edition, J. Sambrook, E. F. Frisch, T. Maniatis, Cold Spring Harbor Laboratory Press, etc.) or the conventional method utilizing the reaction of avidin (or streptavidin) with biotin may be appropriately used.

In addition, various kits for binding (labeling) the above-mentioned labeling substances to proteins are commercially available, so these can be used to label antibody 1 or antibody 2 in accordance with the instructions attached to the kit.

In addition, when performing high performance liquid chromatography, capillary electrophoresis, or capillary chip electrophoresis, in order to separate the antigen-antibody complex and the free labeled antibody more clearly, a separation-enhancing substance, such as a nucleic acid such as DNA or RNA, may be bound (Patent No. 3070418, Patent No. 3531372, etc.).

Furthermore, the solution containing labeled antibody 1 or labeled antibody 2 used in the measurement may contain substances that are typically used as stabilizers in this field, such as sugars, proteins, surfactants, etc., within the concentration ranges typically used in this field.

When antibody 1 and/or antibody 2 are labeled as described above, it is necessary to separate the free antibody labeled with a labeling substance (labeled antibody) from the complex. Therefore, for example, when forming complex 2, it is preferable to label either antibody 1 or antibody 2 with a labeling substance and immobilize the remaining unlabeled antibody 1 or antibody 2 on an insoluble carrier. In this case, it is preferable to immobilize antibody 1 on an insoluble carrier and label antibody 2 with a labeling substance. The free labeled antibody and the complex can be separated by a known B/F separation method.

Any insoluble carrier for immobilizing antibody 1 or antibody 2 can be used as long as it is used in a typical immunological assay. Specific examples include synthetic polymer compounds such as polystyrene, polypropylene, polyacrylic acid, polymethacrylic acid, polyacrylamide, polyglycidyl methacrylate, polyvinyl chloride, polyethylene, polychlorocarbonate, silicone resin, and silicone rubber, and inorganic substances such as porous glass, ground glass, ceramics, alumina, silica gel, activated carbon, and metal oxides. These insoluble carriers can be used in a wide variety of forms, such as microtiter plates, beads, tubes, dedicated trays in which multiple tubes are integrally molded, disk-shaped pieces, and fine particles (latex particles). Among these, microplates and beads are preferred in terms of ease of cleaning and operability when simultaneously processing multiple specimens (samples).

The method for immobilizing antibody 1 or antibody 2 of the present invention on an insoluble carrier is not particularly limited as long as it is performed in accordance with a method normally used in this field. All known immobilization methods normally used in this field can be used, such as chemical binding methods (methods for immobilizing by covalent bonds) and physical adsorption methods.

A preferred example of such a method is to contact an insoluble carrier with a solution containing antibody 1 or antibody 2, usually in the range of 0.1 μg/mL to 20 mg/mL, preferably 1 μg/mL to 5 mg/mL, and to react the solution at an appropriate temperature for a predetermined period of time to obtain an insoluble carrier (solid phase) to which antibody 1 or antibody 2 is bound.

The solvent for preparing the solution of antibody 1 or antibody 2 may be any solvent that does not prevent antibody 1 or antibody 2 from adsorbing or binding to the insoluble carrier, and examples of such solvents include purified water and buffer solutions having a buffering effect near neutral, for example, pH 5.0 to 10.0, preferably pH 6.5 to 8.5 (e.g., phosphate buffer, Tris buffer, Good's buffer, glycine buffer, borate buffer, etc.). The buffer concentration in these buffer solutions is usually selected appropriately from the range of 10 to 500 mM, preferably 10 to 300 mM. The solution may also contain, for example, sugars, salts such as NaCl, surfactants, preservatives, proteins, etc., in amounts that do not prevent antibody 1 or antibody 2 from adsorbing or binding to the insoluble carrier.

In addition, it is desirable to carry out the blocking treatment usually performed in this field, i.e., a treatment in which the insoluble carrier bound to antibody 1 or antibody 2 obtained as described above is further immersed in a solution containing a protein unrelated to human haptoglobin, such as human serum albumin, bovine serum albumin, milk proteins such as skim milk, egg white albumin, or a commercially available blocking agent (e.g. Block Ace (manufactured by DS Pharma Biomedical Co., Ltd.)), in order to prevent nonspecific reactions during measurement.

It is also possible to immobilize antibody 1 or antibody 2 on an insoluble carrier by using a very strong binding reaction such as the avidin-biotin reaction used in this field.

Furthermore, as described above, the insoluble carrier on which antibody 1 or antibody 2 is immobilized can also be used in the publicly known immunoturbidimetric and immunonephelometric methods.

The method for measuring the amount of label in the complex formed as a result of the reaction using labeled antibody 1 or labeled antibody 2 varies depending on the type of labeling substance, but may be performed according to a predetermined method depending on the properties of the labeling substance that can be detected by some method. For example, when the labeling substance is an enzyme, the measurement may be performed according to the standard immunoassay method, such as the method described in "Enzyme Immunoassay Method" (Protein, Nucleic Acid, Enzyme Special Volume No. 31, edited by Kitagawa Tsunehiro, Minamihara Toshio, Tsuji Akio, and Ishikawa Eiji, pp. 51-63, Kyoritsu Shuppan Co., Ltd., 1987). When the labeling substance is a radioactive substance, the measurement may be performed according to the standard method used in RIA, using a measuring device such as a liquid immersion GM counter, liquid scintillation counter, well-type scintillation counter, or HPLC counter, appropriately selected depending on the type and intensity of the radiation emitted by the radioactive substance (see, for example, Medical Chemistry Experiment Lectures, Vol. 8, edited by Yamamura Yuichi, 1st edition, Nakayama Shoten, 1971, etc.). When the labeling substance is a fluorescent substance, the measurement may be performed according to a conventional method performed in FIA using a measuring device such as a fluorometer, for example, the method described in "Illustrated Fluorescent Antibodies, Akira Kawao, 1st Edition, Soft Science Co., Ltd., 1983" or the like. When the labeling substance is a luminescent substance, the measurement may be performed according to a conventional method using a measuring device such as a photocounter, for example, the method described in "Enzyme Immunoassay Method" (Protein, Nucleic Acid, Enzyme, Special Issue No. 31, edited by Tsunehiro Kitagawa, Toshio Minamihara, Akio Tsuji, and Eiji Ishikawa, pp. 252-263, Kyoritsu Shuppan Co., Ltd., 1987) or the like. When the labeling substance is a substance that absorbs in the ultraviolet region, the measurement may be performed according to a conventional method using a measuring device such as a spectrophotometer. When the labeling substance has spin properties, the measurement may be performed according to a standard method using an electron spin resonance apparatus, for example, the method described in "Enzyme Immunoassay" (Protein, Nucleic Acid, Enzyme, Special Issue No. 31, edited by Kitagawa Tsunehiro, Minamihara Toshio, Tsuji Akio, and Ishikawa Eiji, pp. 264-271, Kyoritsu Shuppan Co., Ltd., 1987).

