Methods of examination for ulcerative colitis and primary sclerosing cholangitis

Abstract

The present invention aims to provide a specific test method for ulcerative colitis and primary sclerosing cholangitis. The present invention first provides a test method for ulcerative colitis, providing a test process for detecting antibodies that immunoreact with integrin αVβ6 and / or antibodies that immunoreact with integrin αVβ3 in a sample as an indicator of ulcerative colitis. The present invention secondly provides a test method for primary sclerosing cholangitis, providing a test process for detecting antibodies that immunoreact with integrin αVβ6 in a sample as an indicator of primary sclerosing cholangitis.

Description

Technical Field

[0001] One embodiment of the present invention relates to a method for testing ulcerative colitis (UC) and a test kit for testing UC.

[0002] Another aspect of the present invention relates to a method for testing primary sclerosing cholangitis (PSC) and a test kit for testing PSC. Background Art

[0003] In ulcerative colitis, erosion or inflammatory ulcers of the colon mucosa occur successively from the rectum, accompanied by symptoms such as diarrhea, bloody stools, and abdominal pain. Ulcerative colitis mainly occurs in young people and even the elderly, and because of its repeated active and remission periods, long-term treatment is necessary. In recent years, ulcerative colitis has been increasing worldwide.

[0004] Primary sclerosing cholangitis (PSC) is a progressive chronic liver disease with multiple, diffuse strictures of the bile ducts inside and outside the liver. PSC is considered to be a multifactorial disease including immune abnormalities, but the cause is still unclear. PSC is often complicated by inflammatory bowel disease (especially ulcerative colitis).

[0005] Patent Document 1 describes a mapping method for diagnosis and / or prognosis prediction of ulcerative colitis, which includes classifying the intestinal flora of a subject into 10 intestinal flora groups and performing data processing on each intestinal flora group by self-organizing mapping analysis.

[0006] Patent Document 2 describes measuring the concentration of soluble LR11 in a blood sample from a mammal, and using the concentration as an evaluation index for the severity and prognosis prediction of liver diseases such as primary sclerosing cholangitis and viral hepatitis.

[0007] Prior art literature

[0008] Patent Literature

[0009] Patent Document 1: Japanese Patent Application Publication No. 2017-122603

[0010] Patent Document 2: Japanese Patent Application Publication No. 2014-167446 Summary of the invention

[0011] Technical problem to be solved by the invention

[0012] The existing diagnosis of ulcerative colitis is based on comprehensive judgment of clinical symptoms, endoscopic observation results, enema X-ray examination, and biopsy histological examination. However, molecular biological diagnostic criteria using biomarkers specific to ulcerative colitis have not yet been found. Although a mapping method for diagnosing ulcerative colitis is described in Patent Document 1, it is not easy to implement because it is necessary to collect intestinal flora and perform genetic analysis.

[0013] Similarly, regarding primary sclerosing cholangitis, the existing diagnosis is based on non-specific criteria such as clinical symptoms, and a molecular biological diagnostic criterion using a biomarker specific to primary sclerosing cholangitis has not yet been found. Although the method described in Patent Document 2 is a method for diagnosing liver diseases including primary sclerosing cholangitis and viral hepatitis, it is not a method for distinguishing primary sclerosing cholangitis from other liver diseases and performing a specific diagnosis.

[0014] Therefore, the present invention aims to provide a technique related to the molecular biological diagnosis of ulcerative colitis and primary sclerosing cholangitis.

[0015] Technical solutions for solving technical problems

[0016] The present inventors surprisingly found that the blood concentrations of autoantibodies to integrin αVβ6 and autoantibodies to integrin αVβ3 were significantly increased in patients with ulcerative colitis and primary sclerosing cholangitis, and based on this finding, completed the following invention.

[0017] (1) A method for examining ulcerative colitis,

[0018] The method comprises a detection step of detecting antibodies immunoreactive with a fragment or the whole of integrin αVβ6 and / or antibodies immunoreactive with a fragment or the whole of integrin αVβ3 in a sample as indicators of ulcerative colitis.

[0019] (2) The method according to (1), wherein the detection step includes using a fragment or the whole of integrin αVβ6 as an antigen to detect antibodies that immunoreact therewith and / or using a fragment or the whole of integrin αVβ3 as an antigen to detect antibodies that immunoreact therewith.

[0020] (3) The method according to (1) or (2), wherein the sample is a blood sample.

[0021] (4) A test kit or test reagent for testing ulcerative colitis, comprising a fragment or the whole of integrin αVβ6 and / or a fragment or the whole of integrin αVβ3.

[0022] (5) The test kit or test reagent according to (4), further comprising a detection antibody.

[0023] (6) The test kit or test reagent according to (4) or (5), further comprising a positive standard solution and / or a negative standard solution.

[0024] (7) The test kit or test reagent according to any one of (4) to (6), comprising the fragment or the whole of the integrin αVβ6 and / or the fragment or the whole of the integrin αVβ3 in a form fixed to a solid phase.

[0025] (8) A method for examining primary sclerosing cholangitis,

[0026] The method comprises a detection step of detecting antibodies immunoreactive with a fragment or the whole of integrin αVβ6 and / or antibodies immunoreactive with a fragment or the whole of integrin αVβ3 in a sample as indicators of primary sclerosing cholangitis.

[0027] (9) According to the method described in (8), the detection step includes using a fragment or the whole of integrin αVβ6 as an antigen to detect antibodies that immunoreact therewith and / or using a fragment or the whole of integrin αVβ3 as an antigen to detect antibodies that immunoreact therewith.

[0028] (10) The method according to (8) or (9), wherein the sample is a blood sample.

[0029] (11) A test kit or test reagent for testing primary sclerosing cholangitis, comprising a fragment or the whole of integrin αVβ6 and / or a fragment or the whole of integrin αVβ3.

[0030] (12) The test kit or test reagent according to (11), further comprising a detection antibody.

[0031] (13) The test kit or test reagent according to (11) or (12), further comprising a positive standard solution and / or a negative standard solution.

[0032] (14) The test kit or test reagent according to any one of (11) to (13), comprising the fragment or the whole of the integrin αVβ6 and / or the fragment or the whole of the integrin αVβ3 in a form fixed to a solid phase.

[0033] (15) A method for evaluating the therapeutic effect of ulcerative colitis,

[0034] The method comprises a detection step of detecting antibodies immunoreactive with a fragment or the whole of integrin αVβ6 and / or antibodies immunoreactive with a fragment or the whole of integrin αVβ3 in a sample obtained from a test animal treated for ulcerative colitis.

[0035] (16) A method for evaluating the therapeutic effect of primary sclerosing cholangitis,

[0036] The method comprises the step of detecting antibodies immunoreactive with a fragment or the whole of integrin αVβ6 and / or antibodies immunoreactive with a fragment or the whole of integrin αVβ3 in a sample obtained from a test animal treated for primary sclerosing cholangitis.

[0037] (17) A method for diagnosing ulcerative colitis, comprising:

[0038] detecting antibodies immunoreactive with fragments or the entirety of integrin αVβ6 and / or antibodies immunoreactive with fragments or the entirety of integrin αVβ3 in a sample from the test animal, and

[0039] When the antibody is detected, it is determined that the test animal suffers from ulcerative colitis.

[0040] The subject animal herein is a human or a non-human animal, preferably a human.

[0041] (18) According to the method described in (17), the detection includes using a fragment or the whole of integrin αVβ6 as an antigen to detect antibodies that immunoreact therewith and / or using a fragment or the whole of integrin αVβ3 as an antigen to detect antibodies that immunoreact therewith.

[0042] (19) According to the method of (17) or (18), the sample is a blood sample, preferably a blood sample isolated from the subject animal.

[0043] (20) A biomarker for diagnosing ulcerative colitis, comprising an antibody that immunoreacts with a fragment or the entirety of integrin αVβ6 and / or an antibody that immunoreacts with a fragment or the entirety of integrin αVβ3.

[0044] (21) A method for screening candidate substances for a therapeutic drug for ulcerative colitis, comprising:

[0045] an antibody detection step of detecting an antibody immunoreactive with a fragment or the whole of integrin αVβ6 and / or an antibody immunoreactive with a fragment or the whole of integrin αVβ3 in a sample obtained from an animal to which a test substance has been administered, and

[0046] The selection step is to select the test substance as a candidate substance for the therapeutic drug for ulcerative colitis when the antibody in the sample decreases due to the administration of the test substance.

[0047] (22) A method for producing a non-human model animal of ulcerative colitis, comprising at least one of an antibody administration step and an immunization step,

[0048] The antibody administration step comprises administering to the non-human animal an antibody that immunologically reacts with a fragment or the whole of integrin αVβ6 and / or an antibody that immunologically reacts with a fragment or the whole of integrin αVβ3,

[0049] In the immunization step, a fragment or the whole of integrin αVβ6 and / or a fragment or the whole of integrin αVβ3 are used as antigens to immunize non-human animals.

[0050] (23) A method for obtaining an index of ulcerative colitis, comprising:

[0051] A step of detecting antibodies immunoreactive with a fragment or the whole of integrin αVβ6 and / or antibodies immunoreactive with a fragment or the whole of integrin αVβ3 in a sample.

[0052] (24) According to the method described in (23), the detection step includes using a fragment or the whole of integrin αVβ6 as an antigen to detect antibodies that immunologically react therewith and / or using a fragment or the whole of integrin αVβ3 as an antigen to detect antibodies that immunologically react therewith.

[0053] (25) According to the method according to (23) or (24), the sample is a blood sample.

[0054] (26) Use of a fragment or the whole of integrin αVβ6 and / or a fragment or the whole of integrin αVβ3 in the manufacture of a test kit or test reagent for testing ulcerative colitis.

[0055] (27) The use according to (26), wherein the fragment or the whole of integrin αVβ6 and / or the fragment or the whole of integrin αVβ3 are in a form fixed to a solid phase.

[0056] (28) A fragment or whole of integrin αVβ6 and / or a fragment or whole of integrin αVβ3 for use in the examination of ulcerative colitis.

[0057] (29) The fragment or whole of integrin αVβ6 and / or the fragment or whole of integrin αVβ3 according to (25), which is in a form fixed to a solid phase.

[0058] (30) An antibody that immunoreacts with a fragment or the whole of integrin αVβ6 and / or an antibody that immunoreacts with a fragment or the whole of integrin αVβ3, for use in a method for producing a non-human animal model of ulcerative colitis.

[0059] (31) A fragment or whole of integrin αVβ6 and / or a fragment or whole of integrin αVβ3 for use in a method for preparing a non-human animal model of ulcerative colitis.

[0060] (32) Use of an antibody that immunoreacts with a fragment or the whole of integrin αVβ6 and / or an antibody that immunoreacts with a fragment or the whole of integrin αVβ3 in the manufacture of a reagent for producing a non-human model animal of ulcerative colitis.

[0061] (33) Use of a fragment or the whole of integrin αVβ6 and / or a fragment or the whole of integrin αVβ3 in the manufacture of a reagent for producing a non-human model animal of ulcerative colitis.

