WO2020241617A1 - Anti-human haptoglobin antibody and method for judging inflammatory bowel disease using same - Google Patents

Abstract

The present invention addresses the problem of providing a method for judging inflammatory bowel disease that comprises a simple measurement procedure and correctly reflects clinical conditions.　The present invention pertains to "an anti-human haptoglobin antibody that binds specifically to a region having the amino acid sequence represented by SEQ ID NO:1 in the α-chain of human haptoglobin, a method for judging inflammatory bowel disease with the use of the antibody, and a kit that contains the antibody and is for quantifying human haptoglobin to judge inflammatory bowel disease".

Description

Anti-human haptoglobin antibody and method for determining inflammatory bowel disease using the antibody

Inflammatory bowel disease (IBD) is an intractable chronic inflammatory disease that repeats inflammation in the intestinal tract, which is roughly classified into ulcerative colitis and Crohn's disease, and the number of patients has been increasing in recent years. Diagnosis of inflammatory bowel disease is made by colonoscopy, gastrointestinal angiography, histopathological examination, and the like. Patients with inflammatory bowel disease have a higher risk of carcinogenesis than non-affected patients and should be tested for a long time. However, colonoscopy is highly invasive and places a heavy physical burden on the patient as the subject.

Therefore, in consideration of blood test items such as serum C-reactive protein (CRP) and white blood cell count, which usually reflect the inflammatory reaction, and the disease activity index calculated based on the severity judged by the doctor, etc. Comprehensive diagnosis of diseases.

However, the results of blood tests do not necessarily reflect the pathophysiology of inflammatory bowel disease. In addition, the disease activity index has a problem that the objectivity of diagnosis is poor.

Therefore, diagnosis using biomarkers that can non-invasively diagnose inflammatory bowel disease is also being performed.

For example, fecal calprotectin is known as a non-invasive marker that reflects the degree of inflammation of the intestinal tract. Fecal calprotectin levels are thought to more directly reflect local inflammation of the intestinal tract compared to serum biomarkers. However, in patients with suspected inflammatory bowel disease, loose stools and watery stools are common. There is a problem that an error is likely to occur in the measured value in the measurement using the sample in such a state.

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

Special Table 2009-526233 Gazette Special Table 2012-529508 Special Table 2012-507723

Biomarkers that are highly specific for determining inflammatory bowel disease and that appropriately reflect the disease activity and therapeutic effect of inflammatory bowel disease are not yet known. Under these circumstances, it is required to develop a judgment method that is minimally invasive, reflects the pathophysiology of inflammatory bowel disease, and can make an objective diagnosis.
The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a method for determining inflammatory bowel disease, which is easy to measure and accurately reflects clinical symptoms.

As a result of diligent studies to find an excellent biomarker capable of determining inflammatory bowel disease with high accuracy, the present inventors specifically study the region of the amino acid sequence represented by SEQ ID NO: 1 of the α chain of human haptoglobin. An antibody to bind was established. Then, they have found that inflammatory bowel disease can be determined with high accuracy by measuring human haptoglobin using the antibody, and have completed the present invention.

The present invention has the following configuration.
"[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] Method for determining inflammatory bowel disease, including the following steps:
(1) The amount of human haptoglobin in a sample derived from a subject is measured using antibody 1, which is 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. Process to do,
(2) A step for determining inflammatory bowel disease based on the result of (1) above.
[3] The method for determining inflammatory bowel disease according to [2] above, wherein step (1) includes the following steps:
(1-i) A step of contacting a sample derived from a subject with the antibody 1 to form a complex 1 of human haptoglobin and the antibody 1.
(1-ii) A step of measuring the amount of the complex 1.
[4] The inflammatory property according to the above [2] or [3], wherein the step (1) further uses an antibody 2 that recognizes human haptoglobin, which has an epitope different from that of the antibody 1, and is used in combination. Method for determining intestinal disease.
[5] The method for determining inflammatory bowel disease according to any one of the above [2] to [4], wherein the step (1) includes the following steps:
(1-i) A sample derived from a subject is brought into contact with antibody 1 and antibody 2, which is an antibody that recognizes human haptoglobin and has a different epitope from antibody 1, and human haptoglobin and the antibody 1 are combined. Step of forming a complex 2 with the antibody 2
(1-ii) A step of measuring the amount of the complex 2.
[6] The inflammatory bowel disease according to the above [4] or [5], wherein either one of the antibody 1 and the antibody 2 is immobilized on an insoluble carrier and the other is labeled with a labeling substance. Judgment method.
[7] The method for determining inflammatory bowel disease according to any one of the above [2] to [6], wherein the sample derived from the subject is serum, plasma, or whole blood.
[8] The amount of human haptoglobin in a sample derived from a subject is measured by using 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. A method for obtaining data for determining inflammatory bowel disease.
[9] The amount of human haptoglobin is measured to determine inflammatory bowel disease, which comprises 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. Kit for.
[10] The kit according to the above [9], further comprising an antibody that binds to human haptoglobin, which has 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 for assisting a diagnosis by a doctor or the like.
In addition, all the above methods are carried out in vitro.

According to the determination method of the present invention using the antibody of the present invention, it is possible to obtain an excellent effect that it is possible to determine inflammatory bowel disease with low invasiveness and high accuracy.
In addition, the determination result by the determination method of the present invention is one of the indexes for determining whether or not it is necessary to perform colonoscopy on the patient. Therefore, by implementing the determination method of the present invention, it is possible to avoid over-medical treatment in which the examination is performed even for patients who normally do not need to perform the colonoscopy.

FIG. 1 is a schematic diagram of human haptoglobin. FIG. 2 is an electrophoretogram showing the results of examining the reactivity of the 10-7G2A antibody with respect to Hpt1-1 type and Hpt2-2 type obtained in Example 1. FIG. 3 is an electrophoretogram showing the results of examining the reactivity of the 10-7G2A antibody with respect to the human haptoglobin α-chain fragment obtained in Example 2. FIG. 4 is a list of peptide fragments of the human haptoglobin α chain used in the secondary screening of the 10-7G2A antibody epitope in Example 2. FIG. 5 shows the results of the inhibition experiment of the 10-7G2A antibody obtained in Example 2. In FIG. 5, (a) used a culture supernatant obtained by culturing an established strain in which the human haptoglobin gene was introduced into the colon cancer cell line HCT116 and stably overexpressed as a sample in an L-fucos-added culture medium. The results of the inhibition assay are shown. FIG. 5 (b) shows the results of an inhibition assay using serum from a pancreatic cancer patient as a sample. Further, in FIGS. 5 (a) and 5 (b), (1) is the measurement result using the control peptide solution, (2) is the measurement result using the synthetic peptide 1, and (3) is the synthesis. The measurement results using peptide 3 are shown by solid lines. FIG. 6 shows human haptoglobin obtained by measurement using 10-7G2A antibody and 3-1 antibody using the sera of healthy subjects, ulcerative colitis patients, and Crohn's disease patients obtained in Example 3. Represents a distribution map of a relative unit. FIG. 7 shows the amount of human haptoglobin (relative unit) obtained by measurement using 10-7G2A antibody and 3-1 antibody using the sera of healthy subjects and healthy subjects with high CRP values obtained in Example 4. Represents a distribution map. FIG. 8 is a correlation diagram between the serum CRP value (logarithm) of a healthy person and the serum human haptoglobin amount (logarithm) obtained in Example 4. FIG. 9 is a correlation diagram between the Clinical activity Index (CAI) of an ulcerative colitis patient and the amount of serum human haptoglobin obtained in Example 5.

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

<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. Ulcerative colitis and Crohn's disease are preferred.

<2. Human haptoglobin ＞
Haptoglobin is a liver-derived glycoprotein present in the blood of mammals, and it is generally known that the blood concentration decreases during hemolysis because it binds to hemoglobin released during hemolysis. The haptoglobin according to the present invention refers to a human-derived human haptoglobin.
Human haptoglobin is composed of two subunits, α chain and β chain. The precursor prohaptoglobin in which the α chain and the β chain are bound is cleaved into the α chain and the β chain by the serine protease C1RLP, and both chains become a mature haptoglobin linked via an SS bond.
A schematic diagram of human haptoglobin (αβ) is shown in FIG.

There are two types of α-chains of human haptoglobin, α1 chain and α2 chain. The α1 chain is about 10 kDa and the α2 chain is about 18 kDa. The β chain is about 39 kDa. Therefore, 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 ). To. m and n are integers of 1 or more and may be the same or different.
The three types of β chains are the same. Moreover, since the α2 chain has two SS bonds in the chain, the Hpt2-1 type and the Hpt2-2 type exist as multimers of the α chain β chain.

Examples of the amino acid sequence of the α chain according to 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 (QCKNYY) represented by SEQ ID NO: 1 at the 51st to 56th positions from the N-terminal.
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 the 51st to 56th positions and the 110th to 115th positions from the N-terminal.