Furthermore, when the labeling substance is an enzyme, a method known per se can be used, such as reacting it with a color-developing reagent to induce a color-developing reaction and measuring the amount of pigment produced as a result using a spectrophotometer, etc. To stop the color-developing reaction, a reaction-stopping method normally used in this field can be used, such as adding an enzyme activity inhibitor such as 1-6N sulfuric acid to the reaction solution or a reaction stopper provided in the kit.

Examples of the color-developing reagent include color-developing reagents that are usually used in this field, such as tetramethylbenzidine (TMB), 3,3',5,5'-tetramethylbenzidine (TMBZ), o-phenylenediamine, o-nitrophenyl-β-D-galactoside, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), N-ethyl-N-sulfopropyl-m-anisidine (ADPS), p-nitrophenyl phosphate, etc. The concentration of these may be appropriately set within the concentration range usually used in this field.
The color reaction can be stopped by any reaction stopping method commonly used in this field, for example by adding an enzyme activity inhibitor such as 1 to 6N hydrochloric acid to the reaction solution.

In addition, methods for measuring human haptoglobin using only unlabeled antibodies include, for example, methods that utilize the properties derived from the obtained complex, specifically, methods that measure the enzyme activity, such as protease activity, or fluorescence polarization, possessed by the complex itself as absorbance, or methods using homogeneous immunoassay systems such as surface plasmon resonance.

The concentrations of antibody 1 and antibody 2 used in the method for measuring the amount of human haptoglobin according to the present invention may be appropriately set within the ranges typically used in this field depending on the measurement method.

The reagents and their concentrations during measurement used in the method for measuring the amount of human haptoglobin according to the present invention, as well as the measurement conditions (reaction temperature, reaction time, pH during reaction, measurement wavelength, measurement device, etc.) may all be selected in accordance with the measurement procedures of the above-mentioned publicly known immunological measurement methods. Any automatic analyzer, spectrophotometric system, etc. normally used in this field may be used without exception.

A buffer solution is preferred as a solvent for the solution of antibody 1 and antibody 2 used in the method for measuring the amount of human haptoglobin according to the present invention. The buffer solution is not particularly limited as long as it is one that is normally used in this field, but it is usually one that has a buffering effect near neutral, usually pH 5.0 to 10.0, preferably pH 6.5 to 8.5. Specific examples include all buffer solutions that are normally used in measurement methods that utilize antigen-antibody reactions, such as Tris buffer, phosphate buffer, veronal buffer, borate buffer, and Good's buffer. The buffer concentration of these buffer solutions is usually selected appropriately from the range of 10 to 1000 mM, preferably 10 to 300 mM. The pH is not particularly limited as long as it is in a range that does not inhibit the antigen-antibody reaction, but it is usually preferably in the range of 5 to 9.

As a specific example of a method for measuring human haptoglobin according to the present invention, a method for measuring the concentration of human haptoglobin in a sample using antibody 1 of the present invention immobilized on an insoluble carrier and antibody 2 labeled with POD using peroxidase (POD) as a label is described below.

That is, a subject-derived sample containing human haptoglobin is contacted with an insoluble carrier (containing 0.1 ng to 0.1 mg of antibody 1 of the present invention) on which antibody 1 of the present invention has been immobilized, and reacted at 4 to 40° C. for 3 minutes to 20 hours to form a complex of antibody 1 of the present invention and human haptoglobin on the insoluble carrier. Next, the sample is reacted with 10 to 100 μL of a solution (containing 0.01 ng to 0.1 mg of antibody 2) containing antibody 2 labeled with POD at 4 to 40° C. for 3 minutes to 20 hours to form a complex of antibody 1-human haptoglobin-labeled antibody 2 on the insoluble carrier. Next, a color-developing solution such as a TMBZ solution is added, and the reaction is continued for a certain period of time, followed by the addition of a reaction stop solution such as 1N HCl to stop the reaction, and the absorbance at a wavelength of 450 nm is measured.
On the other hand, a calibration curve of measured values and concentrations of human haptoglobin of known concentrations is prepared by carrying out the same procedure using the same reagent as above. The amount of human haptoglobin according to the present invention is calculated by applying the measured values obtained in the above measurement to the calibration curve.

In addition, a method for measuring the concentration of human haptoglobin in a sample using the 10-7G2A antibody established by the present inventors as antibody 1 of the present invention and the 3-1 antibody disclosed in WO2017/204295 as antibody 2 includes, for example, a method in which a sample derived from a subject is contacted with the 10-7G2A antibody and the 3-1 antibody to form a complex (complex 2) between human haptoglobin, the 10-7G2A antibody, and the 3-1 antibody, and complex 2 is measured. A more specific example of the method is as follows.

That is, human haptoglobin in a sample is contacted with an insoluble carrier (containing 0.1 ng to 0.1 mg of 10-7G2A antibody) on which 10-7G2A antibody has been immobilized, and reacted at 4 to 40°C for 3 minutes to 20 hours to form a complex of 10-7G2A antibody and human haptoglobin on the insoluble carrier. Next, the mixture is reacted with 10 to 100 μL of a solution (containing 0.01 ng to 0.1 mg of 3-1 antibody) containing POD-labeled 3-1 antibody at 4 to 40°C for 3 minutes to 20 hours to form a complex of 10-7G2A antibody-human haptoglobin-POD-labeled 3-1 antibody (complex 2) on the insoluble carrier. Next, a color-developing solution such as TMBZ solution is added, and the reaction is continued for a certain period of time, followed by addition of a reaction stop solution such as 1N HCl to stop the reaction, and the absorbance at a wavelength of 450 nm is measured.
On the other hand, a calibration curve of measured values and concentrations of human haptoglobin of known concentrations is prepared by carrying out the same procedure using the same reagent as above. The amount of human haptoglobin according to the present invention is calculated by applying the measured values obtained in the above measurement to the calibration curve.

The amount of human haptoglobin does not have to be the actual amount of human haptoglobin (protein amount of human haptoglobin). It may be a relative value (relative unit) of the actual measurement value of human haptoglobin obtained by performing a similar measurement using a subject-derived sample, with the actual measurement value (signal value such as fluorescence intensity or absorbance) being used as a reference value obtained by performing a measurement using purified human haptoglobin with a known concentration.

The method for measuring human haptoglobin according to the present invention is not limited to manual methods, but may be performed in a measurement system using an automatic analyzer. There are no particular restrictions on the combination of reagents, etc. when performing measurements using manual methods or an automatic analyzer, and the combination of reagents, etc. that is considered to be the best may be appropriately selected and used according to the environment and model of the automatic analyzer to be used, or taking other factors into consideration.

3) Method for diagnosing inflammatory bowel disease The method for diagnosing inflammatory bowel disease of the present invention is a method in which the amount of human haptoglobin in a sample derived from a subject is measured by the measurement method described above in "2) Method for measuring human haptoglobin", and diagnosing inflammatory bowel disease based on the measurement results.

That is, using antibody 1 of the present invention, the amount of human haptoglobin in a sample from a subject is measured by the method described above in section "2) Method for measuring human haptoglobin," and data relating to human haptoglobin (e.g., information on the presence or absence, concentration, degree of increase in amount, etc. of human haptoglobin) is obtained based on the results to determine inflammatory bowel disease. The obtained data is used to determine (diagnose/test) inflammatory bowel disease, for example, by the method described below.