[0062] (34) A method for diagnosing primary sclerosing cholangitis, comprising:

[0063] detecting antibodies immunoreactive with fragments or the entirety of integrin αVβ6 and / or antibodies immunoreactive with fragments or the entirety of integrin αVβ3 in a sample from the test animal, and

[0064] When the antibody is detected, it is determined that the test animal suffers from primary sclerosing cholangitis.

[0065] The subject animal herein is a human or a non-human animal, preferably a human.

[0066] (35) According to the method described in (34), the detection includes using a fragment or the whole of integrin αVβ6 as an antigen to detect antibodies that immunoreact therewith and / or using a fragment or the whole of integrin αVβ3 as an antigen to detect antibodies that immunoreact therewith.

[0067] (36) According to the method of (34) or (35), the sample is a blood sample, preferably a blood sample isolated from the subject animal.

[0068] (37) A biomarker for diagnosing primary sclerosing cholangitis, comprising an antibody that immunoreacts with a fragment or the entirety of integrin αVβ6 and / or an antibody that immunoreacts with a fragment or the entirety of integrin αVβ3.

[0069] (38) A method for screening candidate substances for a therapeutic drug for primary sclerosing cholangitis, comprising:

[0070] an antibody detection step of detecting an antibody immunoreactive with a fragment or the whole of integrin αVβ6 and / or an antibody immunoreactive with a fragment or the whole of integrin αVβ3 in a sample obtained from an animal to which a test substance has been administered, and

[0071] The selection step is to select the test substance as a candidate substance for the therapeutic drug for primary sclerosing cholangitis when the antibody in the sample is reduced due to the administration of the test substance.

[0072] (39) A method for producing a non-human model animal for primary sclerosing cholangitis, comprising at least one of an antibody administration step and an immunization step,

[0073] The antibody administration step comprises administering to the non-human animal an antibody that immunologically reacts with a fragment or the whole of integrin αVβ6 and / or an antibody that immunologically reacts with a fragment or the whole of integrin αVβ3,

[0074] In the immunization step, a fragment or the whole of integrin αVβ6 and / or a fragment or the whole of integrin αVβ3 are used as antigens to immunize non-human animals.

[0075] (40) A method for obtaining an index of primary sclerosing cholangitis, comprising:

[0076] A step of detecting antibodies immunoreactive with a fragment or the whole of integrin αVβ6 and / or antibodies immunoreactive with a fragment or the whole of integrin αVβ3 in a sample.

[0077] (41) According to the method described in (40), the detection step includes using a fragment or the whole of integrin αVβ6 as an antigen to detect antibodies that immunologically react therewith and / or using a fragment or the whole of integrin αVβ3 as an antigen to detect antibodies that immunologically react therewith.

[0078] (42) According to the method according to (40) or (41), the sample is a blood sample.

[0079] (43) Use of a fragment or the whole of integrin αVβ6 and / or a fragment or the whole of integrin αVβ3 in the manufacture of a test kit or test reagent for testing primary sclerosing cholangitis.

[0080] (44) The use according to (43), wherein the fragment or the whole of integrin αVβ6 and / or the fragment or the whole of integrin αVβ3 are in a form fixed to a solid phase.

[0081] (45) A fragment or whole of integrin αVβ6 and / or a fragment or whole of integrin αVβ3 for use in the examination of primary sclerosing cholangitis.

[0082] (46) The fragment or whole of integrin αVβ6 and / or the fragment or whole of integrin αVβ3 according to (45) are in a form fixed to a solid phase.

[0083] (47) An antibody that immunoreacts with a fragment or the whole of integrin αVβ6 and / or an antibody that immunoreacts with a fragment or the whole of integrin αVβ3, for use in a method for preparing a non-human animal model of primary sclerosing cholangitis.

[0084] (48) A fragment or whole of integrin αVβ6 and / or a fragment or whole of integrin αVβ3 for use in a method for preparing a non-human animal model of primary sclerosing cholangitis.

[0085] (49) Use of an antibody that immunoreacts with a fragment or the whole of integrin αVβ6 and / or an antibody that immunoreacts with a fragment or the whole of integrin αVβ3 in the manufacture of a reagent for preparing a non-human model animal for primary sclerosing cholangitis.

[0086] (50) Use of a fragment or the whole of integrin αVβ6 and / or a fragment or the whole of integrin αVβ3 in the manufacture of a reagent for preparing a non-human model animal for primary sclerosing cholangitis.

[0087] (51) A method for detecting antibodies that immunoreact with a fragment or the whole of integrin αVβ6 and / or antibodies that immunoreact with a fragment or the whole of integrin αVβ3 in a test animal,

[0088] This involves detecting the presence of the antibodies in a sample from the test animal.

[0089] The subject animal is a human or a non-human animal, preferably a human. The human may be a patient with ulcerative colitis or a person suspected of suffering from ulcerative colitis. The human may be a patient with primary sclerosing cholangitis or a person suspected of suffering from primary sclerosing cholangitis. The human may be a patient with primary sclerosing cholangitis complicated with ulcerative colitis or a person suspected of suffering from primary sclerosing cholangitis complicated with ulcerative colitis.

[0090] (52) According to the method described in (51), the detection brings the sample into contact with an antigen which is a fragment or a whole of integrin αVβ6 and / or a fragment or a whole of integrin αVβ3, and detects whether the antibody is present in the sample by detecting the binding of the antigen to the antibody.

[0091] (53) According to the method described in (51), the detection brings the sample into contact with an antigen which is a fragment or a whole of integrin αVβ6 and / or a fragment or a whole of integrin αVβ3, and then brings the sample into contact with anti-human IgG antibody, anti-human IgA antibody, anti-human IgM antibody and / or anti-human IgE antibody, and detects whether the antibody is present in the sample by detecting the binding of the antibody to the anti-human IgG antibody, anti-human IgA antibody, anti-human IgM antibody and / or anti-human IgE antibody.

[0092] Here, the antigen is preferably immobilized on a solid phase.

[0093] (54) The method according to any one of (51) to (53), wherein the sample is a blood sample separated from the test animal. Here, the blood sample is preferably a serum sample, a plasma sample, or whole blood.

[0094] (55) A method for diagnosing ulcerative colitis in a test animal, comprising:

[0095] contacting a sample from a test animal with a solid phase carrier to which an antigen that is a fragment or whole of integrin αVβ6 and / or a fragment or whole of integrin αVβ3 is immobilized, and detecting the presence or absence of the antibody in the sample by detecting the binding of the antigen to the antibody that immunoreacts with the antigen, and

[0096] When the presence of the antibody is detected in the sample, the test animal is diagnosed as suffering from ulcerative colitis.

[0097] The subject animal is a human or non-human animal, preferably a human. The human may be a patient with ulcerative colitis or a person suspected of suffering from ulcerative colitis.

[0098] (56) According to the method described in (55), the detection brings the sample into contact with an antigen which is a fragment or a whole of integrin αVβ6 and / or a fragment or a whole of integrin αVβ3, and then brings the sample into contact with anti-human IgG antibody, anti-human IgA antibody, anti-human IgM antibody and / or anti-human IgE antibody, and detects whether the antibody is present in the sample by detecting the binding of the antibody to the anti-human IgG antibody, anti-human IgA antibody, anti-human IgM antibody and / or anti-human IgE antibody.

[0099] (57) The method according to (55) or (56), wherein the sample is a blood sample separated from the test animal. Here, the blood sample is preferably a serum sample, a plasma sample, or whole blood.

[0100] (58) A method for diagnosing and treating ulcerative colitis in an animal subject, comprising:

[0101] Testing a sample from a test animal for the presence of antibodies that immunoreact with fragments or the entirety of integrin αVβ6 and / or antibodies that immunoreact with fragments or the entirety of integrin αVβ3,

[0102] If the antibody is detected in the sample, the animal is diagnosed as suffering from ulcerative colitis, and

[0103] The test animal diagnosed as suffering from ulcerative colitis is administered an effective amount of a therapeutic drug for ulcerative colitis and / or subjected to blood cell component removal therapy or surgery for treating ulcerative colitis.

[0104] The subject animal is a human or non-human animal, preferably a human. The human may be a patient with ulcerative colitis or a person suspected of suffering from ulcerative colitis.

[0105] (59) According to the method described in (58), the therapeutic drug is selected from one or more of steroid drugs, 5-aminosalicylic acid (5ASA) preparations, immunomodulatory drugs (e.g., azathioprine, mercaptopurine), biological agents (e.g., infliximab, adalimumab, golimumab, tofacitinib, vedolizumab), anti-TNFα drugs and immunosuppressive drugs (e.g., tacrolimus, cyclosporine).

[0106] (60) The method according to (58) or (59), wherein the sample is a blood sample separated from the test animal. Here, the blood sample is preferably a serum sample, a plasma sample, or whole blood.

[0107] (61) A method for diagnosing primary sclerosing cholangitis in a test animal, comprising:

[0108] contacting a sample from a test animal with a solid phase carrier to which an antigen that is a fragment or whole of integrin αVβ6 and / or a fragment or whole of integrin αVβ3 is immobilized, and detecting the presence or absence of the antibody in the sample by detecting the binding of the antigen to the antibody that immunoreacts with the antigen, and

[0109] In case the presence of the antibody is detected in the sample, the subject animal is diagnosed as suffering from primary sclerosing cholangitis.

[0110] The subject animal is a human or a non-human animal, preferably a human. The human may be a primary sclerosing cholangitis patient or a person suspected of suffering from primary sclerosing cholangitis.

[0111] (62) According to the method described in (61), the detection brings the sample into contact with an antigen which is a fragment or a whole of integrin αVβ6 and / or a fragment or a whole of integrin αVβ3, and then brings the sample into contact with anti-human IgG antibody, anti-human IgA antibody, anti-human IgM antibody and / or anti-human IgE antibody, and detects whether the antibody is present in the sample by detecting the binding of the antibody to the anti-human IgG antibody, anti-human IgA antibody, anti-human IgM antibody and / or anti-human IgE antibody.

[0112] (63) According to the method according to (61) or (62), the sample is a blood sample isolated from the subject animal.

[0113] (64) A method for diagnosing and treating primary sclerosing cholangitis in a subject animal, comprising:

[0114] Testing a sample from a test animal for the presence of antibodies that immunoreact with a fragment or the entirety of integrin αVβ6 and / or antibodies that immunoreact with a fragment or the entirety of integrin αVβ3,

[0115] If the presence of the antibody is detected in the sample, the animal is diagnosed as suffering from primary sclerosing cholangitis, and

[0116] An effective amount of a therapeutic agent for primary sclerosing cholangitis is administered to the test animal diagnosed as suffering from primary sclerosing cholangitis.

[0117] The subject animal is a human or a non-human animal, preferably a human. The human may be a primary sclerosing cholangitis patient or a person suspected of suffering from primary sclerosing cholangitis.

[0118] (65) The method according to (64), wherein the sample is a blood sample separated from the test animal. Here, the blood sample is preferably a serum sample, a plasma sample, or whole blood.

[0119] (66) The method according to (54), (57), (60) or (63), further includes separating the blood sample from the subject animal.