Further, as a specific example of the amino acid sequence of the α chain according to the present invention, in addition to the amino acid sequence represented by SEQ ID NO: 2, one or several amino acids in the amino acid sequence represented by SEQ ID NO: 2 described above are substituted. , Which has an amino acid sequence deleted, inserted, or added and has an amino acid sequence represented by SEQ ID NO: 1, or 70% or more, preferably 70% or more of the amino acid sequence represented by SEQ ID NO: 2 described above. Has an amino acid sequence having 80% or more, more preferably 95% or more, and even more preferably 97% or more sequence homology, and has an amino acid sequence represented by SEQ ID NO: 1.

Further, as another specific example of the amino acid sequence of the α chain according to the present invention, in addition to the amino acid sequence represented by SEQ ID NO: 3, one or several amino acids of the amino acid sequence represented by the above-mentioned SEQ ID NO: 3 Has an amino acid sequence represented by substitution, deletion, insertion, or addition and has the amino acid sequence represented by SEQ ID NO: 1, or 70% or more of the amino acid sequence represented by SEQ ID NO: 3 described above. , Preferably those having an amino acid sequence having a sequence homology of 80% or more, more preferably 95% or more, still more preferably 97% or more, and having an amino acid sequence represented by SEQ ID NO: 1. Be done.

<3. Antibody of the present invention (antibody 1)>
The antibody of the present invention is an "anti-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". The antibody may be abbreviated as "antibody 1 of the present invention" or simply "antibody 1" below.

As described above, the amino acid sequence of the α1 chain of human haptoglobin has one amino acid sequence represented by SEQ ID NO: 1. In the amino acid sequence of the α2 chain, there are two amino acid sequences represented by SEQ ID NO: 1. The 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 of the α1 chain and the α2 chain.
That is, the antibody 1 of the present invention recognizes the region of the amino acid sequence represented by SEQ ID NO: 1 of the α1 chain and the region of the amino acid sequence of SEQ ID NO: 1 of the α2 chain (at least one of the two). It is an antibody that binds to Hpt1-1 type, Hpt2-1 type, and Hpt2-2 type.

The antibody 1 of the present invention is represented by "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 "SEQ ID NO: 1 of the α chain of human haptoglobin". "Antibodies that specifically recognize the three-dimensional structure within the region of the amino acid sequence" are included.

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

The antibody 1 of the present invention may be an anti-human haptoglobin antibody having the above-mentioned characteristics, and may be a monoclonal antibody or a polyclonal antibody. Monoclonal antibodies are more preferred. Further, it may be a commercially available product or a product appropriately prepared by a conventional method. Further, in the measurement of human haptoglobin using the antibody 1 of the present invention described later, it is optional to use them alone or in combination as appropriate.

The antibody 1 of the present invention may be an antigen-binding fragment of the antibody 1. The antigen-binding fragment means a fragment of an antibody having an antigen-binding site. Specifically, for example, Fab, Fab', F (ab') 2, Fv, Fd of antibody 1, single chain Fv (scFv), disulfide bond Fv (sdFv), VL, VH, diabody ((VL)). -VH) 2 or (VH-VL) 2), triabodies (trivalent antibodies), tetrabodies (tetravalent antibodies), minibodies ((scFV-CH3) 2), IgG-delta-CH2, scFv-Fc, Examples of the (scFv) 2-Fc fragment include those that specifically bind to the region of the amino acid sequence represented by SEQ ID NO: 1 of the α chain of human haptoglobin.

The amino acid sequence (for example, SEQ ID NO: 2 or SEQ ID NO: 3) of the α chain of human haptoglobin according to the present invention is used as an immunogen (antigen) for obtaining the antibody 1 of the present invention. Amino acid sequence), (b) a partial sequence of the amino acid sequence of the α chain of human haptoglobin according to the present invention (for example, the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3), wherein SEQ ID NO: 1 Examples thereof include a polypeptide containing the amino acid sequence represented by (c) human haptoglobin (full length).

Used as an immunogen to obtain the antibody 1 of the present invention (a) A polypeptide having the amino acid sequence of the α chain of human haptoglobin according to the present invention (for example, the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3). Or (b) A partial sequence of the amino acid sequence of the α chain of human haptoglobin according to the present invention (for example, the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3), and the amino acid sequence represented by SEQ ID NO: 1. The contained polypeptide can be produced by a general chemical method according to its amino acid sequence. For example, the polypeptide can be obtained by a usual chemical synthesis method such as the fluorenylmethyloxycarbonyl method (Fmoc method) and the t-butyloxycarbonyl method (tBoc method). It can also be chemically synthesized using a commercially available peptide synthesizer. Further, it may be a manufactured product outsourced to a manufacturer of a peptide synthetic product.

(C) Human haptoglobin (full length) used as an immunogen to obtain the antibody 1 of the present invention is obtained by introducing the human haptoglobin gene into the human colon cancer cell line HCT116 (ATCC) and stably overexpressing it in culture. It can be obtained by extracting and purifying from a culture solution or culture supernatant of a cancer cell line such as Qing, for example, by a method using an anti-human haptoglobin antibody column. Alternatively, a commercially available refined product of human haptoglobin or the like can be used.

The above-mentioned purification of the antigen protein serving as an immunogen is combined with a method known per se, for example, a combination of several chromatography techniques such as affinity chromatography using Sepharose beads coated with a human haptoglobin antibody or an anti-human haptoglobin α-chain antibody. Just do it.

As a method for obtaining the polyclonal antibody 1 of the present invention, the immunogen obtained by the above method is applied to a conventional method [for example, Introduction to Immunoassay, 2nd Printing, Nao Matsuhashi et al., Society Publishing Center, 1981, etc. According to the described method, etc.], for example, it is prepared by a conventional method immunizing animals such as horses, cows, sheep, rabbits, goats, guinea pigs, rats, mice, etc. An antibody that specifically binds to the region of the amino acid sequence may be selected.

In addition, examples of the method for obtaining the monoclonal antibody 1 of the present invention include the following methods. That is, immunosensitized cells of animals such as rats and mice immunized with the immunogen obtained by the above method, such as splenocytes and lymphocytes, and permanently, such as myeloma cells. Hybridomas were prepared by fusing cells with proliferative properties using a cell fusion technique developed by Keller and Milstein et al. (Nature, 256, 495, 1975), and the sequence number of the α chain of human lymphocytes. A hybridoma that produces a monoclonal antibody that specifically binds to the region of the amino acid sequence represented by 1 is selected. The selected hybridoma may be cultured in a medium or administered intraperitoneally to an animal to produce an antibody in ascites, and the desired monoclonal antibody may be collected from the culture or ascites. Alternatively, a cell producing an antibody having the above-mentioned properties is prepared by a method known per se (Eur.J.Immunol., 6 , 511, 1976) applying a gene recombination technique or the like, and the cell is cultured. The desired monoclonal antibody 1 of the present invention may be collected according to the above.

An example of the method for producing the antibody 1 of the present invention will be described below by taking a method for producing a monoclonal antibody as an example.

That is, immunogen [(a) a polypeptide having an amino acid sequence of the α chain of human haptoglobin according to the present invention (for example, the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3), (b) human haptoglobin according to the present invention. A partial sequence of the amino acid sequence of the α chain of (for example, the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3) and containing the amino acid sequence represented by SEQ ID NO: 1 or (c) human haptoglobin. (Full length)] and an adjuvant such as a complete (or incomplete) Freund's adjuvant are mixed to prepare a suspension. This suspension is applied to a suitable animal as described above, usually in an amount of 1 ng to 10 mg of antigen, usually every 1 to 5 weeks, preferably every 2 to 5 weeks, usually 2 to 10 times, preferably 3 to 3 to. Immunize by administering 8 times subcutaneously, intravenously or intraperitoneally.

Aseptically remove the spleen and lymph nodes from the immunized animal 3-4 days after the final immunization. The spleen is the most preferable organ to be removed. From the removed spleen, splenocytes are prepared by a conventional method. The obtained splenocytes and myeloma cells such as NS-1, Sp2, Sp2 / 0, and X63 are fused according to a conventional method. Examples of the cell fusion method include a method using polyethylene glycol, a cell electrofusion method, and the like, but the method using polyethylene glycol is convenient and preferable.

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

In order to obtain the monoclonal antibody 1 of the present invention from the obtained hybridoma, the hybridoma obtained by the above method may be cultured, and the monoclonal antibody 1 may be purified from the obtained culture supernatant by a conventional method.

Examples of the method for culturing the hybridoma include a conventional method such as an ascites formation method in which the hybridoma is administered into the abdominal cavity of an animal and a cell culture method for culturing the hybridoma.

Purification of the monoclonal antibody from the culture supernatant or mouse ascites may be carried out by appropriately selecting and combining known methods such as ammonium sulfate salt folding method, affinity chromatography, ion exchange chromatography, and molecular sieving chromatography.

The present inventors have established a novel antibody 1 that specifically binds to the region of the amino acid sequence represented by SEQ ID NO: 1 of the α chain of human haptoglobin, and named it "10-7G2A antibody".

<4. Judgment method for inflammatory bowel disease>
The method for determining inflammatory bowel disease of the present invention is
"Method for determining inflammatory bowel disease, including the following steps:
(1) The amount of human haptoglobin in a sample derived from a subject is measured using antibody 1, which is 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. Process to do,
(2) A step for determining inflammatory bowel disease based on the result of (1) above. "
Is.