For example, a reference value (cutoff value) is set in advance, and if the measurement result (measurement value) of human haptoglobin according to the present invention is equal to or greater than the reference value, it is possible to determine that the subject who provided the sample may or may not be suffering from inflammatory bowel disease (e.g., ulcerative colitis or Crohn's disease). If the measurement result (measurement value) of human haptoglobin is less than the reference value, it is possible to determine that the subject may not be suffering from inflammatory bowel disease (negative for inflammatory bowel disease) or is unlikely to be suffering from inflammatory bowel disease.

The above-mentioned reference value may be set based on the boundary value of the amount of human haptoglobin in samples obtained by measuring the amount of human haptoglobin in samples obtained from patients with inflammatory bowel disease and patients with non-inflammatory bowel disease (non-ulcerative colitis and non-Crohn's disease) using the above-mentioned measurement method. The average amount of human haptoglobin in patients with non-inflammatory bowel disease may be set as the reference value.

Another method is to set multiple judgment categories corresponding to the amount of human haptoglobin in the specimen or its quantitative range (measured value or range of measured values). For example, judgment categories are set as follows: (1) no risk of inflammatory bowel disease, (2) low risk of inflammatory bowel disease, (3) symptoms of inflammatory bowel disease, (4) high risk of inflammatory bowel disease, etc. It is then possible to determine whether or not inflammatory bowel disease exists by determining which judgment category the measurement result of the amount of human haptoglobin in a sample derived from the subject falls into.

In addition, the determination can also be made by comparing the measurement result of human haptoglobin in a sample derived from the same subject measured at a certain time point with the measurement result of human haptoglobin in a sample derived from the same subject measured at a different time point, and evaluating the increase or decrease in the measurement result (measurement value) and/or the degree of increase or decrease. For example, if an increase in the measurement result (measurement value) is observed, it can be determined that the subject who provided the sample may have progressed to inflammatory bowel disease.
When no change is observed in the measured value of human haptoglobin, it can be determined that there is no change in the pathological condition of the inflammatory bowel disease of the subject.
When a decrease in the measurement result (measured value) is observed, it can be determined that the pathological condition of the inflammatory bowel disease of the subject has improved.

When the method for diagnosing inflammatory bowel disease of the present invention determines that a subject patient may or is highly likely to be suffering from inflammatory bowel disease, the subject can choose to undergo further invasive testing such as colonoscopy, gastrointestinal imaging, or pathological tissue testing.
On the other hand, if the method for diagnosing inflammatory bowel disease of the present invention determines that the subject patient is likely not to be suffering from inflammatory bowel disease or that the possibility of this being the case is low, a treatment plan can be selected in which the above-mentioned invasive tests are not performed and follow-up observation is performed as necessary.

<5. Method for assisting in the diagnosis of inflammatory bowel disease>
The method for assisting in the diagnosis of inflammatory bowel disease according to the present invention (hereinafter sometimes abbreviated as the assisting method according to the present invention) comprises:
(1) measuring the amount of human haptoglobin in a sample using antibody 1 of the present invention; and
(2) A step of assisting in the diagnosis of inflammatory bowel disease based on the measurement results of (1) above.
The assisting method according to the present invention can be used as a method for assisting doctors and others in diagnosing inflammatory bowel diseases.
The sample, the step of measuring the amount of human haptoglobin, and the step of assisting in the diagnosis of inflammatory bowel disease in the method of obtaining data of the present invention may be performed in accordance with the sample, the method of measuring human haptoglobin, and the method of diagnosing inflammatory bowel disease explained in Section <4. Method of diagnosing inflammatory bowel disease>, and preferred examples, specific examples, etc. thereof are also the same.

6. Device for determining inflammatory bowel disease
The device for diagnosing inflammatory bowel disease according to the present invention (hereinafter sometimes abbreviated as the diagnosis device according to the present invention) comprises at least (1) a measurement unit and (2) a processing unit, and may further comprise (3) a diagnosis unit, (4) an output unit, and (5) an input unit.

The (1) measurement unit in the determination device according to the present invention is configured to measure human haptoglobin in a sample using the antibody 1 of the present invention. Specifically, examples of the measurement device include a measurement device such as a device used in the immunological measurement method in the above-mentioned measurement method.
The (2) processing unit in the determination device according to the present invention is configured to calculate the amount of human haptoglobin measured by the (1) measurement unit.
The (3) determining unit in the determining device according to the present invention is configured to determine inflammatory bowel disease based on the calculation result obtained in the (2) processing unit.
The (4) output unit in the determination device according to the present invention is configured to output the calculation result obtained in the (2) processing unit and/or the determination result obtained in the (3) determination unit.
The (5) input unit in the determination device of the present invention is configured to, upon operation by an operator, send a signal to the (1) measurement unit and/or the (2) processing unit for activating the (1) measurement unit and/or the (2) processing unit.
The measurement, calculation and judgment using the (1) measuring unit, (2) processing unit and (3) judgment unit of the judgment device according to the present invention may be performed in accordance with the method explained in Section <4. Method for diagnosing inflammatory bowel disease>, and preferred examples, specific examples and the like are also performed in accordance therewith.

According to the above-mentioned determination device of the present invention, the determination method of the present invention and/or the method of obtaining data of the present invention can be carried out simply, quickly and accurately.

<7. Reagents for determining inflammatory bowel disease>
The reagent for diagnosing inflammatory bowel disease according to the present invention (hereinafter sometimes abbreviated as the reagent according to the present invention) contains, as a constituent element, the antibody 1 according to the present invention. The reagent according to the present invention may further contain the antibody 2 according to the present invention.
Antibody 1 of the present invention contained in the reagent of the present invention is as explained in Section <3. Antibody of the present invention (Antibody 1)>, and preferred examples, specific examples, etc. are also the same. Antibody 2 is the same as that described in the explanation of antibody 2 in Section "4. Method for determining inflammatory bowel disease", and preferred examples, specific examples, etc. are also the same.

The concentrations of antibody 1 and antibody 2 in the reagent according to the present invention may be appropriately set within the ranges normally used in this field depending on the measurement method. The reagent may also contain reagents normally used in this field, such as buffers, reaction promoters, stabilizers such as sugars, proteins, salts, surfactants, preservatives, etc. These do not inhibit the stability of coexisting reagents and do not inhibit the reaction of antibody 1 according to the present invention. The concentrations of these may also be appropriately selected from the concentration ranges normally used in this field.

<8. Kit for determining inflammatory bowel disease of the present invention>
The kit of the present invention comprises:
"A kit for measuring the amount of human haptoglobin to determine inflammatory bowel disease, comprising an anti-human haptoglobin antibody (antibody 1 of the present invention) that specifically binds to a region of the amino acid sequence represented by sequence number 1 of the α chain of human haptoglobin."

The kit may further contain antibody 2 of the present invention.