[0120] (67) A method for diagnosing and treating primary sclerosing cholangitis complicated by ulcerative colitis in a subject animal, comprising:

[0121] Testing a sample from a test animal for the presence of antibodies that immunoreact with a fragment or the entirety of integrin αVβ6 and / or antibodies that immunoreact with a fragment or the entirety of integrin αVβ3,

[0122] If the antibody is detected in the sample, the animal is diagnosed as suffering from primary sclerosing cholangitis complicated by ulcerative colitis, and

[0123] The test animal diagnosed as suffering from primary sclerosing cholangitis complicated with ulcerative colitis is administered an effective amount of a therapeutic drug for primary sclerosing cholangitis complicated with ulcerative colitis and / or subjected to blood cell component removal therapy or surgery for treating primary sclerosing cholangitis complicated with ulcerative colitis.

[0124] The subject animal is a human or non-human animal, preferably a human. The human may be a patient with primary sclerosing cholangitis complicated with ulcerative colitis or a person suspected of having primary sclerosing cholangitis complicated with ulcerative colitis.

[0125] (68) According to the method described in (67), the therapeutic drug is selected from one or more of steroid drugs, 5-aminosalicylic acid (5ASA) preparations, immunomodulatory drugs (e.g., azathioprine, mercaptopurine), biological agents (e.g., infliximab, adalimumab, golimumab, tofacitinib, vedolizumab), anti-TNFα drugs and immunosuppressive drugs (e.g., tacrolimus, cyclosporine).

[0126] This specification includes the disclosure of Japanese Patent Application No. 2019-000060 which is the basis of priority of this application.

[0127] Effects of the Invention

[0128] According to the inspection method for ulcerative colitis of the present invention and the inspection kit used therefor, ulcerative colitis can be detected with high sensitivity and high specificity, and can be specifically detected by distinguishing it from Crohn's disease and other inflammatory bowel diseases.

[0129] According to the primary sclerosing cholangitis testing method of the present invention and the testing kit used therefor, primary sclerosing cholangitis can be detected with high sensitivity and high specificity, and can be distinguished from IgG4-related sclerosing cholangitis and other hepatobiliary diseases and specifically detected. BRIEF DESCRIPTION OF THE DRAWINGS

[0130] Figure 1-1 The graph shows the concentrations of antibodies against a plurality of human integrins having different combinations of the α-chain and β-chain subunits in sera from patients with ulcerative colitis (UC) and control sera (control).

[0131] Figure 1-2 The graph shows the concentrations of antibodies against a plurality of human integrins having different combinations of the α-chain and β-chain subunits in sera from patients with ulcerative colitis (UC) and control sera (control).

[0132] Figure 1-3 The graph shows the concentrations of antibodies against a plurality of human integrins having different combinations of the α-chain and β-chain subunits in sera from patients with ulcerative colitis (UC) and control sera (control).

[0133] Figure 1-4 The graph shows the concentrations of antibodies against a plurality of human integrins having different combinations of the α-chain and β-chain subunits in sera from patients with ulcerative colitis (UC) and control sera (control).

[0134] Figure 2 The graph shows the concentrations of anti-human integrin αVβ6 antibodies in sera of patients with ulcerative colitis, sera of patients with Crohn's disease, and control sera.

[0135] Figure 3 The concentrations of anti-human integrin αVβ6 antibodies in the sera of patients with primary sclerosing cholangitis, the sera of patients with IgG4-related sclerosing cholangitis, and the control sera are shown.

[0136] Figure 4 The graph shows the concentrations of anti-human integrin αVβ3 antibodies in sera of patients with ulcerative colitis, sera of patients with Crohn's disease, and control sera.

[0137] Figure 5 The concentrations of anti-human integrin αVβ3 antibodies in the sera of patients with primary sclerosing cholangitis, the sera of patients with IgG4-related sclerosing cholangitis, and the control sera are shown.

[0138] Figure 6 The graph shows the concentration of anti-human integrin αVβ6 antibodies in the sera of patients with ulcerative colitis, patients with Crohn's disease, and patients with other intestinal diseases.

[0139] Figure 7 The graph shows the concentration of anti-human integrin αVβ6 antibodies in sera of patients with ulcerative colitis, patients with Crohn's disease, patients with other intestinal diseases, patients with collagen diseases, and normal human sera.

[0140] Fig. 8A The graph shows the concentration of anti-human integrin αVβ6 antibody IgG1 in the sera of patients with ulcerative colitis, patients with Crohn's disease, and patients with other intestinal diseases.

[0141] Figure 8B The graph shows the concentration of anti-human integrin αVβ6 antibody IgG2 in the sera of patients with ulcerative colitis, patients with Crohn's disease, and patients with other intestinal diseases.

[0142] Figure 8C The graph shows the concentration of anti-human integrin αVβ6 antibody IgG3 in the sera of patients with ulcerative colitis, patients with Crohn's disease, and patients with other intestinal diseases.

[0143] Fig.8D The graph shows the concentration of anti-human integrin αVβ6 antibody IgG4 in the sera of patients with ulcerative colitis, patients with Crohn's disease, and patients with other intestinal diseases.

[0144] Fig.9A The graph shows the concentration of anti-human integrin αVβ6 antibody IgA in the sera of patients with ulcerative colitis, patients with Crohn's disease, and patients with other intestinal diseases.

[0145] Fig. 9B The graph shows the concentration of anti-human integrin αVβ6 antibody IgM in the sera of patients with ulcerative colitis, patients with Crohn's disease, and patients with other intestinal diseases.

[0146] Fig. 9C The graph shows the concentration of anti-human integrin αVβ6 antibody IgE in the sera of patients with ulcerative colitis, patients with Crohn's disease, and patients with other intestinal diseases.

[0147] Fig. 10A The partial Mayo scores (partial Mayoscore) (left vertical axis) and the antibody titer of anti-human integrin αVβ6 autoantibodies (IgG) (right vertical axis) of patients with ulcerative colitis at various time points from November 2017 to November 2018 are shown.

[0148] Fig. 10B Partial Mayo scores (left vertical axis) and antibody titers of anti-human integrin αVβ6 autoantibodies (IgG) (right vertical axis) of other patients with ulcerative colitis at various time points from November 2017 to November 2018 are shown.

[0149] Fig.11 The graph shows the concentration of anti-human integrin αVβ3 antibodies in the sera of patients with ulcerative colitis, patients with Crohn's disease, and patients with other intestinal diseases.

[0150] Fig.12 The graph shows the concentration of anti-human integrin αVβ3 antibodies in sera of patients with ulcerative colitis, patients with Crohn's disease, patients with other intestinal diseases, patients with collagen diseases, and normal human sera.

[0151] Fig.13A The graph shows the concentration of anti-human integrin αVβ3 antibody IgG1 in the sera of patients with ulcerative colitis, patients with Crohn's disease, and patients with other intestinal diseases.

[0152] Fig. 13B The graph shows the concentration of anti-human integrin αVβ3 antibody IgG2 in the sera of patients with ulcerative colitis, patients with Crohn's disease, and patients with other intestinal diseases.

[0153] Fig. 13C The graph shows the concentration of anti-human integrin αVβ3 antibody IgG3 in the sera of patients with ulcerative colitis, patients with Crohn's disease, and patients with other intestinal diseases.

[0154] Fig.13D The graph shows the concentration of anti-human integrin αVβ3 antibody IgG4 in the sera of patients with ulcerative colitis, patients with Crohn's disease, and patients with other intestinal diseases.

[0155] Fig.14A The graph shows the concentration of anti-human integrin αVβ3 antibody IgA in the sera of patients with ulcerative colitis, patients with Crohn's disease, and patients with other intestinal diseases.

[0156] Fig. 14B The graph shows the concentrations of anti-human integrin αVβ3 antibody IgM in the sera of patients with ulcerative colitis, patients with Crohn's disease, and patients with other intestinal diseases.

[0157] Fig. 14C The graph shows the concentration of anti-human integrin αVβ3 antibody IgE in the sera of patients with ulcerative colitis, patients with Crohn's disease, and patients with other intestinal diseases. DETAILED DESCRIPTION

[0158] <1. Sample>

[0159] The test animal used in the test method of the present invention is not particularly limited, and may be a human or other non-human mammal, but is preferably a human.

[0160] As samples used in the inspection method of the present invention, body fluids collected from the inspected animals can be cited. Specifically, in addition to blood samples such as serum, plasma, and whole blood, body fluid samples other than blood such as saliva, cerebrospinal fluid, and urine can also be used. In addition, not limited to body fluids, tissues collected from the inspected animals, such as tissues collected from the lesions of ulcerative colitis (e.g., large intestine, rectum) and primary sclerosing cholangitis (e.g., liver) that need to be inspected can also be used as samples. The sample is used in the inspection method of the present invention in a manner separated from the inspected animal.

[0161] <2. Integrin αVβ6>

[0162] Integrins are proteins that are naturally formed of heterodimeric molecules, which are formed of two subunit chains, namely, α chain and β chain. α1 to α11, αV, αX, αM, αL, αD, αE, and αIIb are known as α chains, and β1 to β8 are known as β chains, and there are multiple isotypes with different combinations of the two.

[0163] Integrins are present on the surface of epithelial cells and bind to laminin, fibronectin and other extracellular matrix proteins on the surface of connective tissue.

[0164] Integrin αVβ6 is formed by a heterodimeric molecule containing αV as an α chain and β6 as a β chain. Integrin αVβ6 is hardly expressed in normal tissues, but is expressed on the surface of epithelial cells in response to inflammatory stimulation.

[0165] In the present invention, the origin of the fragment or whole of integrin αVβ6 that immunologically binds to the autoantibody to be detected is not particularly limited, but it is preferably of the same species as the test animal, and particularly preferably a human. The base sequence information of the genes encoding the αV chain and β6 chain of integrins of mammalian species such as humans and the amino acid sequence information of each chain can be obtained from known databases (GenBank, etc.). In particular, the amino acid sequence of the preprotein of human integrin αV chain isoform 1 is registered as GenBank Accession Number NP_002201.2, shown in sequence number 1. The amino acid sequence of the precursor of the human integrin β6 chain is registered as GenBank Accession Number NP_000879.2, shown in sequence number 2. The amino acid sequence information of the integrin αV chain and β6 chain of various mammals other than humans can also be obtained from known databases (GenBank, etc.). It may also be that the αV chain and the β6 chain, which are amino acid sequences formed by post-translational modification and contain one or both of the amino acid sequences of the αV chain and the β6 chain registered in the database, constitute the integrin αVβ6. For example, the partial sequence of positions 1 to 30 of the amino acid sequence of sequence number 1 is a signal peptide sequence, and the amino acid sequence of the mature polypeptide of the human integrin αV chain contains the sequence of positions 31 to 1048 of the amino acid sequence of sequence number 1. Similarly, the partial sequence of positions 1 to 21 of the amino acid sequence of sequence number 2 is a signal peptide sequence, and the amino acid sequence of the mature polypeptide of the human integrin β6 chain contains the sequence of positions 22 to 788 of the amino acid sequence of sequence number 2.

[0166] Furthermore, unless otherwise specified, integrin αVβ6 in the present invention is not limited to the natural type containing a mature or immature amino acid sequence, but may also be a variant having an equivalent form to the natural type integrin αVβ6.

[0167] Integrin αVβ6 is not limited to a form in which both the α chain and the β chain of a natural or variant form containing a mature or immature amino acid sequence include their full length (i.e., the entire integrin αVβ6), but may also be a fragment of integrin αVβ6.