In addition, the method of the present invention obtains data for determining inflammatory bowel disease, which comprises measuring the amount of human haptoglobin in a sample derived from a subject using the antibody 1 of the present invention in this way. Also includes methods for.

1) Samples The samples used in the method for determining inflammatory bowel disease of the present invention include serum, plasma, blood, pancreatic fluid, saliva, lymph, spinal fluid and other body fluids, large intestine, small intestine, etc. derived from humans as subjects. Intestinal tissue, an extract of the tissue, a tissue section of the tissue, a biopsy sample of the tissue, a washing solution of the tissue, or a solution prepared from these. Of these, serum, plasma, or blood is preferred. Serum is particularly preferred.

2) Method for measuring human haptoglobin In the method for determining inflammatory bowel disease of the present invention, the method for measuring the amount of human haptoglobin according to the step of measuring the amount of human haptoglobin is described in <3. Except for using the antibody 1 of the present invention described in the section of the antibody of the present invention (antibody 1)>, it may be carried out by a conventional method for measuring the amount of human haptoglobin.
For example, a sample derived from a subject and an antibody 1 of the present invention are brought into contact with each other to form a complex of human haptoglobin and the antibody 1 (hereinafter, may be abbreviated as complex 1) of the complex 1. A method of measuring the amount can be mentioned.
Above all, 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 more preferably a method in which the antibody 1 of the present invention and an antibody that recognizes human haptoglobin, which has a different epitope from the antibody 1, are used in combination.
The "antibody that recognizes human haptoglobin, which has a different epitope from the antibody 1 of the present invention" may be abbreviated as "antibody 2 according to the present invention" or simply "antibody 2".

The antibody 2 according to the present invention may be a monoclonal antibody or a polyclonal antibody. Monoclonal antibodies are more preferred. It may be a commercially available product or a product appropriately prepared by a conventional method. Further, it is optional to use these alone or in combination as appropriate.

Further, the antibody 2 may be an antigen-binding fragment of the antibody 2, and specifically, for example, Fab, Fab', F (ab') 2, Fv, Fd, and single-chain Fv (scFv) of the antibody 2. , Disulfide-bonded Fv (sdFv), VL, VH, Diabody ((VL-VH) 2 or (VH-VL) 2), Triabody (trivalent antibody), Tetrabody (tetravalent antibody), Minibody ( Examples thereof include (scFV-CH3) 2), IgG-delta-CH2, scFv-Fc, and (scFv) 2-Fc fragments that specifically recognize human haptoglobin.

The origin of antibody 2 is not particularly limited, and examples thereof include those having the above-mentioned properties derived from rabbits, rats, mice, sheep, goats, horses, and the like. Use commercially available products, or those obtained by the method described in, for example, "Introduction to Immunology Experiments, 2nd Printing, Nao Matsuhashi et al., Academic Society Publishing Center, 1981", which have the above-mentioned properties. Just do it.

Antibody 2 can be obtained by immunizing an animal with human haptoglobin or a fragment thereof according to a conventional method for producing a polyclonal antibody or a method for producing a monoclonal antibody, and collecting, purifying, and screening an antibody produced in vivo. Can be done. The human haptoglobin as an antigen can be obtained by extracting from the culture medium or culture supernatant of a cancer cell line by a conventional method, for example, by a method using an anti-human haptoglobin antibody column, and a commercially available product is used. It doesn't matter. Further, as the antibody 2, a commercially available antibody 2 may be used.

The antibody used as the antibody 2 is an antibody that recognizes human haptoglobin and has a different epitope from the antibody 1 of the present invention, and is preferably an antibody that does not compete with the antibody 1 of the present invention for 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) of Immunology Consultants laboratory, Inc.
In addition, 3-1 antibody and 3-5 antibody established by the present inventors disclosed in WO2017 / 204295 can be mentioned. The 3-1 antibody and the 3-5 antibody are antibodies that recognize the β chain of human haptoglobin and do not recognize human haptoglobin in which the SS bond is cleaved.

Specific methods for measuring the amount of human haptoglobin according to the present invention include, for example, enzyme immunoassay (EIA), radioimmunoassay (RIA), enzyme-binding immunoadsorption measurement (ELISA), and fluorescence immunoassay (FIA). ), Measurement method by simple immunochromatography, high performance liquid chromatography (HPLC), electrophoresis method, capillary electrophoresis method, capillary chip electrophoresis method, mass analysis method, immunoassay method, immunoturbidimetric method, etc. Examples thereof include a measurement method according to the above, an immunoblotting method, and the like. Among them, enzyme immunoassay (EIA), radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), fluorescence immunoassay (FIA), immunoassay fistula, and immunoturbidimetric method are preferable, and enzyme immunoassay. The method (EIA) is more preferred.
Examples of these measurement principles include a sandwich method, a competitive method, a two-antibody method, and the like, and the sandwich method is preferable. Further, it is possible to measure by a heterogeneous method in which B / F separation is performed using an insoluble carrier or the like, or by a homogeneous method in which B / F separation is not performed.

[Method for measuring human haptoglobin according to the present invention by the sandwich method]
In the method for measuring human haptoglobin according to the present invention, as a measuring method by the sandwich method, a sample derived from a subject, the antibody 1 and the antibody 2 are brought into contact with each other, and human haptoglobin, the antibody 1 and the antibody 2 are combined. Examples thereof include a method of forming a complex (hereinafter, may be abbreviated as complex 2) and measuring the amount of the complex 2.

The antibody 1 and / or antibody 2 used in the measurement method by the sandwich method is preferably labeled with a labeling substance or the like. For example, when the antibody 1 of the present invention uses an antibody 1 labeled with a labeling substance (labeled antibody 1), the complex 2 may be measured based on the amount of the labeling substance of the labeled antibody 1, for example, the antibody 2 is a labeling substance. When the labeled antibody 2 (labeled antibody 2) is used, the complex 2 may be measured based on the amount of the labeled substance of the labeled antibody 2.

Examples of the labeling substance used for labeling the antibody 1 or the antibody 2 include peroxidase (POD), microperoxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, and glucose-6-phosphorus used in ordinary immunoassays. Enzymes such as acid dehydrogenase, acetylcholinesterase, malic acid dehydrogenase, luciferase; for example, radioactive isotopes such as 99mTc, 131I, 125I, 14C, 3H, 32P, 35S used in radioimmunoassay (RIA). Elements; for example, fluorescein, dancil, fluorescein, coumarin, naphthylamine, fluorescein isothiocyanate (FITC), rhodamine, rhodamine X isothiocyanate, sulfoledamine 101, lucifer yellow, acrydin used in fluorescence immunoassay (FIA). , Fluorescent substances such as aclysine isothiocyanate, riboflavin or derivatives thereof; luminescent substances such as luciferin, isolminol, luminol, bis (2,4,6-trifluorophenyl) oxalate; for example phenol, naphthol, anthracene or Substances with ultraviolet absorption such as these derivatives; for example, 4-amino-2,2,6,6-tetramethylpiperidin-1-oxyl, 3-amino-2,2,5,5-tetramethylpyrrolidin- Oxyl such as 1-oxyl, 2,6-di-t-butyl-α- (3,5-di-t-butyl-4-oxo-2,5-cyclohexadiene-1-ylidene) -p-tolyloxyl Labeling substances such as substances having properties as spin labeling agents typified by compounds having a group; for example, HiLyte dyes such as HiLyte Fluor 647, HiLyte Fluor 488, HiLyte Fluor 555, HiLyte Fluor 680, HiLyte Fluor 750, etc. Also highlights Bioscience (HiLyte Bioscience, Inc.) Product name]; For example, 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 FluorAlexa dyes such as 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, etc. Molecular Probes) trade name]; for example, CyDye dyes such as Cy3, Cy3.5, Cy5, Cy5.5, Cy7 [all are Amersham Biosciences trade names]; for example, Coomassie Brilliant Blue R250, Methyl Orange And the like, all labeling substances usually used in this field are mentioned. Among them, enzymes such as peroxidase, microperoxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, acetylcholinesterase, malic acid dehydrogenase, and luciferase are preferable, and peroxidase is more preferable.

Further, in order to bind (label) the labeling substance as described above to antibody 1 or antibody 2, for example, a self-known labeling method generally used in immunoassays such as EIA, RIA, FIA, etc. It may be used as appropriate.

For example, Enzyme Labeling Method, p.62, by Eiji Ishikawa, Society Publishing Center, 1991; Medical Chemistry Experiment Course, Volume 8, supervised by Yuichi Yamamura, 1st Edition, Nakayama Shoten, 1971; Illustrated Fluorescent Antibody, Akira Kawai Written by, 1st Edition, Soft Science Co., Ltd., 1983; Enzyme Immunoassay, Eiji Ishikawa, Tadashi Kawai, Kiyoshi Muroi, 2nd Edition, Igaku-Shoin, 1982, etc .; Molecular Cloning Laboratory Manual, Second Edition, J. Sambrook, E.I. F. Frisch, T.I. Maniatis, Cold Spring Harbor Laboratory Press, etc.), or a conventional method using the reaction of avidin (or streptavidin) and biotin, etc. may be appropriately used.