That is, specific examples of the kit of the present invention include:
(1) A kit for diagnosing inflammatory bowel disease (Kit 1 of the present invention), comprising a reagent containing the antibody 1 of the present invention (an anti-human haptoglobin antibody that specifically binds to a region of the amino acid sequence represented by SEQ ID NO: 1 of the α-chain of human haptoglobin); or (2) a kit for diagnosing inflammatory bowel disease (Kit 2 of the present invention), comprising a reagent containing the antibody 1 of the present invention and a reagent containing the antibody 2 (an antibody that recognizes human haptoglobin and recognizes a different epitope from that of antibody 1).
Examples include:

The antibody 1 of the present invention contained in the above-mentioned Kit 1 and Kit 2 of the present invention is the same as that described above in the section <3. Antibody of the present invention (Antibody 1)>, and the preferred ones are also the same.
The antibody 2 contained in the kit 2 of the present invention is the same as that described in the explanation of the antibody 2 in the above section <4. Method for determining inflammatory bowel disease>, and preferred examples, specific examples, etc. are also the same.
The antibody 1 or the antibody 2 may be supported on an insoluble carrier or may be labeled with a labeling substance.

The preferred aspects, specific examples, and concentrations of each component of the kit of the present invention are as described above in section 4. Method for determining inflammatory bowel disease.

The concentrations of antibody 1 and antibody 2 in the reagents in Kit 1 and Kit 2 of the present invention may be appropriately set within the ranges typically used in this field depending on the measurement method. These reagents may also contain reagents typically used in this field, such as buffers, reaction promoters, stabilizers such as sugars, proteins, salts, surfactants, preservatives, etc. These do not inhibit the stability of coexisting reagents and do not inhibit the reaction of antibody 1 and antibody 2 according to the present invention. These concentrations may also be appropriately selected from the concentration ranges typically used in this field.

Furthermore, kits 1 and 2 of the present invention may contain, in addition to antibody 1 or antibody 2 of the present invention, the necessary amounts of reagents necessary for measurements such as immunoassays of human haptoglobin levels using the antibodies.

Kit 1 and Kit 2 of the present invention may be combined with a standard human haptoglobin for creating a calibration curve used in measuring the human haptoglobin. The standard may be a commercially available standard or may be produced according to a known method.

Furthermore, the kit of the present invention may contain instructions for use in the method of diagnosing inflammatory bowel disease of the present invention. The "instructions" refers to an instruction manual, package insert, pamphlet (leaflet), etc. for the kit in which the characteristics, principles, operating procedures, and diagnosing procedures of the method are substantially described in text or diagrams.

The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples in any way.

Example 1. Obtaining an anti-human haptoglobin monoclonal antibody (10-7G2A antibody) reactive to the α chain (1) Preparation of human haptoglobin
The established line stably overexpressing human haptoglobin was obtained by introducing the human haptoglobin gene into the human colon cancer cell line HCT116 (ATCC) by the method described in Oncotarget, 9(16), 12732-44., 2018. The line was cultured in RPMI with L-glutamine and _NaHCO3 (Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C under 5% _CO2 conditions. IWAKI culture plates 10 cm and 15 cm (IWAKI, Tokyo, Japan) were used for the culture.
After the culture, the cells were cultured for 96 hours using FBS-free RPMI supplemented with 1 mM L-fucose, and the culture supernatant was then collected.
The resulting culture supernatant was applied to a human haptoglobin antibody column using a PERISTA bio-mini-pump (ATTO, Tokyo, Japan) (0.5 mL/min, 4°C, overnight). The human haptoglobin antibody column was prepared by coupling 7.5 mg of anti-human haptoglobin polyclonal antibody (Dako) to Hi-Trap-NHS-activated HP (GE Healthcare).
Next, Column Washing Buffer (50 mM _Na2HPO4 , 50 mM _{NaH2PO4 ,} 0.5 M NaCl, _pH 7.4) was applied to the antibody column at 1.0 mL/min for 15 minutes to wash the antibody column, and then Elution buffer (0.1 M Glycine, pH 2.7) was applied to the antibody column at 0.5 mL/min for 20 minutes to elute human haptoglobin from the antibody column.
The eluate was neutralized by adding 1.0 mL of neutralization buffer (2 M Tris-HCl, pH 8.0) to the eluate, and then concentrated approximately 200-fold using an Amicon Column (Millipore, Massachusetts, USA) and further desalted to purify human haptoglobin.
All of the above column treatments were carried out at 4°C.

(2) Preparation of anti-human haptoglobin monoclonal antibody BALB/c mice were immunized with 200 μg of the human haptoglobin prepared above together with Freund's complete adjuvant. The mice were then immunized twice with 50 μg of human haptoglobin at 2-week intervals, and finally with 100 μg of human haptoglobin. The spleen was then removed, and the spleen cells were fused with myeloma cells (SP2/0) by a standard method using polyethylene glycol (described in JP-A-5-244983), and the fused cells were cultured in GIT medium (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.).

(3) Primary screening of anti-human haptoglobin monoclonal antibodies Anti-mouse IgG antibody (rabbit) (Sigma-Aldrich) was immobilized at 0.25 μg per well on a microplate, followed by blocking with bovine serum albumin (BSA) or casein.
50 μL of the supernatant of the cell culture obtained in (2) above or the culture medium (control) was added to the wells and left to stand for 60 minutes, after which the wells were washed three times with a washing solution (PBS-T) containing 0.1% Tween 20 in PBS.
Next, the human haptoglobin obtained in (1) above was dissolved in PBS to a concentration of 250 ng/mL, and 50 μL of the solution was added to the wells and allowed to stand for 60 minutes.The wells were then washed three times with PBS-T.
Next, POD-labeled anti-human haptoglobin polyclonal antibody [polyclonal antibody was purchased from DAKO and labeled with POD by the standard method (Eiji Ishikawa, "Enzyme Labeling Method", Academic Press Center, 1991, p. 62)] was added and left to stand for 30 minutes, after which the wells were washed three times with PBS-T.
Further, 50 μL of substrate solution (o-phenylenediamine (OPD) (FUJIFILM Wako Pure Chemical Industries, Ltd.)) was added, and color was developed for 30 minutes. 100 μL of 1M sulfuric acid solution was added to stop the reaction. Then, the absorbance of the obtained solution at 492 nm was measured using an absorption spectrometer. From the results, the anti-human haptoglobin monoclonal antibody that emitted light was selected as the primary candidate.