[0168] In addition, the autoantibodies to be detected are not limited to antibodies that immunologically bind to substances formed only by the amino acid sequence of a fragment or the whole of integrin αVβ6, but may also be antibodies that immunologically bind to substances in which other peptides are added to each chain of a fragment or the whole of integrin αVβ6 (especially substances added to the C-terminal side).

[0169] As a fragment of integrin αVβ6, at least one of the αV chain and β6 chain constituting the dimer of integrin can be listed as a substance shorter than the mature or immature natural type or its variant. Specifically, a dimer containing the following αV chain and / or β6 chain can be exemplified as a fragment of integrin αVβ6: as an αV chain, an αV chain containing a partial sequence of Phe from the 31st to Val from the 992nd in the amino acid sequence of the αV chain shown in sequence number 1; as a β6 chain, a β6 chain containing a partial sequence of Gly from the 22nd to Asn from the 707th in the amino acid sequence of the β6 chain shown in sequence number 2. The fragment of integrin αVβ6 preferably forms a dimer, and preferably has binding activity with extracellular matrix proteins such as laminin and fibronectin. The binding activity of the fragment of integrin αVβ6 with extracellular matrix proteins can be confirmed by ELISA (enzyme-linked immunosorbent assay) method and the like.

[0170] Whether the whole integrin αVβ6 or its fragments form dimers can be confirmed in the following manner: when the whole integrin αVβ6 or its fragments are subjected to SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) in the absence of 2-mercaptoethanol, a band corresponding to the molecular weight of the dimer can be detected, and when it is subjected to SDS-PAGE in the presence of 2-mercaptoethanol, the band corresponding to the molecular weight of the dimer disappears.

[0171] As a commercially available integrin αVβ6, recombinant human integrin αVβ6 (R&D Systems, Minnesota, USA, product number 3817-AV) can be exemplified. The recombinant human integrin αVβ6 is a dimer of the following αV chain and β6 chain: the αV chain is formed by a partial sequence of Phe at position 31 to Val at position 992 in the amino acid sequence of the αV chain shown in sequence number 1, a linker sequence added to its C-terminus, and an acidic tail sequence, and the β6 chain is formed by a partial sequence of Gly at position 22 to Asn at position 707 in the amino acid sequence of the β6 chain shown in sequence number 2, a linker sequence added to its C-terminus, and a basic tail sequence.

[0172] More specific embodiments of the αV chain constituting the fragment or the whole of integrin αVβ6 include:

[0173] (I) a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a partial sequence of the amino acid sequence of SEQ ID NO: 1 from Phe at position 31 to Thr at position 1048,

[0174] (II) a polypeptide comprising a partial sequence of the amino acid sequence shown in SEQ ID NO: 1 and being functionally equivalent to the polypeptide of (I),

[0175] (III) a polypeptide comprising an amino acid sequence having 85% or more sequence homology to the amino acid sequence of SEQ ID NO: 1 or a partial sequence thereof and being functionally equivalent to the polypeptide of (I), and

[0176] (IV) A polypeptide comprising an amino acid sequence represented by SEQ ID NO: 1 or a partial sequence thereof in which one or more amino acids are replaced, deleted and / or added and which is functionally equivalent to the polypeptide of (I)

[0177] The peptides are selected from the group consisting of

[0178] The polypeptides of (I) to (IV) may also be polypeptides comprising another amino acid sequence added to at least one of the N-terminal and C-terminal sides of the amino acid sequence or partial sequence defined by (I) to (IV), preferably the amino acid sequence on the C-terminal side.

[0179] Among the above (II), (III) and (IV), the polypeptides that are functionally equivalent to the polypeptide of (I) can be exemplified by the following polypeptides: polypeptides that can form a dimer with the integrin β6 chain (particularly preferably a polypeptide chain formed by the amino acid sequence shown in sequence number 2, a polypeptide chain formed by a partial sequence from Gly at position 22 to Cys at position 788 in the amino acid sequence shown in sequence number 2, or a polypeptide chain formed by a partial sequence from Gly at position 22 to Asn at position 707 in the amino acid sequence shown in sequence number 2), and the dimer formed has the ability to bind to extracellular matrix proteins such as laminin and fibronectin, and the extracellular matrix proteins can bind to natural integrin αVβ6 or commercially available integrin αVβ6.

[0180] As the partial sequence of (II), the partial sequence from Phe at position 31 to Val at position 992 in the amino acid sequence shown in SEQ ID NO: 1 can be cited. As the partial sequence of (III) and (IV), the partial sequence from Phe at position 31 to Thr at position 1048 in the amino acid sequence shown in SEQ ID NO: 1 or the partial sequence from Phe at position 31 to Val at position 992 in the amino acid sequence shown in SEQ ID NO: 1 can be cited.

[0181] The sequence homology of (III) is preferably 90% or more, more preferably 95% or more, even more preferably 96% or more, particularly preferably 97% or more, and most preferably 98% or more or 99% or more.

[0182] The "one or more" in (IV) is, for example, 1 to 100, preferably 1 to 50, preferably 1 to 30, preferably 1 to 20, preferably 1 to 15, preferably 1 to 10, preferably 1 to 5, preferably 1 to 4, preferably 1 to 3, preferably 1 to 2, and preferably 1.

[0183] As a more specific embodiment of the β6 chain constituting the fragment or the whole of integrin αVβ6, there can be mentioned

[0184] (V) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a partial sequence of the amino acid sequence of SEQ ID NO: 2 from Gly at position 22 to Cys at position 788,

[0185] (VI) a polypeptide comprising a partial sequence of the amino acid sequence shown in SEQ ID NO: 2 and being functionally equivalent to the polypeptide of (V),

[0186] (VII) a polypeptide comprising an amino acid sequence having 85% or more sequence homology to the amino acid sequence of SEQ ID NO: 2 or a partial sequence thereof and being functionally equivalent to the polypeptide of (V), and

[0187] (VIII) A polypeptide having one or more amino acids substituted, deleted and / or added in the amino acid sequence of SEQ ID NO: 2 or a partial sequence thereof and being functionally equivalent to the polypeptide of (V)

[0188] The peptides are selected from the group consisting of

[0189] The polypeptides of (V) to (VIII) may also be polypeptides comprising another amino acid sequence added to at least one of the N-terminal and C-terminal sides, preferably the amino acid sequence on the C-terminal side, of the amino acid sequence or partial sequence defined by (V) to (VIII).

[0190] The polypeptides functionally equivalent to the polypeptide of (V) in (VI), (VII) and (VIII) can be exemplified by the following polypeptides: polypeptides capable of forming a dimer with the integrin αV chain (particularly preferably a polypeptide chain formed by the amino acid sequence shown in SEQ ID NO: 1, a polypeptide chain formed by a partial sequence from Phe at position 31 to Thr at position 1048 in the amino acid sequence shown in SEQ ID NO: 1, or a polypeptide chain formed by a partial sequence from Phe at position 31 to Val at position 992 in the amino acid sequence shown in SEQ ID NO: 1), and the dimer formed has the ability to bind to extracellular matrix proteins such as laminin and fibronectin, and the extracellular matrix proteins can bind to natural integrin αVβ6 or commercially available integrin αVβ6.

[0191] As the partial sequence of (VI), there can be cited a partial sequence from Gly at position 22 to Asn at position 707 in the amino acid sequence shown in SEQ ID NO: 2. As the partial sequence of (VII) and (VIII), there can be cited a partial sequence from Gly at position 22 to Cys at position 788 in the amino acid sequence shown in SEQ ID NO: 2 or a partial sequence from Gly at position 22 to Asn at position 707 in the amino acid sequence shown in SEQ ID NO: 2.

[0192] The sequence homology of (VII) is preferably 90% or more, more preferably 95% or more, further preferably 96% or more, particularly preferably 97% or more, and most preferably 98% or more or 99% or more.

[0193] The "one or more" in (VIII) is, for example, 1 to 100, preferably 1 to 50, preferably 1 to 30, preferably 1 to 20, preferably 1 to 15, preferably 1 to 10, preferably 1 to 5, preferably 1 to 4, preferably 1 to 3, preferably 1 to 2, and preferably 1.

[0194] In (III), (VII) and (XI) described later, the sequence homology of the amino acid sequence can be obtained using methods well known to those skilled in the art, sequence analysis software, etc. As sequence analysis software, for example, the blastp program of the BLAST algorithm and the fasta program of the FASTA algorithm can be cited.

[0195] <3. Integrin αVβ3>

[0196] Integrin αVβ3 is formed by a heterodimeric molecule containing αV as an α chain and β3 as a β chain.

[0197] In the present invention, the origin of the fragment or whole of integrin αVβ3 that immunologically binds to the autoantibody to be detected is not particularly limited, but is preferably of the same species as the test animal, and is particularly preferably human. The base sequence information of the gene encoding the αV chain and β3 chain of integrin of mammalian species such as humans and the amino acid sequence information of each chain can be obtained from known databases (GenBank, etc.).

[0198] The αV chain is as described above, and therefore the description thereof is omitted.

[0199] The amino acid sequence of the precursor of human integrin β3 chain is registered as GenBank Accession Number AAA52589.1 and is shown in SEQ ID NO: 3. Amino acid sequence information of integrin β3 chain of various mammals other than humans can also be obtained from known databases (GenBank, etc.).

[0200] It may also be that the αV chain and the β3 chain, which are amino acid sequences formed by post-translational modification and contain one or both of the amino acid sequences of the αV chain and the β3 chain registered in the database, constitute the integrin αVβ3. For example, the amino acid sequence of the mature polypeptide of the human integrin αV chain contains the sequence of positions 31 to 1048 of the amino acid sequence of sequence number 1. Similarly, the partial sequence of positions 1 to 26 of the amino acid sequence of sequence number 3 is a signal peptide sequence, and the amino acid sequence of the mature polypeptide of the human integrin β3 chain contains the sequence of positions 27 to 788 of the amino acid sequence of sequence number 3.

[0201] Furthermore, unless otherwise specified, integrin αVβ3 in the present invention is not limited to the natural type containing a mature or immature amino acid sequence, but may also be a variant having an equivalent form to the natural type integrin αVβ3.

[0202] Integrin αVβ3 is not limited to a form in which both the α chain and the β chain of a natural or variant form containing a mature or immature amino acid sequence include their full length (i.e., the entire integrin αVβ3), but may also be a fragment of integrin αVβ3.

[0203] In addition, the autoantibodies to be detected are not limited to antibodies that immunologically bind to substances formed only by the amino acid sequence of a fragment or the whole of integrin αVβ3, but may also be antibodies that immunologically bind to substances in which other peptides are added to each chain of a fragment or the whole of integrin αVβ3 (especially substances added to the C-terminal side).

[0204] As fragments of integrin αVβ3, at least one of the αV chain and β3 chain constituting the dimer of integrin can be listed as a substance shorter than the mature or immature natural type or its variant. Specifically, a dimer containing the following αV chain and / or β3 chain can be exemplified as a fragment of integrin αVβ3: as an αV chain, an αV chain containing a partial sequence of Phe from the 31st position to Val from the 992nd position in the amino acid sequence of the αV chain shown in sequence number 1; as a β3 chain, a β3 chain containing a partial sequence of Gly from the 27th position to Asp from the 718th position in the amino acid sequence of the β3 chain shown in sequence number 3. The fragment of integrin αVβ3 preferably forms a dimer, and more preferably has binding activity with extracellular matrix proteins such as laminin and fibronectin. The binding activity of the fragment of integrin αVβ3 with extracellular matrix proteins can be confirmed by ELISA method, etc.