In addition, since various kits for binding (labeling) the labeling substance to the protein as described above are commercially available, they may be used to label antibody 1 or antibody 2 according to the instruction manual attached to the kit. ..

In addition, when performing high performance liquid chromatography, capillary electrophoresis, or capillary chip electrophoresis, in order to more clearly separate the antigen-antibody complex from the free labeled antibody, for example, nucleic acids such as DNA and RNA are used. Separation improving substances may be combined (Patent No. 3070418, Patent No. 3531372, etc.).

In the solution containing the labeled antibody 1 or the labeled antibody 2 used for the measurement, those usually used as stabilizers in this field, such as saccharides, proteins, surfactants and the like, are usually used in this field. It may be contained within the concentration range used.

When labeling antibody 1 and / or antibody 2 as described above, it is necessary to separate the antibody (labeled antibody) labeled with the free labeling substance and the complex. Therefore, for example, when forming a 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 fix the antibody 1 on an insoluble carrier and label the antibody 2 with a labeling substance. Separation of the free labeled antibody and the complex can be performed by a known B / F separation method.

As the insoluble carrier for immobilizing antibody 1 or antibody 2, any of those used in ordinary immunological measurement methods can be used. Specifically, synthetic polymer compounds such as polystyrene, polypropylene, polyacrylic acid, polymethacrylic acid, polyacrylamide, polyglycidyl methacrylate, polyvinyl chloride, polyethylene, polychlorocarbonate, silicone resin, and silicone rubber, such as porous glass. , Sri glass, ceramics, alumina, silica gel, activated carbon, inorganic substances such as metal oxides, etc. In addition, these insoluble carriers can be used in a wide variety of forms such as microtiter plates, beads, tubes, dedicated trays in which a large number of tubes are integrally molded, disc-shaped pieces, fine particles (latex particles), and the like. Of these, microplates and beads are preferable from the viewpoints of ease of washing and operability when processing a large number of samples (samples) at the same time.

The method for immobilizing the antibody 1 or antibody 2 of the present invention on an insoluble carrier is not particularly limited as long as it is usually carried out according to the method used in this field. All known immobilization methods usually used in this field are mentioned, and examples thereof include a chemical bond method (immobilization method by covalent bond), a method of physically adsorbing, and the like.

As a preferred example thereof, for example, 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 is brought into contact with an insoluble carrier at an appropriate temperature. Examples thereof include a method of obtaining an insoluble carrier (solid phase) to which antibody 1 or antibody 2 is bound by reacting for a predetermined time.

The solvent for preparing the solution of the antibody 1 or the antibody 2 may not have a property of preventing the antibody 1 or the antibody 2 from adsorbing or binding on the insoluble carrier, for example, purified water, for example, pH 5.0 to. 10.0, preferably a buffer solution having a buffering action near neutral pH 6.5 to 8.5 (for example, phosphate buffer solution, Tris buffer solution, Good's buffer solution, glycine buffer solution, borate buffer solution, etc.) is preferable. The buffer concentration in these buffer solutions is usually appropriately selected from the range of 10 to 500 mM, preferably 10 to 300 mM. Further, this solution contains, for example, saccharides, salts such as NaCl, surfactants, preservatives, proteins and the like as long as they do not prevent antibody 1 or antibody 2 from adsorbing or binding on the insoluble carrier. It may be.

It should be noted that the blocking treatment usually performed in this field, that is, the insoluble carrier to which antibody 1 or antibody 2 obtained as described above is bound, is further added to a protein unrelated to human haptoglobin, for example, human serum albumin, bovine. Immersing in a solution containing milk protein such as serum albumin and skim milk, egg white albumin, and a commercially available blocking agent (for example, Block Ace (manufactured by DS Pharma Biomedical Co., Ltd.)) is nonspecific at the time of measurement. It is desirable from the viewpoint of preventing a target reaction.

It is also possible to immobilize antibody 1 or antibody 2 on an insoluble carrier 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 immunoturbidimetric methods and immunomodulation methods known per se.

The method for measuring the labeled amount in the complex produced as a result of the reaction using the labeled antibody 1 or the labeled antibody 2 differs depending on the type of the labeling substance, but has properties that can be detected by some method possessed by the labeling substance. Depending on the situation, it may be carried out according to a predetermined method. For example, when the labeling substance is an enzyme, a conventional method of immunoassay, for example, "enzyme immunoassay" (protein, nucleic acid, enzyme, separate volume No. 31, edited by Tsunehiro Kitagawa, Toshio Minamihara, Akio Tsuji, Eiji Ishikawa, 51- The measurement may be performed according to the method described in 63, Kyoritsu Publishing Co., Ltd., 1987). When the labeling substance is a radioactive substance, for example, according to the conventional method used in RIA, a immersion type GM counter, a liquid scintillation counter, a well type scintillation counter, depending on the type and intensity of radiation emitted by the radioactive substance, Measurements may be performed by appropriately selecting and using a measuring device such as an HPLC counter (see, for example, Medical Chemistry Experiment Course, Volume 8, supervision by Yuichi Yamamura, 1st Edition, Nakayama Shoten, 1971, etc.). When the labeling substance is a fluorescent substance, for example, the conventional method used in FIA using a measuring device such as a fluorometer, for example, "Illustration Fluorescent antibody, by Akira Kawao, 1st edition, Soft Science Co., Ltd." The measurement may be performed according to the method described in "Company, 1983" or the like. When the labeling substance is a luminescent substance, a conventional method using a measuring device such as a photo counter, for example, "enzyme immunoassay" (protein, nucleic acid, enzyme, separate volume No. 31, Tsunehiro Kitagawa, Toshio Minamihara, Akio Tsuji, Eiji Ishikawa The measurement may be performed according to the method described in Edit, 252 to 263, Kyoritsu Publishing Co., Ltd., 1987). When the labeling substance is a substance having absorption in the ultraviolet, the measurement may be carried out by a conventional method using a measuring device such as a spectrophotometer. If the labeling substance has spin properties, a conventional method using an electron spin resonance device, for example, "enzyme immunoassay" (protein, nucleic acid, enzyme, separate volume No. 31, edited by Tsunehiro Kitagawa, Toshio Minamihara, Akio Tsuji, Eiji Ishikawa) , 264 to 271, Kyoritsu Publishing Co., Ltd., 1987), etc., respectively.

Further, when the labeling substance is an enzyme, a method known per se, such as a method of reacting this with a color-developing reagent to induce a color-developing reaction and measuring the amount of dye produced as a result with a spectrophotometer or the like, can be mentioned. In order to stop the color development reaction, for example, an enzyme activity inhibitor such as 1 to 6N sulfuric acid or a reaction stop agent attached to the kit is added to the reaction solution, which is a reaction stop method usually used in this field. May be used.

Examples of the coloring reagent include tetramethylbenzidine (TMB), 3,3', 5,5'-tetramethylbenzidine (TMBZ), o-phenylenediamine, o-nitrophenyl-β-D-galactoside, 2,2. '-Azino-bis (3-ethylbenzthiazolin-6-sulfonic acid) (ABTS), N-ethyl-N-sulfopropyl-m-anisidine (ADPS), p-nitrophenyl phosphate, etc., usually used in this field Color-developing reagents that are used. In addition, these used concentrations may be appropriately set from the concentration range usually used in this field.
Further, in order to stop the color development reaction, a reaction stop method usually used in this field may be used, for example, adding an enzyme activity inhibitor such as 1 to 6N hydrochloric acid to the reaction solution.

Further, as a method for measuring human haptoglobin using only an unlabeled antibody, for example, a method for measuring using the property derived from the obtained complex, specifically, the protease activity possessed by the complex itself. Examples thereof include a method of measuring the enzyme activity and fluorescence deflection as absorbance, or a homogenia swimnoassay system 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 range usually used in this field, depending on the measuring method.

Reagents used in the method for measuring the amount of human haptoglobin according to the present invention, the concentration at the time of measurement, measurement conditions at the time of measurement, etc. (reaction temperature, reaction time, pH at the time of reaction, measurement wavelength, measuring device, etc.) All may be selected according to the measurement operation method of the immunological measurement method as described above, which is known per se. Any of the automatic analyzers, spectrophotometric systems, etc. that are usually used in this field can be used without exception.

A buffer solution is preferable as the solvent used in 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 usually used in this field, but examples thereof include those having a buffering action at a pH of 5.0 to 10.0, preferably around pH 6.5 to 8.5. Specifically, for example, all the buffer solutions usually used for the measurement method using the antigen-antibody reaction, such as Tris buffer solution, phosphate buffer solution, veronal buffer solution, borate buffer solution, and Good's buffer solution, can be mentioned. .. The buffer concentration of these buffer solutions is usually appropriately selected from the range of 10 to 1000 mM, preferably 10 to 300 mM. The pH is not particularly limited as long as it does not suppress the antigen-antibody reaction, but is usually preferably in the range of 5 to 9.