(4) Secondary Screening of Anti-Human Haptoglobin Monoclonal Antibodies Western blotting was performed using several types of anti-human haptoglobin monoclonal antibodies obtained in (3) above, and antibodies reactive to the human haptoglobin α-chain were selected from among them.
That is, first, human haptoglobin type 1-1 purified product (haptoglobin, phenotype 1-1, Hpt1-1 type, manufactured by Sigma-Aldrich) and human haptoglobin type 2-2 purified product (haptoglobin, phenotype 2-2, Hpt2-2 type, manufactured by Sigma-Aldrich) were each prepared at 100 μg/mL using 50 mM phosphate buffer, and mixed with sample buffer 1 (0.25 M Tris-HCl pH 6.8, 8% SDS, 40% glycerol, 0.02% BPB, 20% 2-mercaptoethanol) in a ratio of 3:1, and treated at 95° C. for 5 minutes to prepare a sample.
Next, 4 μL of the sample was electrophoresed on a 12.5% polyacrylamide gel. The resulting electrophoretic gel was semi-dry blotted onto a PVDF membrane using a Bio-Rad blotting system according to the protocol. After transfer, the PVDF membrane was blocked with a phosphate buffer solution containing 4% Block Ace (DS Pharma Biomedical Co., Ltd.).
The membrane was immersed in a solution of the primary candidate anti-human haptoglobin monoclonal antibody labeled with POD, diluted 200-fold with phosphate buffer containing 4% Block Ace, and reacted at room temperature for 1 hour. After the reaction, the membrane was washed three times with phosphate buffer containing 0.05% Tween 20. After washing, the membrane was immersed for 10 to 30 minutes in 10 mL of phosphate buffer (50 mM PB, pH 7.5) containing 20 mg of β-NADH (manufactured by Oriental Yeast Co., Ltd.), 3 mg of Nitro-TB (manufactured by Dojindo Chemical Industries Co., Ltd.), and 50 μL of 0.2% hydrogen peroxide solution, to develop a blue color. After color development, the membrane was washed with purified water to stop the reaction.

From these results, an antibody was selected that was reactive with both the α1 chain of human haptoglobin type 1-1 (band around 10 KDa) and the α2 chain of human haptoglobin type 2-2 (band around 18 KDa). In other words, an antibody that has the ability to bind to the α chain was obtained. This antibody was named "10-7G2A antibody."
The electrophoretic results when the 10-7G2A antibody was used are shown in Figure 2. In Figure 2, lane 1 shows the results when Hpt1-1 type was used as a sample, and lane 2 shows the results when Hpt2-2 type was used as a sample.

Example 2. Confirmation of antigen-binding site of anti-human haptoglobin monoclonal antibody (10-7G2A antibody) reactive to α-chain (1) Preparation of human haptoglobin α-chain fragment 1) Creation of cDNA and PCR cloning cDNA of human haptoglobin (Hpt2) was cloned from total RNA of human hepatoma cell line HepG2 (ATCC) by standard RT-PCR.
The cloned cDNA was subcloned into pGEM-T Easy vector (Promega, Wisconsin, USA). Escherichia coli was transformed with the resulting recombinant vector, and the bacteria were grown in liquid medium, after which the cells were harvested by centrifugation, and the plasmid was harvested using a plasmid purification kit.
A portion of the purified plasmid was digested with restriction enzymes EcoRV and XhoI and cleaved. The resulting DNA fragment was excised from the gel, purified, and ligated into the EcoRV-XhoI site of pcDNA3.1-Hyg(+) (Invitrogen, CA, USA) to obtain a recombinant plasmid.

2) Preparation of a cDNA fragment encoding a human haptoglobin α-chain fragment Using the recombinant plasmid obtained in 1) above as a template, a cDNA fragment encoding an α-chain fragment was prepared by the following method.

First, PCR was carried out using the primer pair Hpt-α-F1 and Hpt-α-R1 shown in Table 1 below to amplify cDNA encoding the full-length amino acid sequence of the human haptoglobin α2 chain fragment.
Furthermore, PCR was carried out using the primer pairs shown in combinations 3 to 7 in Table 1 below to amplify various cDNAs encoding the amino acid sequences of fragments of the human haptoglobin α2 chain fragment.
In the above PCR, the initial denaturation was performed at 95°C for 3 minutes, followed by 40 cycles of reaction at [98°C, 10 seconds → 57°C, 15 seconds → 68°C, 35 seconds], and finally a reaction at 68°C for 7 minutes.
The primer pair used in the above PCR and the amino acid sequence of the human haptoglobin α-chain fragment encoded by the base sequence of the cDNA amplified with the primer pair are summarized in Table 1 below.

3) Expression of haptoglobin α-chain fragment peptide Each cDNA fragment encoding the amino acid sequence fragment of the haptoglobin α-chain obtained above was subcloned into the pGEM-Teasy vector. E. coli was transformed with each subcloned vector, and the transformed E. coli was grown in a liquid medium, after which each vector was purified using a plasmid purification kit. A part of the purified vector was digested with restriction enzymes NheI and EcoRI, electrophoresed, and the cut pieces were purified from the electrophoretic gel, and ligated into the NheI-EcoRI site of the pCIneo expression vector (Promega) to obtain each recombinant plasmid.
The resulting recombinant plasmid was used to transfect HEK293T cells by standard methods, and the transfected HEK293T cells were cultured in OPTI-MEM medium for 2 days to transiently express the human haptoglobin α-chain fragment of interest, and the supernatant containing the fragment was collected.

Separately, the full-length Hpt2 gene and the C1rlp gene obtained by PCR cloning were co-transfected into HEK293T cells, and the resulting transfected cells were cultured to obtain human haptoglobin α2 chain fragment by co-expression of full-length human haptoglobin and C1RLP.
Incidentally, C1RLP is an enzyme that cleaves full-length haptoglobin into α1 chain and β chain or α2 chain and β chain.

(2) Primary screening of 10-7G2A antibody epitope The culture supernatant containing each human haptoglobin α-chain fragment from the transfected HEK293T cells obtained above was used as a sample for SDS-PAGE, and then transferred to a PVDF membrane. After blocking the PVDF membrane, the 10-7G2A antibody was reacted at room temperature for one hour. The PVDF membrane was washed three times with PBS-T, and then reacted with HRP (Horseradish peroxidase)-labeled anti-mouse IgG (secondary antibody) at room temperature for one hour. The PVDF membrane was washed three times with PBS-T again, and then luminescence was generated using ImmunoStar ^TM Zeta (FUJIFILM Wako Pure Chemical Industries, Ltd.) and imaged using a FUSION Chemiluminescence imaging system (Vilber-Lourmat, Marne la Vallee, France).

The results are shown in Figure 3.

In FIG. 3, each lane shows the results of Western blotting of cell lysates with 10-7G2A or anti-β-actin antibody.
Lane 1: Culture supernatant of untransfected HEK293T cells Lane 2: Human haptoglobin α2 chain fragment obtained by co-expressing full-length human haptoglobin and C1RLP Lane 3: Full-length α2 chain fragment Lane 4: Peptide consisting of the amino acid sequence represented by SEQ ID NO:13 Lane 5: Peptide consisting of the amino acid sequence represented by SEQ ID NO:14 Lane 6: Peptide consisting of the amino acid sequence represented by SEQ ID NO:15 Lane 7: Peptide consisting of the amino acid sequence represented by SEQ ID NO:16 Lane 8: Peptide consisting of the amino acid sequence represented by SEQ ID NO:17

The "full length α2 chain fragment" in lane 3 is a polypeptide having an amino acid sequence of 142 amino acids from the 19th V to the 160th Q of the amino acid sequence represented by SEQ ID NO:3, with the signal peptide from the N-terminus to the 18th amino acid and the C-terminus amino acid truncated.