[0205] Whether the whole integrin αVβ3 or its fragments form dimers can be confirmed in the following manner: when the whole integrin αVβ3 or its fragments are subjected to SDS-PAGE in the absence of 2-mercaptoethanol, a band corresponding to the molecular weight of the dimer can be detected, and when it is subjected to SDS-PAGE in the presence of 2-mercaptoethanol, the band corresponding to the molecular weight of the dimer disappears.

[0206] As a commercially available integrin αVβ3, recombinant human integrin αVβ3 (R&D Systems, Minnesota, USA, product number 3050-AV) can be exemplified. The recombinant human integrin αVβ3 is a dimer of the following αV chain and β3 chain: the αV chain is formed by the partial sequence of Phe from the 31st position to Val from the 992nd position in the amino acid sequence of the αV chain shown in sequence number 1, and a linker sequence and an acidic tail sequence added to its C-terminus, and the β3 chain is formed by the partial sequence of Gly from the 27th position to Asp from the 718th position in the amino acid sequence of the β3 chain shown in sequence number 3, and a linker sequence and a basic tail sequence added to its C-terminus.

[0207] More specific embodiments of the αV chain constituting the entire or fragment of integrin αVβ3 include polypeptides selected from (I) to (IV) above.

[0208] As a more specific embodiment of the β3 chain constituting the fragment or the whole of integrin αVβ3, there can be mentioned:

[0209] (IX) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3, or a partial sequence of the amino acid sequence set forth in SEQ ID NO: 3 from Gly at position 27 to Asp at position 718, or a partial sequence of the amino acid sequence set forth in SEQ ID NO: 3 from Gly at position 27 to Thr at position 788,

[0210] (X) a polypeptide comprising a partial sequence of the amino acid sequence shown in SEQ ID NO: 3 and being functionally equivalent to the polypeptide of (IX),

[0211] (XI) a polypeptide comprising an amino acid sequence having 85% or more sequence homology to the amino acid sequence of SEQ ID NO: 3 or a partial sequence thereof and being functionally equivalent to the polypeptide of (IX), and

[0212] (XII) A polypeptide comprising an amino acid sequence represented by SEQ ID NO: 3 or a partial sequence thereof in which one or more amino acids are replaced, deleted and / or added and which is functionally equivalent to the polypeptide of (IX)

[0213] The peptides are selected from the group consisting of

[0214] The polypeptides of (IX) to (XII) may also be polypeptides comprising another amino acid sequence added to at least one of the N-terminal and C-terminal sides, preferably the amino acid sequence on the C-terminal side, of the amino acid sequence or partial sequence defined by (IX) to (XII).

[0215] Among the above (X) to (XII), the polypeptide that is functionally equivalent to the polypeptide of (IX) can be exemplified by the following polypeptides: a polypeptide that can form a dimer with the integrin αV chain (particularly preferably a polypeptide chain formed by the amino acid sequence shown in SEQ ID NO: 1, a polypeptide chain formed by a partial sequence from Phe at position 31 to Thr at position 1048 in the amino acid sequence shown in SEQ ID NO: 1, or a polypeptide chain formed by a partial sequence from Phe at position 31 to Val at position 992 in the amino acid sequence shown in SEQ ID NO: 1), and the dimer formed has the ability to bind to extracellular matrix proteins such as laminin and fibronectin, and the extracellular matrix proteins can bind to natural integrin αVβ3 or commercially available integrin αVβ3.

[0216] As the partial sequence of (X), there can be cited a partial sequence from Gly at position 27 to Asp at position 718 in the amino acid sequence shown in SEQ ID NO: 3. As the partial sequence of (XI) and (XII), there can be cited a partial sequence from Gly at position 27 to Asp at position 718 in the amino acid sequence shown in SEQ ID NO: 3 or a partial sequence from Gly at position 27 to Thr at position 788 in the amino acid sequence shown in SEQ ID NO: 3.

[0217] The sequence homology of (XI) is preferably 90% or more, more preferably 95% or more, further preferably 96% or more, particularly preferably 97% or more, and most preferably 98% or more or 99% or more.

[0218] The "one or more" in (XII) is, for example, 1 to 100, preferably 1 to 50, preferably 1 to 30, preferably 1 to 20, preferably 1 to 15, preferably 1 to 10, preferably 1 to 5, preferably 1 to 4, preferably 1 to 3, preferably 1 to 2, and preferably 1.

[0219] <4. Examination methods for ulcerative colitis or primary sclerosing cholangitis>

[0220] The inspection method of the present invention first relates to

[0221] A method for detecting ulcerative colitis.

[0222] The method comprises a detection step of detecting antibodies immunoreactive with a fragment or the whole of integrin αVβ6 and / or antibodies immunoreactive with a fragment or the whole of integrin αVβ3 in a sample as indicators of ulcerative colitis.

[0223] The inspection method of the present invention secondly relates to

[0224] A method for detecting primary sclerosing cholangitis.

[0225] The method comprises a detection step of detecting antibodies immunoreactive with a fragment or the whole of integrin αVβ6 and / or antibodies immunoreactive with a fragment or the whole of integrin αVβ3 in a sample as indicators of primary sclerosing cholangitis.

[0226] In the following description, “a fragment or the entirety of integrin αVβ6” is collectively referred to as “integrin αVβ6”, and “a fragment or the entirety of integrin αVβ3” is collectively referred to as “integrin αVβ3”.

[0227] The first testing method of the present invention is accomplished based on the surprising discovery that antibodies (autoantibodies) to integrin αVβ6 and antibodies (autoantibodies) to integrin αVβ3 in samples obtained from test animals serve as indicators of ulcerative colitis.

[0228] The second testing method of the present invention is accomplished based on the surprising discovery that antibodies (autoantibodies) to integrin αVβ6 and antibodies (autoantibodies) to integrin αVβ3 in samples obtained from test animals serve as indicators of primary sclerosing cholangitis.

[0229] The detection step in the inspection method of the present invention is a step of detecting antibodies that immunoreact with integrin αVβ6 and / or antibodies that immunoreact with integrin αVβ3 in a sample. The "detection" in the present invention includes not only confirming whether antibodies that immunoreact with integrin αVβ6 and / or antibodies that immunoreact with integrin αVβ3 are present in a sample, but also includes measuring the content of the antibodies in the sample, i.e., the concept of "quantification".

[0230] The detection process in the inspection method of the present invention can be a method that can quantitatively or qualitatively detect the antibody in the sample, and the specific method is not particularly limited. For example, immunological techniques can be used, such as antibody enzyme method (ELISA method), immunoprecipitation method (IPP method), immunoblot method (IB method), latex agglutination method, immunochromatography, indirect fluorescent antibody method (IF method), radioimmunoassay (RIA method) and other techniques. The ELISA method that can handle many samples is particularly preferred. In the ELISA method, specifically, the integrin αVβ6 and / or integrin αVβ3 described in detail above as the antigen of the antibody is fixed to a solid phase, and the sample is contacted with the antigen fixed to the solid phase. By using enzyme-labeled detection antibodies and the like to detect the immune complex of the antigen and the antibody that may be contained in the sample, the antibody can be detected. The method for suppressing nonspecific reactions accompanying the implementation of the ELISA method, the labeling substance that can be used during detection, the measuring instrument, etc. are not particularly limited. As a solid phase for fixing the antigen, a solid phase of any shape such as a plate, a bead, and a tube can be used.

[0231] In immunological methods such as ELISA, the process of contacting the sample that may contain the antibody with the antigen, the necessary washing process, the necessary labeling process, and the necessary detection process can be carried out in an appropriate buffer, but it is preferably carried out in a buffer containing one or more metal ions selected from calcium ions, magnesium ions, manganese ions, sodium, lithium, etc. The metal ions are preferably divalent metal ions such as calcium ions, magnesium ions, and manganese ions, and one or two of calcium ions, magnesium ions, and manganese ions are particularly preferred. The concentration of the metal ions in the buffer is not particularly limited, and can be exemplified as a total of 0.02mM to 200mM, preferably 0.2mM to 20mM, and particularly preferably 0.4mM to 10mM. The detection sensitivity is increased in a buffer containing these metal ions, so it is preferred.

[0232] In the detection step, the amount of the antibody immunoreactive with integrin αVβ6 and / or the amount of the antibody immunoreactive with integrin αVβ3 can be obtained as a measured value of the labeled amount of the detection antibody bound to the immune complex of the antibody and the antigen. It is also possible to establish a calibration curve using a positive standard solution containing the antibody of known concentration, and calculate the amount of the antibody in the test sample from the measured value of the labeled amount using the calibration curve.

[0233] In the detection process, the determination of the presence or absence of antibodies that immunoreact with integrin αVβ6 and / or antibodies that immunoreact with integrin αVβ3 (positive / negative determination) can be performed by comparing the measured value of the antibodies in a sample obtained from a test animal individual (e.g., a person suspected of suffering from ulcerative colitis or primary sclerosing cholangitis) with the measured value of the antibodies in a sample obtained from an animal individual that has never suffered from ulcerative colitis or primary sclerosing cholangitis (e.g., a normal person). Here, the measured value of the antibodies in a sample obtained from an individual that has never suffered from ulcerative colitis or primary sclerosing cholangitis may be a value measured simultaneously or a value measured in advance. When the measured value of the antibodies in the sample obtained from the test animal individual is significantly greater than the measured value of the antibodies in the sample obtained from an animal individual that has never suffered from ulcerative colitis or primary sclerosing cholangitis, it can be determined to be positive.

[0234] The isotype of the antibody immunoreacting with integrin αVβ6 and / or the antibody immunoreacting with integrin αVβ3 detected in the detection step is not particularly limited, and may be an isotype of IgG antibody, IgA antibody, IgM antibody, IgE antibody, etc., and IgG antibody or IgA antibody is particularly preferred. As an antibody immunoreacting with integrin αVβ6, it is particularly preferred to detect IgG or IgA. As an antibody immunoreacting with integrin αVβ3, it is particularly preferred to detect IgG antibody. The subtype of IgG antibody is not particularly limited, for example, IgG1 antibody, IgG2 antibody, IgG3 antibody or IgG4 antibody can be detected. As an IgG antibody immunoreacting with integrin αVβ6, it is more preferred to detect IgG1 antibody, IgG2 antibody or IgG4 antibody, and it is most preferred to detect IgG1 antibody. As an IgG antibody immunoreacting with integrin αVβ3, it is more preferred to detect IgG1 antibody, IgG2 antibody or IgG3 antibody, and it is most preferred to detect IgG1 antibody.

[0235] <5. Test kits or test reagents for ulcerative colitis or primary sclerosing cholangitis>

[0236] The test kit or test reagent of the present invention firstly relates to a test kit or test reagent for testing ulcerative colitis, comprising a fragment or the whole of integrin αVβ6 and / or a fragment or the whole of integrin αVβ3.