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

That is, a sample derived from a subject containing human haptoglobin is brought into contact with an insoluble carrier on which the antibody 1 of the present invention is immobilized (containing 0.1 ng to 0.1 mg of the antibody 1 of the present invention), and the temperature is 4 to 40 ° C. for 3 minutes. The reaction is carried out for about 20 hours to generate a complex of the antibody 1 of the present invention and human haptoglobin on an insoluble carrier. Next, antibody 1-human haptoglobin-labeled antibody was reacted with 10 to 100 μL of a solution containing antibody 2 labeled with POD (containing 0.01 ng to 0.1 mg of antibody 2) at 4 to 40 ° C. for 3 minutes to 20 hours. The complex of 2 is formed on an insoluble carrier. Subsequently, after adding a color-developing solution such as TMBZ solution, the reaction is carried out for a certain period of time, a reaction stop solution such as 1N HCl is added to stop the reaction, and the absorbance at a wavelength of 450 nm is measured.
On the other hand, for human haptoglobin having a known concentration, the same procedure is performed using the same reagent as above to prepare a calibration curve of the measured value and the concentration. The amount of human haptoglobin according to the present invention is determined by applying the measured value obtained by the above measurement to the calibration curve.

Further, as a method for measuring the human haptoglobin concentration in a sample, the 10-7G2A antibody established by the present inventors is used as the antibody 1 of the present invention, and the 3-1 antibody disclosed in WO2017 / 204295 is used as the antibody 2. For example, a sample derived from a subject is brought into contact with a 10-7G2A antibody and a 3-1 antibody to form a complex (complex 2) of human haptoglobin, 10-7G2A antibody and 3-1 antibody, and the complex is formed. A method of measuring the body 2 can be mentioned. The more specific method is as follows, for example.

That is, human haptoglobin in the sample is brought into contact with an insoluble carrier on which a 10-7G2A antibody is immobilized (containing 0.1 ng to 0.1 mg of 10-7G2A antibody) and reacted at 4 to 40 ° C. for 3 minutes to 20 hours to become insoluble. A complex of 10-7G2A antibody and human haptoglobin is generated on the carrier. Next, 10 to 100 μL of a solution containing POD-labeled 3-1 antibody (containing 0.01 ng to 0.1 mg of 3-1 antibody) was reacted at 4 to 40 ° C. for 3 minutes to 20 hours to obtain a 10-7 G2A antibody. -Human haptoglobin-POD-labeled 3-1 antibody complex (complex 2) is generated on an insoluble carrier. Subsequently, after adding a color-developing solution such as TMBZ solution, the reaction is carried out for a certain period of time, a reaction stop solution such as 1N HCl is added to stop the reaction, and the absorbance at a wavelength of 450 nm is measured.
On the other hand, for human haptoglobin having a known concentration, the same procedure is performed using the same reagent as above to prepare a calibration curve of the measured value and the concentration. The amount of human haptoglobin according to the present invention is determined by applying the measured value obtained by the above measurement to the calibration curve.

The amount of human haptoglobin does not have to be the actual amount of human haptoglobin (the amount of protein of human haptoglobin). The measured measurement value (signal value such as fluorescence intensity, absorbance, etc.) measured using purified human haptoglobin whose concentration is known is used as a reference value, and the same measurement is performed using a sample derived from the subject for that value. It may be a relative value (relative unit) of the measured value of human haptoglobin performed.

The method for measuring human haptoglobin according to the present invention is not limited to the method used, and may be performed by a measurement system using an automatic analyzer. In addition, there are no particular restrictions on the combination of reagents, etc. when measuring using the method or automatic analyzer, depending on the environment and model of the automatic analyzer to be applied, or considering other factors. The best combination of reagents and the like may be appropriately selected and used.

3) Method for determining inflammatory bowel disease The method for determining inflammatory bowel disease of the present invention measures the amount of human haptoglobin in a sample derived from a subject as described in "2) Method for measuring human haptoglobin" described above. It is a method of making a judgment based on the measurement result.

That is, using the antibody 1 of the present invention, the amount of human haptoglobin in the sample derived from the subject was measured by the method described in the above-mentioned "2) Method for measuring human haptoglobin", and based on the result, inflammation Obtain data on human haptoglobin for determining sexual bowel disease (for example, information on the presence or absence of human haptoglobin, concentration, degree of increase in amount, etc.). Using the obtained data, for example, the determination (diagnosis / examination) of inflammatory bowel disease is performed by the following method.

For example, if a reference value (cutoff value) is set in advance and the measurement result (measured value) of human haptoglobin according to the present invention is equal to or higher than the reference value, the subject who provided the sample has inflammatory bowel disease. It is possible to determine whether or not there is a possibility of suffering from (for example, ulcerative colitis or Crohn's disease). If the measurement result (measured value) of human haptoglobin is less than the standard value, the subject may not have inflammatory bowel disease (negative for inflammatory bowel disease) or may have. Is low, etc. can be determined.

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

Further, there is a method of setting a plurality of judgment categories according to the amount of human haptoglobin in the sample or its quantitative range (measured value or range of measured value). For example, [(1) No risk of inflammatory bowel disease, (2) Low risk of inflammatory bowel disease, (3) Signs of inflammatory bowel disease, (4) High risk of inflammatory bowel disease Etc.] to set the judgment classification. Then, it is possible to determine inflammatory bowel disease by determining which determination category the measurement result of the amount of human haptoglobin of the sample derived from the subject falls into.

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

If it is determined by the method for determining inflammatory bowel disease of the present invention that a patient who is a subject may or is likely to have inflammatory bowel disease, colonoscopy is further performed. You can choose to perform invasive examinations such as colonoscopy, gastrointestinal angiography, and histopathological examination.
On the other hand, when it is determined by the method for determining inflammatory bowel disease of the present invention that the patient who is the subject may not have inflammatory bowel disease or is unlikely to have inflammatory bowel disease, the above-mentioned invasive It is possible to choose a treatment policy in which follow-up is performed as needed without testing.

<5. How to help determine inflammatory bowel disease>
The method for assisting the determination of inflammatory bowel disease according to the present invention (hereinafter, may be abbreviated as the auxiliary method according to the present invention).
It includes (1) a step of measuring the amount of human haptoglobin in a sample using the antibody 1 of the present invention, and (2) a step of assisting the determination of inflammatory bowel disease based on the measurement result of (1) above.
The assisting method according to the present invention can be used as a method for assisting a doctor or the like in diagnosing inflammatory bowel disease.
Regarding the sample in the method for obtaining the data of the present invention, the step of measuring the amount of human haptoglobin, and the step of assisting the determination of inflammatory bowel disease, <4. It may be performed according to the sample described in the section of> Method for determining inflammatory bowel disease>, the method for measuring human haptoglobin, and the method for determining inflammatory bowel disease, and preferred examples and specific examples thereof are also the same.

<6. Device for determining inflammatory bowel disease>
The device for determining the inflammatory bowel disease according to the present invention (hereinafter, may be abbreviated as the determination device according to the present invention) includes at least (1) a measuring unit and (2) a processing unit. .. Further, it may include (3) a determination unit, (4) an output unit, and (5) an input unit.

The (1) measuring 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, a measuring device such as a device used for an immunological measuring method in the above measuring method can be mentioned.
The (2) processing unit in the determination device according to the present invention is configured to calculate the amount of human haptoglobin measured by (1) the measuring unit.
The (3) determination unit in the determination device according to the present invention is configured to determine inflammatory bowel disease based on the calculation result obtained by (2) the processing unit.
The (4) output unit in the determination device according to the present invention is configured to output (2) the calculation result obtained by the processing unit and / and (3) the determination result obtained by the determination unit.
The (5) input unit in the determination device according to the present invention receives the operation of the operator and sends the (1) measurement unit or / and (2) processing unit to the (1) measurement unit or / and (2) processing. It is configured to send a signal to activate the unit.
Regarding the measurement, calculation and determination using the (1) measurement unit, (2) processing unit and (3) determination unit of the determination device according to the present invention, <4. The method may be performed according to the method described in the section of> Method for determining inflammatory bowel disease>, and preferable examples, specific examples, and the like are also performed according to the method.

According to the determination device according to the present invention, the determination method of the present invention and / and the method of obtaining the data of the present invention can be performed easily, in a short time, and with high accuracy.

<7. Reagents for determining inflammatory bowel disease>
The reagent for determining inflammatory bowel disease according to the present invention (hereinafter, may be abbreviated as the reagent according to the present invention) includes the antibody 1 of the present invention as a constituent requirement. The reagent according to the present invention may further contain the antibody 2 according to the present invention.
Regarding the antibody 1 of the present invention contained in the reagent according to the present invention, <3. As described in the section of the antibody (antibody 1)> of the present invention, preferred examples, specific examples and the like are also the same. The antibody 2 is the same as that described in the description of the antibody 2 in the section “4. Method for determining inflammatory bowel disease”, and preferred examples, specific examples, and the like 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 a range usually used in this field, depending on the measurement method. In addition, the reagents may contain reagents usually used in this field, such as buffers, reaction accelerators, sugars, proteins, salts, stabilizers such as surfactants, preservatives and the like. These do not inhibit the stability of coexisting reagents and do not inhibit the reaction of antibody 1 according to the present invention. In addition, these concentrations may be appropriately selected from the concentration range usually used in this field.