Furthermore, the results in Figure 3 show that Western blot analysis of β-actin revealed that each protein was present at approximately the same concentration.

Based on the results in Figure 3, it was estimated that the region of the amino acid sequence represented by sequence number 18 (DDGCPKPPEIAHGYVEHSVRYQCKNYYKLRTEGDGVYTLNNE), which is common to sequence numbers 13 to 17, contains the epitope of the 10-7G2A antibody, and was selected.

(3) Secondary Screening of 10-7G2A Antibody Epitope 1) Preparation of Human Haptoglobin α Chain Fragment Peptide Library A peptide library was designed for the amino acid sequence of the region of the human haptoglobin α chain selected above. That is, based on the position of the amino acid sequence shown in SEQ ID NO: 18 on the amino acid sequence shown in SEQ ID NO: 3 (amino acid sequence of the α2 chain), peptides Pep#1 to Pep#6 shown in Figure 4 were designed and synthesized.
The amino acid sequence of each peptide and its sequence number in this specification are shown in Table 2 below.

Peptide synthesis was performed by Fmoc solid-phase peptide synthesis (SPPS) using Rink Amide resin (50-80 μmol scale, WATANABE CHEMICAL INDUSTRIES, LTD.) and a peptide synthesizer Prelude (Protein Technologies, Inc.) or a high-speed flow peptide synthesizer developed by Pentelute et al.

When using the peptide synthesizer Prelude, peptide synthesis was performed according to the following method (I. Sakamoto, K. Tezuka, K. Fukae, K. Ishii, K. Taduru, M. Maeda, M. Ouchi, K. Yoshida, Y. Nambu, J. Igarashi, N. Hayashi, T. Tsuji, Y. Kajihara, Chemical synthesis of homogeneous human glycosyl-interferon-beta that exhibits potent antitumor activity in vivo, Journal of the American Chemical Society 134(12) (2012) 5428-31.).
That is, Fmoc-AA-OH (4.6 equiv.) was coupled to the resin by reaction with HCTU (4.0 equiv.) and N-methylmorpholine (16 equiv.) in dimethylformamide (DMF) for 15 min. This coupling step was carried out twice. After washing the resin with DMF, the Fmoc group was deprotected by reaction with 20% piperidine in DMF (5 mL) for 5 min. The above coupling reaction was repeated until the peptide elongation was completed.

On the other hand, when using a high-speed flow peptide synthesizer, Fmoc-AA-OH (1.0 mmol) was reacted with 0.38 M HBTU (a solution containing 2.5 mL of DMF and 0.5 mL of N,N-diisopropylethylamine (DIEA)) to couple to the resin. When Fmoc-Cys and Fmoc-His were combined to suppress epimerization, a solution containing Fmoc-AA-OH in which the amount of DIEA was changed to 0.19 mL was used. Deprotection of the Fmoc group was performed using the flow system of the synthesizer.
Three-minute cycles (coupling, washing, deprotection, and washing) were manually repeated at 70°C until peptide elongation was complete (MD Simon, PL Heider, A. Adamo, AA Vinogradov, SK Mong, X. Li, T. Berger, RL Policarpo, C. Zhang, Y. Zou, X. Liao, AM Spokoyny, KF Jensen, BL Pentelute, Rapid flow-based peptide synthesis, Chembiochem : a European journal of chemical biology 15(5) (2014) 713-20.).

After coupling of all amino acids by synthesis using a peptide synthesizer or a fast flow peptide synthesizer, the peptidyl resin was treated with 10 mL of a solution containing 82.5% TFA, 5% phenol, 5% water, 5% thioanisole, and 2.5% 1,2-ethanedithiol, or 10 mL of a solution containing 95% trifluoroacetic acid (TFA), 2.5% water, and 2.5% triisopropylsilane to remove all side chain protecting groups and cleave the amino acids from the resin, yielding a mixture containing the crude peptide. To the resulting mixture, ice-cold ether was added to obtain a precipitate, which was dissolved in 10% acetonitrile containing 0.1% TFA. The resulting mixture containing the crude peptide was lyophilized.

2) Secondary screening of epitopes of 10-7G2A antibody The mixture containing the crude peptides Pep#1 to Pep#6 obtained in (3) 1) above was immobilized on a 96-well plate (Pierce ^™ , Amine-binding, Maleic Anhydride 96-well Plates, Thermo Scientific) according to the manufacturer's instructions. Then, the mixture was reacted with POD-labeled 10-7G2A antibody to verify the binding properties of the 10-7G2A antibody to each synthetic peptide.
As a result, it was confirmed that the 10-7G2A antibody binds to Pep#2 and Pep#3.

Based on the above verification results, Pep#7 to Pep#19 were designed based on the amino acid sequences of Pep#2 and Pep#3. Next, peptides having the amino acid sequences of Pep#7 to Pep#19 shown in Table 2 were synthesized by the method described in (3) 1) above.
Each of the synthetic peptides obtained was immobilized on a 96-well plate (Pierce ^™ , Amine-binding, Maleic Anhydride 96-well Plates, Thermo Scientific) according to the manufacturer's instructions. Then, the peptides were reacted with the POD-labeled 10-7G2A antibody to verify the binding properties of the 10-7G2A antibody to each synthetic peptide.
As a result, it was confirmed that the 10-7G2A antibody specifically binds to Pep#18.

Based on the above verification results, the amino acid sequences of Pep#22 to Pep#29 were designed based on the amino acid sequence of Pep#18 in order to perform epitope analysis of the 10-7G2A antibody by alanine scanning mutagenesis. Next, peptides having the amino acid sequences of Pep#22 to Pep#29 shown in Table 2 were synthesized by the method described in (3) 1) above.
Each of the synthetic peptides obtained was immobilized on a 96-well plate (Pierce ^™ , Amine-binding, Maleic Anhydride 96-well Plates, Thermo Scientific) according to the manufacturer's instructions. Then, the peptides were reacted with the POD-labeled 10-7G2A antibody to verify the binding properties of the 10-7G2A antibody to each synthetic peptide.

As a result of the verification, the 10-7G2A antibody was shown to be reactive with Pep#22, Pep#23, and Pep#26. Based on this, QCKNYY (SEQ ID NO: 1), a sequence common to Pep#22 and Pep#23, was identified as a candidate epitope for the 10-7G2A antibody.

3) Inhibition experiment with 10-7G2A antibody
(i) Samples The colon cancer cell line HCT116 was introduced with the human haptoglobin gene, and the established cell line was stably overexpressed. The culture supernatant obtained by culturing the cell line in a culture medium containing L-fucose, or serum from a pancreatic cancer patient was diluted to 1/100 and used as a sample. MOPS buffer containing 2% BSA was used as a control.

(ii) Synthetic Peptide Solution Crude peptides of peptide 1 (QCKNYY, SEQ ID NO: 1), peptide 2 (YYNKCQ, SEQ ID NO: 46), and control peptide (LPECEA, SEQ ID NO: 47) were obtained by the method described in (3) 1) above.
The amino acid sequence of peptide 2 is the reverse sequence of peptide 1.
Then, reverse-phase HPLC was carried out using a reverse-phase HPLC column, Proteonavi.