[0237] The test kit or test reagent of the present invention secondly relates to a test kit or test reagent for testing primary sclerosing cholangitis, comprising a fragment or the whole of integrin αVβ6 and / or a fragment or the whole of integrin αVβ3.

[0238] In the following description, “a fragment or the entirety of integrin αVβ6” is collectively referred to as “integrin αVβ6”, and “a fragment or the entirety of integrin αVβ3” is collectively referred to as “integrin αVβ3”.

[0239] The test kit or test reagent of the present invention can be used in the above-mentioned test method of the present invention.

[0240] The test kit of the present invention may contain reagents necessary for immunoassay as necessary, for example, one or more metal salts selected from buffer, calcium salt, magnesium salt, manganese salt, sodium salt, lithium salt, etc.

[0241] The test reagent of the present invention may be a liquid or solid composition containing integrin αVβ6 and / or integrin αVβ3 and a carrier such as a solvent or excipient accepted for the purpose of formulation.

[0242] In the test kit or test reagent of the present invention, the form of integrin αVβ6 and / or integrin αVβ3 is not particularly limited, and may be in a solution state, a dry state, or a form fixed to a solid phase. When integrin αVβ6 and / or integrin αVβ3 is in a dry state, the test kit or test reagent of the present invention may contain a buffer or a solvent for converting it into a solution state before use.

[0243] The test kit or test reagent containing integrin αVβ6 and / or integrin αVβ3 in a form fixed to a solid phase can be used to test ulcerative colitis or primary sclerosing cholangitis by ELISA. The form of the solid phase can be any form such as a plate, a bead, a tube, etc. The kit used to test ulcerative colitis or primary sclerosing cholangitis by ELISA can contain a positive standard solution, a negative standard solution, a blocking solution, a cleaning solution, a sample diluent, a detection antibody, a matrix solution, etc. in addition to the solid phase. The detection antibody can be labeled with a marker that can be detected by an enzyme, etc. In addition to the serum dilution of an ulcerative colitis or primary sclerosing cholangitis patient containing autoantibodies to integrin αVβ6 and / or integrin αVβ3, the positive standard solution also uses a solution to which antibodies to integrin αVβ6 and / or integrin αVβ3 are added. As the antibody added to the solution, IgG antibody, IgA antibody, IgM antibody, IgE antibody, etc. are preferably the same isotype as the autoantibody to be measured. The negative standard solution uses a serum dilution of a person who does not suffer from ulcerative colitis or primary sclerosing cholangitis, a serum of a patient with other intestinal diseases medically distinguished from ulcerative colitis, a serum of a patient with other hepatobiliary diseases medically distinguished from primary sclerosing cholangitis, a normal person's serum (control serum), and a dilution thereof.

[0244] <6. Method for evaluating the therapeutic effect of ulcerative colitis or primary sclerosing cholangitis>

[0245] The method for evaluating the therapeutic effect of the present invention first relates to

[0246] A method for evaluating the effect of treatment of ulcerative colitis,

[0247] The method comprises the step of detecting antibodies immunoreactive with a fragment or the whole of integrin αVβ6 and / or antibodies immunoreactive with a fragment or the whole of integrin αVβ3 in a sample obtained from a test animal treated for ulcerative colitis.

[0248] The second method for evaluating the therapeutic effect of the present invention relates to

[0249] Methods for evaluating the effectiveness of treatment for primary sclerosing cholangitis,

[0250] The method comprises the step of detecting antibodies immunoreactive with a fragment or the whole of integrin αVβ6 and / or antibodies immunoreactive with a fragment or the whole of integrin αVβ3 in a sample obtained from a test animal treated for primary sclerosing cholangitis.

[0251] In the following description, “a fragment or the entirety of integrin αVβ6” is collectively referred to as “integrin αVβ6”, and “a fragment or the entirety of integrin αVβ3” is collectively referred to as “integrin αVβ3”.

[0252] As described above, regarding the inspection method of the present invention, antibodies that immunoreact with integrin αVβ6 and antibodies that immunoreact with integrin αVβ3 in a sample are useful as indicators of ulcerative colitis or primary sclerosing cholangitis, respectively. Therefore, the amount of the antibodies in a sample obtained from a test animal that has been treated for ulcerative colitis or primary sclerosing cholangitis decreases when the treatment is effective and the disease is cured, and remains unchanged or increases compared to before the start of treatment when the treatment is not fully effective. Therefore, by detecting the antibodies in a sample obtained from a test animal that has been treated for ulcerative colitis or primary sclerosing cholangitis, the effect of the treatment can be evaluated.

[0253] Examples of the test animals include humans and non-human animals that have been treated for ulcerative colitis or primary sclerosing cholangitis.

[0254] Specific aspects of the sample, integrin αVβ6, integrin αVβ3, antibody, detection step, etc. in the method for evaluating the therapeutic effect of the present invention are the same as those described in relation to the testing method of the present invention.

[0255] The quantitative result of the antibody in the sample obtained from the test animal treated with ulcerative colitis or primary sclerosing cholangitis obtained by the detection step in the method for evaluating the therapeutic effect of the present invention is compared with the quantitative result of the antibody in the sample obtained from the same animal individual before the start of the treatment. If the former is smaller than the latter, it can be evaluated as effective treatment. On the other hand, if the former is the same as the latter or larger than the latter, it can be evaluated as ineffective or insufficient treatment. Based on the evaluation result, it can be judged to continue treatment, change treatment policy, stop treatment, etc.

[0256] In addition, the quantitative results of the antibodies in the sample obtained from the test animal treated for ulcerative colitis or primary sclerosing cholangitis obtained by the detection step in the method for evaluating the therapeutic effect of the present invention are compared with the quantitative results of the antibodies in the sample obtained from a normal individual of the same animal species, and when the former is the same as the latter or smaller than the latter, it can be evaluated that the treatment is effective. On the other hand, when the former is larger than the latter, it can be evaluated that the treatment is ineffective or insufficient. Based on the evaluation results, it can be judged whether to continue the treatment, change the treatment policy, stop the treatment, etc.

[0257] <Method for screening candidate substances for therapeutic drugs for ulcerative colitis or primary sclerosing cholangitis>

[0258] The screening method of the present invention first relates to

[0259] A method for screening candidate substances for treating ulcerative colitis, comprising:

[0260] an antibody detection step of detecting antibodies immunoreactive with a fragment or the whole of integrin αVβ6 and / or antibodies immunoreactive with a fragment or the whole of integrin αVβ3 in a sample obtained from an animal to which a test substance has been administered, and

[0261] A step of selecting the test substance as a candidate substance for a therapeutic drug for ulcerative colitis when the antibody in the sample decreases due to administration of the test substance.

[0262] The screening method of the present invention secondly relates to

[0263] A method for screening candidate substances for therapeutic drugs for primary sclerosing cholangitis, comprising:

[0264] an antibody detection step of detecting antibodies immunoreactive with a fragment or the whole of integrin αVβ6 and / or antibodies immunoreactive with a fragment or the whole of integrin αVβ3 in a sample obtained from an animal to which a test substance has been administered, and

[0265] A step of selecting the test substance as a candidate substance for a therapeutic drug for primary sclerosing cholangitis when the antibody in the sample decreases due to administration of the test substance.

[0266] As described above, antibodies immunoreactive with a fragment or the whole of integrin αVβ6 and / or antibodies immunoreactive with a fragment or the whole of integrin αVβ3 in a sample are useful as indicators of ulcerative colitis or primary sclerosing cholangitis. Therefore, when the antibodies in the sample are reduced by administration of the test substance, the test substance can be selected as a candidate substance for a therapeutic drug for ulcerative colitis or primary sclerosing cholangitis.

[0267] The specific embodiments of the sample, fragment or whole of integrin αVβ6, fragment or whole of integrin αVβ3, antibody, antibody detection step, etc. in the embodiment of the antibody detection step of the screening method of the present invention are the same as those described in relation to the inspection method of the present invention.

[0268] In the screening method of the present invention, the test substance is not particularly limited as long as it is a test substance that may become a candidate for a new drug. The animal to which the test substance is administered is typically an animal such as a non-human model animal of ulcerative colitis or primary sclerosing cholangitis in which the amount of antibodies immunoreactive with a fragment or the whole of integrin αVβ6 and / or a fragment or the whole of integrin αVβ3 in the sample is larger than that of a normal individual.

[0269] In the selection step of the screening method of the present invention, the quantitative result of the antibody in the sample obtained from the animal administered with the test substance obtained by the antibody detection step is compared with the quantitative result of the antibody in the sample obtained from the same animal individual before the administration of the test substance. When the former is smaller than the latter, it is judged that the antibody in the sample has decreased due to the administration of the test substance, and the test substance can be selected as a candidate substance for the treatment of ulcerative colitis or primary sclerosing cholangitis.

[0270] In the selection step of the screening method of the present invention, the quantitative result of the antibody in the sample obtained from the animal to which the test substance is administered, obtained by the antibody detection step, is compared with the quantitative result of the antibody in the sample obtained from a normal individual of the same animal species, for example. When the former is the same as the latter or smaller than the latter, it is judged that the antibody in the sample has decreased due to the administration of the test substance, and the test substance can be selected as a candidate substance for the treatment of ulcerative colitis or primary sclerosing cholangitis.

[0271] <Method for producing non-human animal model of ulcerative colitis or primary sclerosing cholangitis>

[0272] The method for producing the non-human model animal of the present invention first relates to

[0273] A method for producing a non-human model animal of ulcerative colitis, comprising at least

[0274] an antibody administration step of administering an antibody immunoreactive with a fragment or the whole of integrin αVβ6 and / or an antibody immunoreactive with a fragment or the whole of integrin αVβ3 to a non-human animal, and

[0275] Immunization process of immunizing non-human animals with a fragment or the whole of integrin αVβ6 and / or a fragment or the whole of integrin αVβ3 as an antigen

[0276] at least one of the parties.

[0277] The method for making the non-human model animal of the present invention secondly relates to

[0278] A method for preparing a non-human model animal of primary sclerosing cholangitis, comprising at least

[0279] an antibody administration step of administering an antibody immunoreactive with a fragment or the whole of integrin αVβ6 and / or an antibody immunoreactive with a fragment or the whole of integrin αVβ3 to a non-human animal, and

[0280] Immunization process of immunizing non-human animals with a fragment or the whole of integrin αVβ6 and / or a fragment or the whole of integrin αVβ3 as an antigen

[0281] at least one of the parties.

[0282] Specific aspects of the fragment or whole of integrin αVβ6, the fragment or whole of integrin αVβ3, antibodies, etc. in the method for producing a non-human model animal of the present invention are the same as those in the testing method of the present invention.

[0283] A non-human model animal for ulcerative colitis or primary sclerosing cholangitis can be prepared by an antibody administration step of administering an antibody that immunoreacts with a fragment or whole of integrin αVβ6 and / or a fragment or whole of integrin αVβ3 to a non-human animal.

[0284] Examples of non-human animals to which antibodies are administered include mice such as BALB / c mice and B6 mice, and other non-human animals.