<8. Kit for determining inflammatory bowel disease of the present invention>
The kit of the present invention
"To determine inflammatory bowel disease, which comprises an anti-human haptoglobin antibody (antibody 1 of the present invention) that specifically binds to the region of the amino acid sequence represented by SEQ ID NO: 1 of the α chain of human haptoglobin. A kit for measuring the amount of haptoglobin. "

The kit may further contain the antibody 2 according to the present invention.

That is, as a specific example of the kit of the present invention,
(1) Determine inflammatory bowel disease including a reagent containing the antibody 1 of the present invention (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). (Kit 1 of the present invention), or (2) a reagent containing the antibody 1 of the present invention and a reagent containing antibody 2 (an antibody that recognizes human haptoglobin, which has a different epitope from antibody 1). Kit for determining inflammatory bowel disease including (Kit 2 of the present invention),
Can be mentioned.

The antibody 1 of the present invention contained in the kit 1 and the kit 2 of the present invention is described in <3. It is the same as that described in the section of the antibody (antibody 1)> of the present invention, and the preferred one is also the same.
The antibody 2 contained in the kit 2 of the present invention is described in <4. It is the same as that described in the description of antibody 2 in the section> Method for determining inflammatory bowel disease>, and preferred examples, specific examples, and the like are also the same.
Antibody 1 or antibody 2 may be supported on an insoluble carrier. Further, it may be labeled with a labeling substance.

For preferable aspects, specific examples, concentrations, etc. of each component of the kit of the present invention, refer to <4. Method for determining inflammatory bowel disease> As described in the section.

The concentrations of antibody 1 and antibody 2 in the reagents in the kit 1 and kit 2 of the present invention may be appropriately set within a range usually used in this field depending on the measurement method. In addition, these reagents may contain reagents usually used in this field, such as buffers, reaction accelerators, sugars, proteins, salts, stabilizers such as surfactants, preservatives and the like. 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. In addition, these concentrations may be appropriately selected from the concentration range usually used in this field.

Further, the kits 1 and 2 of the present invention may include, in addition to the antibody 1 and the antibody 2 of the present invention, a required amount of reagents necessary for measurement such as immunoassay of the amount of human haptoglobin using the antibody.

Kit 1 and Kit 2 of the present invention may be combined with a standard product of human haptoglobin for preparing a calibration curve used when measuring the human haptoglobin. As the standard product, a commercially available standard product may be used, or a product manufactured according to a known method may be used.

Furthermore, the kit of the present invention may include an instruction manual or the like for use in the method for determining inflammatory bowel disease of the present invention. The "instruction manual" is an instruction manual, package insert, pamphlet (leaflet), etc. of the kit in which the features, principles, operating procedures, judgment procedures, etc. of the method are substantially described in sentences or charts. Means.

Hereinafter, the present invention will be specifically described based on Examples, but the present invention is not limited to these Examples.

Example 1. Acquisition of anti-human haptoglobin monoclonal antibody (10-7G2A antibody) reactive with α chain (1) Preparation of human haptoglobin Human colon cancer cell line by the method described in Oncotarget, 9 (16), 12732-44., 2018. The established strain that stably overexpresses human haptoglobin obtained by introducing the human haptoglobin gene into HCT116 (ATCC) is 10% fetal bovine serum (FBS; HyClone, Logan, UT), 100 U / mL penicillin, and 100 μg. The cells were cultured in RPMI with L-glutamine and LVDS ₃ (manufactured by nacalai tesque) supplemented with / mL streptomycin under 37 ° C. and 5% CO ₂ conditions. IWAKI culture plates 10 cm and 15 cm (IWAKI, Tokyo, Japan) were used as culture plates.
After culturing, the cells were cultured for 96 hours using RPMI containing no FBS plus 1 mM L-fucose, and then the culture supernatant was collected.
The obtained culture supernatant was applied to a human haptoglobin antibody column using PERISTA bio-mini-pump (ATTO, Japan, Tokyo) (0.5 mL / min, 4 ° C, overnight). The human haptoglobin antibody column was prepared by coupling 7.5 mg of an anti-human haptoglobin polyclonal antibody (manufactured by Dako) to Hi-Trap-NHS-activated HP (manufactured by GE Healthcare).
Then, Column Washing Buffer (50 mM Na ₂ HPO ₄ , 50 mM NaH ₂ PO ₄ , 0.5 M NaCl, pH 7.4) was applied to the antibody column at 1.0 mL / min for 15 minutes, and the antibody column was washed. Further, Elution buffer (0.1 M Glycine, pH 2.7) was applied to the antibody column at 0.5 mL / min for 20 minutes, and human haptoglobin was eluted from the antibody column.
Neutralization buffer (2M Tris-HCl, pH 8.0) was added to the obtained eluate in an amount of 1.0 mL to neutralize the eluate. The eluate was 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 performed at 4 ° C.

(2) Preparation of anti-human haptoglobin monoclonal antibody 200 μg of the human haptoglobin prepared above was immunized with BALB / c mice together with Freund's complete adjuvant. Then, 50 μg of human haptoglobin was immunized twice at 2-week intervals, and finally 100 μg of human haptoglobin was immunized. After that, the spleen was removed, and the spleen cells and myeloma cells (SP2 / 0) were fused by a conventional method using polyethylene glycol (described in Japanese Patent Application Laid-Open No. 5-244983), and this was fused with GIT medium (Fuji Film Wako Pure Chemical Industries, Ltd.). ) Was cultured.

(3) Primary screening of anti-human haptoglobin monoclonal antibody 0.25 μg of anti-mouse IgG antibody (rabbit) (manufactured by Sigma-Aldrich) was immobilized per well of a microplate. Then, it was blocked with bovine serum albumin (BSA), casein, or the like.
The supernatant of the cell culture solution obtained in (2) above or 50 μL of the culture solution (control) was added to the well and allowed to stand for 60 minutes. Then, the wells were washed 3 times with a washing solution (PBS-T) in which 0.1% Tween 20 was added to PBS.
Next, the human haptoglobin obtained in (1) above was dissolved in PBS to a concentration of 250 ng / mL, 50 μL thereof was added to a well, and the mixture was allowed to stand for 60 minutes. The wells were then washed 3 times with PBS-T.
Next, POD-labeled anti-human haptoglobin polyclonal antibody [polyclonal antibody was purchased from DAKO and labeled with POD by a conventional method (Eiji Ishikawa, "Enzyme Labeling Method", Society Publishing Center, 1991, p.62). ] Was added, and the mixture was allowed to stand for 30 minutes. The wells were then washed 3 times with PBS-T.
Further, 50 μL of a substrate solution (o-phenylenediamine (OPD) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)) was added to develop color for 30 minutes, and 100 μL of a 1 M sulfuric acid solution was added to terminate the reaction. Then, the absorbance of the obtained solution at 492 nm was measured using an absorptiometer. Based on this result, the luminescent anti-human haptoglobin monoclonal antibody was selected as a primary candidate.

(4) Secondary screening of anti-human haptoglobin monoclonal antibody Using the several types of anti-human haptoglobin monoclonal antibody obtained in (3) above, the following Western blotting was performed, and the antibody having reactivity with the human haptoglobin α chain was performed. Was selected.
That is, first, using 50 mM phosphate buffer, human haptoglobin type 1-1 refined product (Haptoglobin, Phenotype 1-1, Hpt1-1 type, manufactured by Sigma Aldrich) and human haptoglobin type 2-2 refined product (Haptoglobin). , Phenotype 2-2, Hpt2-2 type, manufactured by Sigma Aldrich Co., Ltd.) were prepared to 100 μg / mL each, and sample buffer 1 (0.25 M Tris-HCl pH 6.8, 8% SDS, 40% glycerol, A sample was prepared by mixing 0.02% BPB, 20% 2-mercaptoethanol at a ratio of 3: 1 and treating at 95 ° C. for 5 minutes.
Then, 4 μL of the sample was electrophoresed on a 12.5% polyacrylamide gel. The obtained electrophoresis gel was blotting on a PVDF membrane semi-dry according to the protocol using a blotting system manufactured by Bio-Rad. The PVDF membrane after transfer was blocked with a phosphate buffer solution containing 4% of Block Ace (manufactured by DS Pharma Biomedical Co., Ltd.).
The POD-labeled primary candidate anti-human haptoglobin monoclonal antibody was immersed in a solution diluted 200-fold with a phosphate buffer containing 4% of Block Ace, and the membrane was allowed to react at room temperature for 1 hour. After the reaction, the membrane was washed 3 times with phosphate buffer containing 0.05% Tween 20. After washing, the membrane was dissolved in β-NADH (manufactured by Oriental Yeast Co., Ltd.) 20 mg, Nitro-TB (manufactured by Dojin Chemical Co., Ltd.) 3 mg, and 50 μL of 0.2% hydrogen peroxide solution (50 mM PB, pH 7.5) Immersed in 10 mL for 10 to 30 minutes to develop a blue color. After color development, the membrane was washed with purified water to stop the reaction.