Fractions containing the desired products were pooled and lyophilized to give isolated yields of 57% peptide 1, 47% peptide 2, and 50% control peptide.
Each purified peptide was dissolved in PBS and used as a synthetic peptide solution.

(iii) ELISA (inhibition assay)
The amount of human haptoglobin in the samples was measured using an iMark™ Microplate Reader (Bio-Rad) by the following method.
First, Fab fragments of anti-human haptoglobin antibodies (manufactured by DAKO) were immobilized on a 96-well ELISA plate, which was then blocked with PBS containing 3% BSA.
The samples prepared in (i) above were added to each well and incubated at room temperature (RT) for 1 h. Each well was washed three times with PBS containing 0.1% Tween 20 (PBS-T). A mixture of POD-labeled 10-7G2A antibody and synthetic peptide solution was added to each well and incubated at room temperature for 1 h. Each well was washed three times with PBS-T. Tetramethylbenzidine (TMBZ, Sigma-Aldrich Co. LLC, St Louis, USA) was then added to each well and incubated for 15 min to develop color. 1N sulfuric acid was added to each well to stop the reaction.
The absorbance was measured at a wavelength of 450 nm.

The synthetic peptide solutions were added so that the concentrations of the synthetic peptides upon reaction with the POD-labeled 10-7G2A antibody were 50 μg/mL, 150 μg/mL, 250 μg/mL, 400 μg/mL, or 500 μg/mL, respectively.
The POD-labeled 10-7G2A antibody used above was the 10-7 antibody obtained in Example 1, which was labeled with horseradish peroxidase by a standard method (see Eiji Ishikawa, "Enzyme Labeling Method," Academic Press Center, 1991, p. 62).

On the other hand, the amount of human haptoglobin was measured in the same manner as above, except that a synthetic peptide solution containing a control peptide was used instead of the synthetic peptide solution.

(iv) Results The results obtained are shown in FIG.
Figure 5(a) shows the results of an inhibition assay using a culture supernatant obtained by culturing a colon cancer cell line HCT116 that stably overexpresses the human haptoglobin gene in an L-fucose-containing medium as a sample, and Figure 5(b) shows the results of an inhibition assay using serum from a pancreatic cancer patient as a sample.
In addition, in Figures 5(a) and 5(b), (1) shows the measurement results using a control peptide solution, (2) shows the measurement results using synthetic peptide 1 (dashed line), and (3) shows the measurement results using synthetic peptide 3 (solid line).
In addition, in FIG. 5, the vertical axis indicates the percentage (%) of absorbance obtained by measurement in the presence of each concentration of synthetic peptide, relative to the absorbance obtained by measurement using the control peptide, which is taken as 100.

5(a) and 5(b), synthetic peptide 1 inhibited the binding of 10-7G2A antibody to human haptoglobin. Moreover, synthetic peptide 2, which has the reverse amino acid sequence of synthetic peptide 1, inhibited the binding of 10-7G2A antibody to human haptoglobin, similar to synthetic peptide 1.
From the above results, the amino acid sequence shown in SEQ ID NO: 1 was identified as the epitope of the 10-7G2A antibody.

Example 3. Measurement of human haptoglobin using 10-7G2A antibody and diagnosis of inflammatory bowel disease (1) Preparation of samples and reagents 1) Samples Sera from ulcerative colitis patients (n=45), Crohn's disease patients (n=20), and healthy subjects (n=67) were diluted 1/5,000 to prepare samples. Culture supernatant of liver cancer cell line HepG2 was used as a control.

2) Antibodies Used Antibody 1: The 10-7G2A antibody, which is an anti-human haptoglobin monoclonal antibody whose recognition site was identified in Example 2, was used.
Antibody 2: 3-1 antibody obtained by the method described in Experimental Example 3 of WO2017/204295 was used.

3) Antibody-immobilized plate
10-7G2A antibody 5 μg/mL (50 mM sodium carbonate buffer, pH 9.6) was immobilized on a NUNC-IMMUNO MODULE plate (Cosmo Bio Co., Ltd.) by standard methods, and then blocked with phosphate-buffered saline (PBS) containing 1% Block Ace (DS Pharma Biomedical Co., Ltd.) to obtain a plate on which the 10-7G2A antibody was immobilized.

4) POD-labeled 3-1 antibody solution
The 3-1 antibody was converted into a Fab' fragment by a standard method, and then labeled with peroxidase (POD) by a standard method (Ishikawa Eiji, "Enzyme Labeling Method", Academic Press Center, 1991, p. 62). It was then diluted with TBS containing 10% Block Ace (DS Pharma Biomedical Co., Ltd.) to obtain a POD-labeled 3-1 antibody solution (1.06 μmol/L). When used, it was diluted 5000-fold with TBS containing 10% Block Ace (DS Pharma Biomedical Co., Ltd.). The concentration of the POD-labeled 3-1 antibody solution when used was 2.12×10 ^-1 pmol/L.

5) Coloring solutions, etc. The following test solutions were prepared.
Coloring solution: 5 mmol/L 3,3',5,5'-tetramethylbenzidine (TMBZ) solution (Sigma-Aldrich)
Stop solution: 1N HCl
Washing solution: Tris-buffered saline containing 0.05% polyoxyethylene (20) sorbitan monolaurate (Tween 20)

(2) Measurement of human haptoglobin
The amount of human haptoglobin in the samples was measured using an iMark™ Microplate Reader (Bio-Rad) by the following method.

50 μL of sample was added to the wells of the antibody-immobilized plate obtained in 3) above, and reacted at room temperature for 60 minutes. After the reaction, 300 μL/well of washing solution was added to the wells, and the wells were washed three times. Next, 50 μL/well of POD-labeled 3-1 antibody solution was added to the wells, and reacted at room temperature for 60 minutes. After the reaction, 300 μL/well of washing solution was added to the wells, and the wells were washed three times. Next, 50 μL/well of color-developing solution was added to the wells, and reacted at room temperature for 20 minutes. 50 μL/well of reaction stop solution was added to stop the reaction. The absorbance was measured at a wavelength of 450 nm.

On the other hand, purified human haptoglobin type 2-2 (Haptoglobin, Phenotype 2-2, Hpt2-2, Sigma-Aldrich) was diluted to 0, 0.001, 0.004, 0.016, 0.063, 0.250, and 1 μg/mL with MOPS buffer containing 2% BSA, and the amount of human haptoglobin was measured in the same manner as above, and the absorbance at a wavelength of 450 nm was measured to create a calibration curve.

The absorbance value for 1 μg/mL of the Hpt2-2 purified product was defined as 1 (standard). The absorbance obtained in the above measurement using the subject-derived sample was calculated as a relative value to the standard. This relative value is expressed as a "relative unit."
Significance tests represent all pairwise non-parametric comparisons performed by Wilcoxon test using the predictive analysis software JMP pro 14.
The results obtained are shown in FIG.
The measurement results are shown in Table 3.