[0285] The administration route of the antibody immunoreactive with a fragment or the whole of integrin αVβ6 and / or the antibody immunoreactive with a fragment or the whole of integrin αVβ3 in the antibody administration step is not particularly limited, and examples thereof include subcutaneous administration, intraperitoneal administration, intravenous administration, etc. The antibody does not have to be administered to the non-human animal as a purified antibody, and may be administered to the non-human animal as, for example, a serum containing the antibody.

[0286] The antibody administered in the antibody administration step is selected to be an antibody that can immunologically react with integrin αVβ6 and / or integrin αVβ3 possessed by the non-human animal to which the antibody is administered.

[0287] On the other hand, a non-human model animal for ulcerative colitis or primary sclerosing cholangitis can be prepared by an immunization step of immunizing a non-human animal with a fragment or the whole of integrin αVβ6 and / or a fragment or the whole of integrin αVβ3 as an antigen.

[0288] Examples of non-human animals to which an antigen is administered include mice such as BALB / c mice and B6 mice, and other non-human animals.

[0289] The method in the immunization step is not particularly limited. Non-human animals can be immunized by administering a fragment or whole of integrin αVβ6 and / or a fragment or whole of integrin αVβ3 together with an appropriate adjuvant such as Freund's complete adjuvant to the non-human animal via subcutaneous administration, intravenous administration, or intraperitoneal administration.

[0290] In the immunization step, the fragment or whole of integrin αVβ6 and / or the fragment or whole of integrin αVβ3 administered as an antigen is selected in such a way that its antibody can react immunologically with integrin αVβ6 and / or integrin αVβ3 possessed by the non-human animal to be immunized through autoimmunity.

[0291] Example

[0292] <Experiment 1: Identification of specific autoantibodies in patients with ulcerative colitis>

[0293] In order to identify the antigens of ulcerative colitis patient-specific autoantibodies, the present inventors fixed extracellular matrix proteins known in the literature as antigen candidates on a solid phase, and confirmed whether the sera of ulcerative colitis patients contained antibodies binding to the extracellular matrix proteins by ELISA (Enzyme-Linked ImmunoSorbent Assay).

[0294] In the ELISA method, human IgG contained in the serum of ulcerative colitis patients and bound to the candidate antigen immobilized on the solid phase is used as the autoantibody to be detected, and rabbit anti-human IgG polyclonal antibody labeled with HRP (horseradish peroxidase) is used as the detection antibody.

[0295] 1. Antigen candidates

[0296] As antigen candidates, the following proteins known as extracellular matrix proteins were used.

[0297] Human integrin α2bβ3 (7148-A2, R&D system)

[0298] Human integrin α1β1 (7064-AB, R&D system)

[0299] Human integrin α2β1 (5698-A2, R&D system)

[0300] Human integrin α3β1 (2840-A3, R&D system)

[0301] Human integrin α4β1 (5668-A4, R&D system)

[0302] Human integrin α4β7 (5397-A3, R&D system)

[0303] Human integrin α5β1 (3230-A5, R&D system)

[0304] Human integrin α6β1 (7809-A6, R&D system)

[0305] Human integrin α6β4 (5497-A6, R&D system)

[0306] Human integrin α9β1 (5438-A9, R&D system)

[0307] Human integrin α10β1 (5895-AB, R&D system)

[0308] Human integrin α11β1 (6357-AB, R&D system)

[0309] Human integrin αEβ7 (5850-A3, R&D system)

[0310] Human integrin αLβ2 (3868-AV, R&D system)

[0311] Human integrin αMβ2 (4047-AM, R&D system)

[0312] Human integrin αVβ1 (6579-AVB, R&D system)

[0313] Human integrin αVβ3 (3050-AV, R&D system)

[0314] Human integrin αVβ5 (2528-AV, R&D system)

[0315] Human integrin αVβ6 (3817-AV, R&D system)

[0316] Human integrin αVβ8 (4135-AV, R&D system)

[0317] Human integrin αXβ2 (5755-AX, R&D system)

[0318] 2. Serum samples

[0319] Serum samples were obtained from 8 patients diagnosed with ulcerative colitis.

[0320] As patients with ulcerative colitis, patients without concurrent primary sclerosing cholangitis were selected.

[0321] As control serum samples, serum samples were obtained from three normal subjects.

[0322] 3. Process

[0323] In the following experiments, ELISA Starter Accessory Kit (E101, Bethyl Laboratories) was used unless otherwise specified.

[0324] ELISA coating buffer, ELISA washing solution, ELISA blocking buffer and conjugate diluent were prepared according to the instructions of the kit.

[0325] All processes were carried out at room temperature unless otherwise stated.

[0326] 3.1. Coating of antigen candidates

[0327] (1) Dilute one of the above-mentioned antigen candidates with the ELISA coating buffer of the kit to a solution with a concentration of 2 μg / ml, and add 100 μl of this solution to each well of the microplate of the kit.

[0328] (2) The above microplate was incubated at 4°C for 60 minutes.

[0329] (3) After incubation, remove the solution in each well by aspiration.

[0330] (4) Wash each well with the ELISA washing solution of the kit. Specifically, repeat the following washing operation three times: fill each well with the ELISA washing solution, and then remove the ELISA washing solution by aspiration.

[0331] 3.2. Closure

[0332] (1) 200 μl of the ELISA blocking buffer of the kit was added to each well.

[0333] (2) Incubate for 30 minutes.

[0334] (3) After incubation, the ELISA blocking buffer was removed and each well was washed three times.

[0335] 3.3. Test samples

[0336] (1) The ulcerative colitis patient serum sample (n=8) or the control serum sample (n=3) was diluted with the conjugate diluent of the kit at a ratio of 1:100.

[0337] (2) 100 μl of the serum dilution obtained by the dilution was added to each well of the blocked microplate.

[0338] (3) Incubate for 60 minutes.

[0339] (4) After incubation, the serum dilution solution was removed and each well was washed five times.

[0340] 3.4. HRP-conjugated detection antibodies

[0341] (1) The detection antibody (abcam6759, rabbit anti-human IgG H&L (HRP)) conjugated with HRP (horseradish peroxidase) was diluted at a ratio of 1:50,000 using the conjugate diluent of the kit.

[0342] (2) 100 μl of the detection antibody dilution solution obtained by the dilution was added to each well of the microplate that had come into contact with the serum.

[0343] (3) Incubate for 60 minutes.

[0344] (4) After incubation, the detection antibody diluent was removed and each well was washed five times.

[0345] 3.5. Enzyme substrate reaction

[0346] (1) Prepare TMB (3,3',5,5'-tetramethylbenzidine) solution according to the manufacturer's recommended conditions.

[0347] (2) 100 μl of the TMB solution was added to each well of the microplate that had been in contact with the detection antibody.

[0348] (3) Incubate for 7 to 8 minutes.

[0349] (4) To stop the oxidation of TMB, 100 μl of 0.18 MH 2 SO 4 Add to each well.

[0350] (5) The color development of the product of the oxidation reaction is measured at a wavelength of 450 nm using an ELISA reader.

[0351] 4. Results and Discussion

[0352] The results are shown in Figure 1-1 to Figure 1-4 The combination of the model numbers of the α-subunit and β-subunit of the integrin as the antigen candidate is shown in the title portion of each graph.

[0353] Figure 1-1 to Figure 1-4The middle vertical axis represents the relative value of absorbance at 450 nm, which is proportional to the amount of the detection antibody on the solid phase. UC represents serum samples from patients with ulcerative colitis (n=8), and control represents control serum samples (n=3).

[0354] When human integrin αVβ6 and human integrin αVβ3 were used as antigens, it was confirmed that the antibody concentrations were higher in the four samples of ulcerative colitis patient serum than in the control serum sample ( Figure 1-3 , Figure 1-4 ). This indicates that an increase in the concentration of human integrin αVβ6 autoantibodies in serum and an increase in the concentration of human integrin αVβ3 autoantibodies in serum can serve as diagnostic indicators for ulcerative colitis. When other human integrins were used as antigens, no significant difference in antibody concentration was observed between ulcerative colitis patient serum samples and control serum samples.

[0355] <Experiment 2: Diagnosis of ulcerative colitis based on anti-human integrin αVβ6 antibody>

[0356] In Experiment 1, it was confirmed that the concentration of anti-human integrin αVβ6 antibody was high in the sera of patients with ulcerative colitis.

[0357] Therefore, in order to explore the possibility that ulcerative colitis can be diagnosed based on the concentration of anti-human integrin αVβ6 antibodies in serum, the number of samples in this experiment was increased, and the number of samples was increased, including serum samples from patients with ulcerative colitis, n=63, serum samples from patients diagnosed with Crohn's disease, n=48, and control serum samples from normal people or patients diagnosed with Behcet's disease, eosinophilic gastroenteritis, infectious enteritis, ischemic enteritis, polyposis-pigmentation-alopecia-nail dystrophy (Cronkhite-Canada) syndrome, and IBDU (inflammatory bowel disease unclassified), n=11.

[0358] In this experiment, patients with ulcerative colitis but not complicated with primary sclerosing cholangitis were selected.

[0359] The concentration of anti-human integrin αVβ6 antibody in each serum sample was measured by ELISA method in the same manner as described in Experiment 1. 2 and MgCl 2 Add to ELISA washing solution, ELISA blocking buffer and conjugate diluent to a final concentration of 1 mM.

[0360] The results are shown in Figure 2 . Figure 2The vertical axis in the middle represents the relative value of absorbance at 450 nm, which is proportional to the amount of detection antibody on the solid phase. "Ulcerative colitis" represents serum samples of patients with ulcerative colitis (n=63), "Crohn's disease" represents serum samples of patients with Crohn's disease (n=48), and "control" represents control serum samples (n=11). The cutoff value represented by the dotted line is the value obtained by adding 3 times the standard deviation (SD) to the mean value of the relative absorbance values ​​of the control serum samples.

[0361] Depend on Figure 2 It was found that 58 of 63 serum samples from patients with ulcerative colitis were positive for anti-human integrin αVβ6 antibodies, with a sensitivity of 92%. On the other hand, 45 of 48 serum samples from patients with Crohn's disease were negative for anti-human integrin αVβ6 antibodies, and 11 of 11 control serum samples were negative, with a specificity of 95%. This result supports the ability to diagnose ulcerative colitis based on the blood concentration of autoantibodies to human integrin αVβ6.

[0362] <Experiment 3: Diagnosis of primary sclerosing cholangitis based on anti-human integrin αVβ6 antibody>

[0363] In this experiment, the possibility of diagnosing primary sclerosing cholangitis (PSC) based on the concentration of anti-human integrin αVβ6 antibodies in serum was investigated. The number of samples used was n=24 serum samples from PSC patients, n=5 serum samples from patients diagnosed with IgG4-related sclerosing cholangitis, and n=6 control serum samples from normal subjects.

[0364] Among the 24 cases of primary sclerosing cholangitis, 18 cases were complicated by ulcerative colitis.

[0365] The concentration of anti-human integrin αVβ6 antibody in each serum sample was measured by ELISA method in the same manner as described in Experiment 1. 2 and MgCl 2 Add to ELISA washing solution, ELISA blocking buffer and conjugate diluent to a final concentration of 1 mM.