From this result, an antibody having reactivity with both the α1 chain of human haptoglobin type 1-1 (band near 10KDa) and the α2 chain of human haptoglobin type 2-2 (band around 18KDa) was selected. That is, an antibody having an ability to bind to the α chain was obtained. The antibody was named "10-7G2A antibody".
FIG. 2 shows the migration results when the 10-7G2A antibody was used. In FIG. 2, lane 1 shows the result of using Hpt1-1 type as a sample, and lane 2 shows the result of using Hpt2-2 type as a sample.

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

2) Preparation of cDNA Fragment Encoding Human Haptoglobin α Chain Fragment Using the recombinant plasmid obtained in 1) above as a template, a cDNA fragment encoding the α chain fragment was prepared by the following method.

First, PCR was performed using the primer pairs of Hpt-α-F1 and Hpt-α-R1 shown in Table 1 below to amplify the cDNA encoding the full-length amino acid sequence of the human haptoglobin α2 chain fragment.
In addition, PCR was performed using the primer pairs shown in combinations 3 to 7 in Table 1 below to amplify various cDNAs encoding the amino acid sequences of the fragments of the human haptoglobin α2 chain fragment.
In the above PCR, the first denaturation was performed at 95 ° C. for 3 minutes, then the reaction [98 ° C., 10 seconds → 57 ° C., 15 seconds → 68 ° C., 35 seconds] was performed for 40 cycles, and finally 68 ° C. for 7 minutes. It was reacted.
The amino acid sequences of the primer pair used in the above PCR and the α-chain fragment fragment of human haptoglobin encoded by the nucleotide sequence of the cDNA amplified by 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 a pGEM-Teasy vector. Escherichia coli was transformed using the subcloned individual vectors, and the transformed Escherichia coli was grown using a liquid medium, and then each vector was purified using a plasmid purification kit. A part of the purification vector is digested with restriction enzymes NheI and EcoRI, and after electrophoresis, the fragments are purified from the electrophoresis gel and ligated to the NheI-EcoRI site of the pCIneo expression vector (Promega). A replacement plasmid was obtained.
Using the obtained recombinant plasmid, HEK293T cells were transfected by a conventional method, and the transfected HEK293T cells were cultured in OPTI-MEM medium for 2 days to transiently express the target human haptoglobin α chain fragment. The supernatant containing the fragment was collected.

Separately, the full length of the Hpt2 gene and the C1rlp gene obtained by PCR cloning were introduced into HEK293T cells by co-transfection, the obtained transduced cells were cultured, and human haptoglobin α2 was co-expressed with full-length human haptoglobin and C1RLP. A chain fragment was obtained.
C1RLP is an enzyme that cleaves the full-length haptoglobin into α1 chain and β chain or α2 chain and β chain.

(2) Primary screening of 10-7G2A antibody epitopes After performing SDS-PAGE using the culture supernatant containing each human haptoglobin α chain fragment of the transfected HEK293T cells obtained above as a sample, PVDF membrane Transferred to. After blocking the PVDF membrane, the 10-7G2A antibody was reacted at room temperature for 1 hour. After washing the PVDF membrane with PBS-T three times, react with HRP (Horseradish peroxidase) -labeled anti-mouse IgG (secondary antibody) for 1 hour at room temperature, wash the PVDF membrane again with PBS-T three times, and then ImmunoStar ^TM. It was made to emit light using Zeta (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and imaged using the FUSION Chemiluminescence imaging system (Vilber-Lourmat, Marne la Vallee, France).

The results are shown in Fig. 3.

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

The "total length of the α2 chain fragment" in lane 3 is the signal peptide from the N-terminal to the 18th amino acid and the 19th V in which the C-terminal amino acid is cleaved in the amino acid sequence represented by SEQ ID NO: 3. It is a polypeptide having an amino acid sequence of 142 amino acids up to the 160th Q.

In addition, from the results shown in FIG. 3, it was found that each protein had almost the same concentration by Western blot of β-actin.

Based on the results of FIG. 3, the region of the amino acid sequence (DDGCPKPPEIAHGYVEHSVRYQCKNYYKLRTEGDGVYTLNNE) common to SEQ ID NOs: 13 to 17 and represented by SEQ ID NO: 18 is a region containing an epitope of the 10-7G2A antibody. I presumed that it was selected.

(3) Secondary screening of 10-7G2A antibody epitope 1) Preparation of human haptoglobin α-chain fragment peptide library A peptide library of the amino acid sequence of the region selected above in the α-chain of human haptoglobin was designed. That is, the peptides of Pep # 1 to Pep # 6 shown in FIG. 4 are based on the position of the amino acid sequence represented by SEQ ID NO: 18 on the amino acid sequence represented by SEQ ID NO: 3 (amino acid sequence of α2 chain). Was designed and synthesized.
The relationship between the amino acid sequence of each peptide and the SEQ ID NO: in the present specification is shown in Table 2 below.

For peptide synthesis, use Rink Amide resin (50-80 μmol scale, WATANABE CHEMICAL INDUSTRIES, LTD.), Peptide synthesizer Prelude (Protein Technologies, Inc.), or a high-speed flow peptide synthesizer developed by Pentelute, etc. The Fmoc peptide solid phase synthesis method (SPPS) was used.

When using the peptide synthesizer Prelude, peptide synthesis was performed by 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 peptides potent antitumor activity in vivo, Journal of the American Chemical Society 134 (12) ( 2012) 5428-31.).
That is, in dimethylformamide (DMF), Fmoc-AA-OH (4.6 eq) was reacted with HCTU (4.0 eq) and N-methylmorpholine (16 eq) for 15 minutes and coupled to the resin. .. This coupling step was performed twice. The resin was washed with DMF and then reacted with DMF (5 mL) containing 20% piperidine for 5 minutes to deprotect the Fmoc groups. The above coupling reaction was repeated until the peptide elongation was completed.

On the other hand, when using a high-speed fluid peptide synthesizer, Fmoc-AA-OH (1.0 mmol) is reacted with 0.38 M HBTU (a solution containing 2.5 mL of DMF and 0.5 mL of N, N-diisopropylethylamine (DIEA)). And coupled to the resin. When Fmoc-Cys and Fmoc-His were bound to suppress epimerization, an Fmoc-AA-OH-containing solution 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.
A 3-minute cycle (coupling, washing, deprotection, and washing) was repeated by the method 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 all amino acids by synthesis using a peptide synthesizer or a high-speed fluid peptide synthesizer, peptidyl resin is added to 82.5% TFA, 5% phenol, 5% water, 5% thioanisole, 2.5% 1,2-ethane. Treat with 10 mL of dithiol-containing solution or 10 mL of solution containing 95% trifluoroacetic acid (TFA), 2.5% water, and 2.5% triisopropylsilane to remove all side chain protecting groups and remove amino acids from the resin. Cleavage gave a mixture containing the crude peptide. Ice-cold ether was added to the obtained mixture to obtain a precipitate, and the precipitate 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 A mixture containing the crude peptides of Pep # 1 to Pep # 6 obtained in (3) 1) above was placed on a 96-well plate (Pierce ^TM , Amine-binding, Maleic). Immobilized on Anhydride 96-well Plates (manufactured by Thermo SCIENTIFIC) according to the manufacturer's instructions. The 10-7G2A antibody was then reacted with a 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. Then, the peptides having the Pep # 7 to Pep # 19 amino acid sequences shown in Table 2 were synthesized by the method of (3) 1) above.
Each of the obtained synthetic peptides was immobilized on 96-well plates (Pierce ^TM , Amine-binding, Maleic Anhydride 96-well Plates, manufactured by Thermo SCIENTIFIC) according to the manufacturer's instructions. The 10-7G2A antibody was then reacted with a 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 particularly binds to Pep # 18.

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

As a result of verification, the 10-7G2A antibody showed reactivity with Pep # 22, Pep # 23, and Pep # 26. From this, QCKNYY (SEQ ID NO: 1), which is a sequence common to Pep # 22 and Pep # 23, was identified as an epitope candidate for the 10-7G2A antibody.

3) Inhibition experiment of 10-7G2A antibody
(i) Sample 1/100 of the culture supernatant obtained by introducing the human haptoglobin gene into the colorectal cancer cell line HCT116 and culturing the established strain with stable overexpression in the L-fucos-added culture medium, or the serum of a pancreatic cancer patient. Diluted with and used as a sample. MOPS buffer containing 2% BSA was used as the control.

(Ii) Synthetic Peptide Solution The crude peptide of peptide 1 (QCKNYY, SEQ ID NO: 1), peptide 2 (YYNKCQ, SEQ ID NO: 46), and control peptide (LPECEA, SEQ ID NO: 47) was prepared by the method of (3) 1) above. Obtained.
The amino acid sequence of peptide 2 is the reverse sequence of peptide 1.
Next, reverse phase HPLC was performed using a reverse phase HPLC column Proteonavi.