As is clear from Figure 6 and Table 3, the amount of human haptoglobin measured using a combination of 10-7G2A antibody and 3-1 antibody was significantly higher in patients with ulcerative colitis and Crohn's disease compared to healthy subjects.
From the above results, it was found that the determination method of the present invention makes it possible to determine inflammatory bowel disease.

Example 4. Examination of the relationship between serum human haptoglobin amount and serum CRP level in healthy subjects The correlation between serum human haptoglobin amount and serum CRP level, which is known as an inflammatory marker, was examined by the following method.

First, serum from healthy individuals (n=70) who had been determined to have high serum CRP levels in clinical tests was used to measure the amount of human haptoglobin in the serum using the same method as in Example 3.

FIG. 7 shows the results of the measurement of serum human haptoglobin levels in healthy individuals with high serum CRP levels (healthy individuals with high CRP levels).
7 also shows the results of measuring the serum human haptoglobin levels in healthy subjects (n=20) obtained in Example 3. The serum CRP levels of the healthy subjects in Example 3 were all normal.

Furthermore, the relationship between serum human haptoglobin levels and serum CRP levels was statistically analyzed in healthy subjects with high serum CRP levels and healthy subjects with normal serum CRP levels (n=90). The analysis was performed using the predictive analysis software JMP pro 14, with Pearson's product-moment correlation coefficient test.

The results are shown in Figure 8. That is, Figure 8 is a correlation diagram between serum CRP values (logarithm) and serum human haptoglobin levels (logarithm) in healthy individuals. As a result of the analysis, the relationship between serum human haptoglobin levels and CRP values was analytically correlated with a coefficient of correlation ρ of 0.139 and a p-value of 0.223.

As is clear from the results in FIG. 7, the serum human haptoglobin levels were not elevated in healthy subjects with high CRP values, as compared with healthy subjects with normal CRP values.
Furthermore, as is clear from the results in FIG. 8, no correlation was observed between the serum human haptoglobin level and the serum CRP level.

Both haptoglobin and CRP are known to increase in inflammatory diseases, but CRP has been thought to be more sensitive than haptoglobin. However, the above results show that the amount of human haptoglobin does not correlate with the CRP level, and that in subjects without inflammatory bowel disease, the amount of human haptoglobin does not increase even when the serum CRP level increases. This result indicates that an increase in CRP level does not reflect intestinal inflammation.
On the other hand, as is clear from the results of Example 3 and FIG. 6, the serum human haptoglobin level was significantly increased in patients with inflammatory bowel disease.

From the above, it was found that the increase in the amount of human haptoglobin in the blood confirmed in this example does not simply reflect systemic inflammation, but reflects inflammation specific to the intestinal tract. In other words, it was confirmed that the amount of human haptoglobin in the blood increases specifically in inflammatory bowel disease by the method for measuring the amount of human haptoglobin using antibody 1 of the present invention.

Example 5. Study of the relationship between serum human haptoglobin level and CAI in ulcerative colitis patients The relationship between serum human haptoglobin level in ulcerative colitis patients obtained by the measurement in Example 3 and clinical activity (abbreviated as CAI), which is one of the ulcerative colitis activity indexes determined by clinical findings in the same ulcerative colitis patients, was statistically analyzed. The analysis was performed using JMP pro 14, a predictive analysis software, by Pearson's product-moment correlation coefficient test, as in Example 1.

The results are shown in Figure 9. That is, Figure 9 is a correlation diagram between CAI and serum human haptoglobin levels in patients with ulcerative colitis.
As a result of the analysis, the correlation coefficient ρ between serum human haptoglobin levels and CAI was 0.574 and p-value was 0.0001.
That is, it was confirmed that in ulcerative colitis, the serum human haptoglobin level is positively correlated with CAI, which is used clinically as an activity index of ulcerative colitis.

The above results also support the idea that the assessment method of the present invention makes it possible to objectively assess inflammatory bowel disease, reflecting the local pathological condition of inflammatory bowel disease.

Claims (8)

A method for assisting in the diagnosis of inflammatory bowel disease, comprising the steps of:
(1) measuring the amount of human haptoglobin in a sample derived from a subject using Antibody 1, which is an anti-human haptoglobin antibody that specifically binds to a region of the amino acid sequence represented by SEQ ID NO:1 of the α-chain of human haptoglobin;
(2) determining whether or not the patient has inflammatory bowel disease based on the results of (1) above;
the inflammatory bowel disease is ulcerative colitis or Crohn's disease;
The subject-derived sample is serum, plasma, or whole blood;
The antibody 1 is capable of binding to all of Hpt1-1, Hpt2-1, and Hpt2-2, and further capable of binding to pro-human haptoglobin;
A method for determining that a subject may be suffering from ulcerative colitis or Crohn's disease when the amount of human haptoglobin is higher than a reference value. The method for assisting in the diagnosis of inflammatory bowel disease according to claim 1, wherein step (1) comprises the following steps:
(1-i) contacting a sample derived from a subject with the antibody 1 to form a complex 1 between human haptoglobin and the antibody 1;
(1-ii) Measuring the amount of complex 1. The method for assisting in the diagnosis of inflammatory bowel disease according to claim 1, characterized in that step (1) further uses in combination with antibody 2, which recognizes human haptoglobin and recognizes an epitope different from that of antibody 1. The method for assisting in the diagnosis of inflammatory bowel disease according to claim 1, wherein step (1) comprises the following steps:
(1-i) contacting a sample derived from a subject with the antibody 1 and with an antibody 2 that recognizes human haptoglobin and recognizes a different epitope from that recognized by the antibody 1, thereby forming a complex 2 between the human haptoglobin and the antibody 1 and the antibody 2;
(1-ii) Measuring the amount of complex 2. The method for assisting in the diagnosis of inflammatory bowel disease according to claim 3, wherein either the antibody 1 or the antibody 2 is immobilized on an insoluble carrier, and the other is labeled with a labeling substance. A method for obtaining data for diagnosing inflammatory bowel disease, comprising measuring the amount of human haptoglobin in a sample derived from a subject using an anti-human haptoglobin antibody that specifically binds to a region of the amino acid sequence represented by SEQ ID NO:1 of the α chain of human haptoglobin, wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease, the sample derived from the subject is serum, plasma, or whole blood, and the anti-human haptoglobin antibody is capable of binding to all of Hpt1-1 type, Hpt2-1 type, and Hpt2-2 type, and further capable of binding to pro-human haptoglobin . A kit for measuring the amount of human haptoglobin in order to determine the presence of inflammatory bowel disease, comprising an anti-human haptoglobin antibody that specifically binds to a region of the amino acid sequence represented by SEQ ID NO: 1 of the α chain of human haptoglobin, wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease , and the anti-human haptoglobin antibody is capable of binding to all of Hpt1-1 type, Hpt2-1 type, and Hpt2-2 type, and further capable of binding to pro-human haptoglobin . The kit according to claim 7, further comprising an antibody that binds to human haptoglobin and recognizes an epitope different from that of the anti-human haptoglobin antibody.

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