Fractions containing the desired product were collected and lyophilized. Peptide 1 was obtained in 57%, peptide 2 in 47%, and control peptide in 50% isolation yield.
Each purified peptide was dissolved in PBS and used as a synthetic peptide solution.

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

The synthetic peptide solution was added so that the concentration of the synthetic peptide during the reaction with the POD-labeled 10-7G2A antibody was 50 μg / mL, 150 μg / mL, 250 μg / mL, 400 μg / mL, or 500 μg / mL, respectively. It was.
The POD-labeled 10-7G2A antibody used above is the same as the 10-7 antibody obtained in Example 1 by a conventional method (Eiji Ishikawa, "Enzyme Labeling Method", Society Publishing Center, 1991, p.62). It is labeled with horseradish peroxidase (method of).

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

(Iv) Results The obtained results are shown in Fig. 5.
FIG. 5 (a) shows inhibition using a culture supernatant obtained by culturing an established strain in which the human haptoglobin gene was introduced into the colon cancer cell line HCT116 and stably overexpressed in a culture solution containing L-fucos as a sample. The results of the assay are shown. FIG. 5 (b) shows the results of an inhibition assay using serum from a pancreatic cancer patient as a sample.
Further, in FIGS. 5 (a) and 5 (b), (1) is a measurement result using a control peptide solution, (2) is a measurement result (broken line) using synthetic peptide 1, and (3) is. The measurement results (solid line) using the synthetic peptide 3 are shown respectively.
Further, in FIG. 5, the vertical axis shows the ratio (%) of the absorbance obtained by the measurement in the presence of the synthetic peptide at each concentration when the absorbance obtained by the measurement using the control peptide is 100. ..

As is clear from the results of FIGS. 5 (a) and 5 (b), synthetic peptide 1 inhibited the binding of the 10-7G2A antibody to human haptoglobin. In addition, the synthetic peptide 2 having the reverse amino acid sequence of the synthetic peptide 1 inhibited the binding of the 10-7G2A antibody to human haptoglobin, similarly to the synthetic peptide 1.
From the above results, the amino acid sequence represented by 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 determination of inflammatory bowel disease (1) Preparation of samples and reagents 1) Samples Ulcerative colitis patients (n = 45), Crohn's disease patients (n) = 20) and the serum of a healthy person (n = 67) were diluted to 1 / 5,000 to prepare a sample. The culture supernatant of the liver cancer cell line HepG2 was used as a control.

2) Antibodies used Antibodies 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: The 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 a conventional method, and then Block Ace. It was blocked with phosphate buffered saline (PBS) containing 1% (manufactured by DS Pharma Biomedical Co., Ltd.) to obtain a plate on which the 10-7G2A antibody was immobilized.

4) POD-labeled 3-1 antibody solution 3-1 After making the antibody into a Fab'fragment by a conventional method, the conventional method (Eiji Ishikawa, "Enzyme Labeling Method", Society Publishing Center, 1991, p.62) Labeled with peroxidase (POD). Then, it was diluted with TBS containing 10% of Block Ace (manufactured by DS Pharma Biomedical Co., Ltd.) to obtain a POD-labeled 3-1 antibody solution (1.06 μmol / L). At the time of use, it was diluted 5000 times with TBS containing 10% of Block Ace (manufactured by DS Pharma Biomedical Co., Ltd.). The concentration of the POD-labeled 3-1 antibody solution at the time of use is 2.12 × 10 ^-1 pmol / L.

5) Coloring solution, etc. The following test solutions were prepared.
Color-developing solution: 5 mmol / L 3,3', 5,5'-tetramethylbenzidine (TMBZ) solution (manufactured by Sigma-Aldrich)
Reaction terminator: 1N HCl
Washing solution: Tris-buffered saline containing 0.05% polyoxyethylene (20) sorbitan monolaurate (Tween20)

(2) Measurement of human haptoglobin Using iMark TM Microplate Reader (Biorad), the amount of human haptoglobin in the sample was measured by the following method.

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

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

The absorbance value in the case of Hpt2-2 type refined product 1 μg / mL was set to 1 (reference). Then, the relative value of the absorbance obtained by the above measurement using the sample derived from the subject was determined with respect to the standard. The relative value is expressed as "relative unit".
The significant difference test represents a nonparametric comparison of all pairs performed by the Wilcoxon test using the predictive analysis software JMP pro 14.
The obtained results are shown in FIG.
The measurement results are shown in Table 3.

As is clear from FIG. 6 and Table 3, the amount of human haptoglobin obtained by measuring the amount of human haptoglobin using the combination of 10-7G2A antibody and 3-1 antibody was ulcerative colitis as compared with healthy subjects. It was significantly higher in patients and patients with Crohn's disease.
From the above results, it was found that the determination method of the present invention can determine inflammatory bowel disease.

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

First, the amount of human haptoglobin in the serum was measured by the same method as in Example 3 using the serum (n = 70) of a healthy person whose serum CRP value was determined to be high by clinical examination.

FIG. 7 shows the measurement results of the serum human haptoglobin amount of a healthy person having a high serum CRP value (healthy person with a high CRP value) obtained by the above measurement.
In addition, the measurement results of the serum human haptoglobin amount of the healthy person (n = 20) obtained in Example 3 are also shown in FIG. The serum CRP values of the healthy subjects in Example 3 were all normal values.

Furthermore, the relationship between the amount of serum human haptoglobin and the serum CRP value of healthy subjects with high serum CRP value and healthy subjects with normal serum CRP value (n = 90) was statistically analyzed. The analysis was performed by Pearson's product moment correlation coefficient test using JMPpro14, a predictive analysis software.

The obtained results are shown in FIG. That is, FIG. 8 is a correlation diagram between the serum CRP value (logarithm) of a healthy person and the serum human haptoglobin amount (logarithm). As a result of the analysis, the relationship between the serum human haptoglobin amount and the CRP value was an analysis correlation coefficient ρ = 0.139 and a p value = 0.223.

As is clear from the results of FIG. 7, the serum human haptoglobin level was not increased in the healthy subjects having a high CPR value as compared with the healthy subjects having a normal CRP value.
Moreover, as is clear from the results of FIG. 8, no correlation was observed between the serum human haptoglobin amount and the serum CRP value.

Both haptoglobin and CRP are known to increase in inflammatory diseases, but CRP has been considered to be superior to haptoglobin in terms of sensitivity. However, from the above results, the amount of human haptoglobin does not correlate with the CRP level, and in subjects who do not have inflammatory bowel disease, the amount of human haptoglobin does not increase even if the serum CRP level increases. Became clear. This result indicates that elevated CRP levels do not reflect intestinal inflammation.
On the other hand, as is clear from the results of Examples 3 and 6, the serum human haptoglobin amount was significantly increased in inflammatory bowel disease.

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

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

The obtained results are shown in FIG. That is, FIG. 9 is a correlation diagram between the CAI of a patient with ulcerative colitis and the amount of serum human haptoglobin.
As a result of the analysis, the relationship between the amount of serum human haptoglobin and CAI was an analysis correlation coefficient ρ = 0.574 and a p-value = 0.0001.
That is, in ulcerative colitis, it was confirmed that the amount of serum human haptoglobin has a positive correlation with CAI, which is clinically used as an activity index of ulcerative colitis.

From the above results, it was confirmed that the determination method of the present invention can objectively determine inflammatory bowel disease, reflecting the local pathological condition of inflammatory bowel disease.

Claims (10)

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. Method for determining inflammatory bowel disease, including the following steps:
   (1) The amount of human haptoglobin in a sample derived from a subject is measured using antibody 1, which is 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. Process to do,
   (2) A step for determining inflammatory bowel disease based on the result of (1) above.
3. The method for determining inflammatory bowel disease according to claim 2, wherein the step (1) includes the following steps:
   (1-i) A step of contacting a sample derived from a subject with the antibody 1 to form a complex 1 of human haptoglobin and the antibody 1.
   (1-ii) A step of measuring the amount of the complex 1.
4. The method for determining inflammatory bowel disease according to claim 2, wherein the step (1) further uses an antibody 2 that recognizes human haptoglobin, which has an epitope that is different from that of antibody 1, in combination.
5. The method for determining inflammatory bowel disease according to claim 2, wherein the step (1) includes the following steps:
   (1-i) A sample derived from a subject is brought into contact with antibody 1 and antibody 2, which is an antibody that recognizes human haptoglobin and has a different epitope from antibody 1, and human haptoglobin and the antibody 1 are combined. Step of forming a complex 2 with the antibody 2
   (1-ii) A step of measuring the amount of the complex 2.
6. The method for determining inflammatory bowel disease according to claim 4, wherein either one of the antibody 1 and 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 according to claim 2, wherein the sample derived from the subject is serum, plasma, or whole blood.
8. 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 is used to measure the amount of human haptoglobin in a sample derived from a subject. A method for obtaining data for determining inflammatory bowel disease.
9. A kit for measuring the amount of human haptoglobin for determining inflammatory bowel disease, which comprises 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. ..
10. The kit according to claim 9, further comprising an antibody that binds to human haptoglobin, which has an epitope different from that of the anti-human haptoglobin antibody.

Images