[0366] The results are shown in Figure 3 . Figure 3The middle vertical axis represents the relative value of absorbance at 450 nm, which is proportional to the amount of detection antibody on the solid phase. "Primary sclerosing cholangitis" represents serum samples of PSC patients (n=24), "IgG4-related sclerosing cholangitis" represents serum samples of IgG4-related sclerosing cholangitis patients (n=5), and "control" represents control serum samples (n=6). The cutoff value represented by the dotted line is the value obtained by adding 3 times the standard deviation (SD) to the mean value of the relative absorbance values ​​of the control serum samples.

[0367] Depend on Figure 3 It was found that 23 out of 24 serum samples of PSC patients were positive for anti-human integrin αVβ6 antibodies, with a sensitivity of 96%. On the other hand, 4 out of 5 serum samples of IgG4-related sclerosing cholangitis patients and 6 out of 6 control serum samples were negative for anti-human integrin αVβ6 antibodies, with a specificity of 91%. This result supports the diagnosis of primary sclerosing cholangitis (PSC) based on the blood concentration of autoantibodies to human integrin αVβ6.

[0368] <Experiment 4: Diagnosis of ulcerative colitis based on anti-human integrin αVβ3 antibody>

[0369] In Experiment 1, it was confirmed that the concentration of anti-human integrin αVβ3 antibodies was high in the sera of patients with ulcerative colitis.

[0370] Therefore, in order to explore the possibility of diagnosing ulcerative colitis based on the concentration of anti-human integrin αVβ3 antibodies in serum, the number of samples in this experiment was increased, and the number of samples used was n=15: serum samples from patients with ulcerative colitis, n=4: serum samples from patients diagnosed with Crohn's disease, and n=9: control serum samples from normal subjects.

[0371] In this experiment, patients with ulcerative colitis but not complicated with primary sclerosing cholangitis were selected.

[0372] The concentration of anti-human integrin αVβ3 antibody in each serum sample was measured by ELISA method in the same manner as described in Experiment 1. 2 and MgCl 2 Add to ELISA washing solution, ELISA blocking buffer and conjugate diluent to a final concentration of 1 mM.

[0373] The results are shown in Figure 4 . Figure 4The middle vertical axis represents the relative value of absorbance at 450 nm, which is proportional to the amount of detection antibody on the solid phase. "Ulcerative colitis" represents serum samples of patients with ulcerative colitis (n=15), "Crohn's disease" represents serum samples of patients with Crohn's disease (n=4), and "control" represents control serum samples (n=9). The cutoff value represented by the dotted line is the value obtained by adding 3 times the standard deviation (SD) to the mean value of the relative absorbance values ​​of the control serum samples.

[0374] Depend on Figure 4 It was found that 58 of 63 serum samples from patients with ulcerative colitis were positive for anti-human integrin αVβ3 antibodies, with a sensitivity of 93%. On the other hand, 4 of 4 serum samples from patients with Crohn's disease were negative for anti-human integrin αVβ3 antibodies, and 9 of 9 control serum samples were negative for anti-human integrin αVβ3 antibodies, with a specificity of 100%. This result supports the ability to diagnose ulcerative colitis based on the blood concentration of autoantibodies to human integrin αVβ3.

[0375] <Experiment 5: Diagnosis of primary sclerosing cholangitis based on anti-human integrin αVβ3 antibody>

[0376] In this experiment, the possibility of diagnosing primary sclerosing cholangitis (PSC) based on the concentration of anti-human integrin αVβ3 antibodies in serum was investigated. Serum samples from PSC patients were n=24, serum samples from patients diagnosed with IgG4-related sclerosing cholangitis were n=5, and control serum samples from normal persons or patients diagnosed with bile duct cancer, primary biliary cholangitis, cirrhosis, or hepatocellular carcinoma were n=6.

[0377] Among the 24 cases of primary sclerosing cholangitis, 18 cases were complicated by ulcerative colitis.

[0378] The concentration of anti-human integrin αVβ3 antibody in each serum sample was measured by ELISA method in the same manner as described in Experiment 1. 2 and MgCl 2 Add to ELISA washing solution, ELISA blocking buffer and conjugate diluent to a final concentration of 1 mM.

[0379] The results are shown in Figure 5 . Figure 5The middle vertical axis represents the relative value of absorbance at 450 nm, which is proportional to the amount of detection antibody on the solid phase. "Primary sclerosing cholangitis" represents serum samples of PSC patients (n=24), "IgG4-related sclerosing cholangitis" represents serum samples of IgG4-related sclerosing cholangitis patients (n=5), and "control" represents control serum samples (n=6). The cutoff value represented by the dotted line is the value obtained by adding 3 times the standard deviation (SD) to the mean value of the relative absorbance values ​​of the control serum samples.

[0380] Depend on Figure 5 It was found that 15 of 24 serum samples of PSC patients were positive for anti-human integrin αVβ3 antibodies, with a sensitivity of 63%. On the other hand, 5 of 5 serum samples of IgG4-related sclerosing cholangitis patients were negative for anti-human integrin αVβ3 antibodies, and 6 of 6 control serum samples were negative, with a specificity of 100%. This result supports the diagnosis of primary sclerosing cholangitis (PSC) based on the blood concentration of autoantibodies to human integrin αVβ3.

[0381] <Experiment 6: Diagnosis of ulcerative colitis based on anti-human integrin αVβ6 antibody>

[0382] In this experiment, it was confirmed that the method for diagnosing ulcerative colitis based on the concentration of anti-human integrin αVβ6 antibodies in serum can specifically diagnose ulcerative colitis as positive. In this experiment, serum samples of patients with ulcerative colitis were n=66, serum samples of patients with Crohn's disease were n=47, and serum samples of patients with other intestinal diseases (infectious enteritis, ischemic enteritis, Behcet's disease, eosinophilic gastroenteritis, polyposis-pigmentation-alopecia-nail dystrophy syndrome or IBDU) were n=9.

[0383] In this experiment, patients with ulcerative colitis but not complicated with primary sclerosing cholangitis were selected.

[0384] The concentration of anti-human integrin αVβ6 antibody in each serum sample was measured by ELISA in the same manner as described in Experiment 2.

[0385] The results are shown in Figure 6 . Figure 6 The middle vertical axis represents the relative value of absorbance at 450 nm, which is proportional to the amount of detection antibody on the solid phase. "Ulcerative colitis" represents serum samples of patients with ulcerative colitis, "Crohn's disease" represents serum samples of patients with Crohn's disease, and "Other intestinal diseases" represents serum samples of patients with other intestinal diseases. The cutoff value represented by the dotted line is the value obtained by adding 3 times the standard deviation (SD) to the average value of the relative absorbance values ​​of serum samples of patients with other intestinal diseases.

[0386] In terms of anti-human integrin αVβ6 antibodies, 62 out of 66 serum samples from patients with ulcerative colitis were positive, with a sensitivity of 94%. On the other hand, in terms of anti-human integrin αVβ6 antibodies, 44 out of 47 serum samples from patients with Crohn's disease and 9 out of 9 serum samples from patients with other intestinal diseases were negative, with a specificity of 95%. This result shows that the blood concentration of autoantibodies to human integrin αVβ6 is specifically high in patients with ulcerative colitis.

[0387] <Experiment 7: Diagnosis of ulcerative colitis based on anti-human integrin αVβ6 antibody>

[0388] In this experiment, it was confirmed that the method for diagnosing ulcerative colitis based on the concentration of anti-human integrin αVβ6 antibodies in serum can specifically diagnose ulcerative colitis as positive. In this experiment, the number of samples used was n=51 serum samples from patients with ulcerative colitis, n=26 serum samples from patients with Crohn's disease, n=24 serum samples from patients with other intestinal diseases (infectious enteritis, ischemic enteritis, Behcet's disease, eosinophilic gastroenteritis, polyposis-pigmentation-alopecia-nail dystrophy syndrome or IBDU), n=27 serum samples from patients with collagen diseases, and n=22 serum samples from normal people.

[0389] In this experiment, patients with ulcerative colitis but not complicated with primary sclerosing cholangitis were selected.

[0390] The concentration of anti-human integrin αVβ6 antibody in each serum sample was measured by ELISA in the same manner as described in Experiment 2.

[0391] The results are shown in Figure 7 . Figure 7 The middle vertical axis represents the relative value of absorbance at 450 nm, which is proportional to the amount of detection antibody on the solid phase. "Ulcerative colitis" represents serum samples of patients with ulcerative colitis, "Crohn's disease" represents serum samples of patients with Crohn's disease, "other intestinal diseases" represents serum samples of patients with other intestinal diseases, "collagen disease" represents serum samples of patients with collagen disease, and "normal person" represents serum samples of normal people. The cutoff value represented by the dotted line is the value obtained by adding 3 times the standard deviation (SD) to the average value of the relative absorbance values ​​of normal human serum samples.

[0392] For anti-human integrin αVβ6 antibodies, 43 out of 51 serum samples from patients with ulcerative colitis were positive, with a sensitivity of 84%. On the other hand, for anti-human integrin αVβ6 antibodies, 24 out of 26 serum samples from patients with Crohn's disease were negative, 23 out of 24 serum samples from patients with other intestinal diseases were negative, 26 out of 27 serum samples from patients with collagen diseases were negative, and 22 out of 22 serum samples from normal people were negative, with a specificity of 96%. This result also shows that the blood concentration of autoantibodies to human integrin αVβ6 is specifically high in patients with ulcerative colitis, as in Experiment 6.

[0393] <Experiment 8: IgG subtypes of anti-human integrin αVβ6 autoantibodies in patients with ulcerative colitis>

[0394] In this experiment, the IgG subtype of anti-human integrin αVβ6 autoantibodies in the sera of patients with ulcerative colitis was confirmed.

[0395] In this experiment, the number of samples used was n=47, including serum samples from patients with ulcerative colitis, n=8, and serum samples from patients with Crohn's disease, n=8, and serum samples from patients with other intestinal diseases (infectious enteritis, ischemic enteritis, Behcet's disease, eosinophilic gastroenteritis, and polyposis-pigmentation-alopecia-onychia dystrophy syndrome or IBDU).

[0396] In this experiment, patients with ulcerative colitis but not complicated with primary sclerosing cholangitis were selected.

[0397] In order to distinguish and detect IgG1, IgG2, IgG3, and IgG4 that specifically bind to human integrin αVβ6, in the ELISA method described in Experiment 1 using human integrin αVβ6 as an antigen, in addition to using HRP-conjugated anti-human IgG1 antibody (Anti-IgG1-HRP, AP006, Binding Site), HRP-conjugated anti-human IgG2 antibody (Anti-IgG2-HRP, AP007, Binding Site), HRP-conjugated anti-human IgG3 antibody (Anti-IgG3-HRP, AP008, Binding Site), or HRP-conjugated anti-human IgG4 antibody (Anti-IgG4-HRP, AP009, Binding Site) as detection antibodies, CaCl was added to each of the two ELISAs. 2 and MgCl 2 ELISA was performed in the same manner as in Experiment 1 except that the ELISA washing solution, ELISA blocking buffer, and conjugate diluent were added to a final concentration of 1 mM.

[0398] The detection results of IgG1 are shown in Fig. 8A The detection results of IgG2 are shown in Figure 8B The detection results of IgG3 are shown in Figure 8C The detection results of IgG4 are shown in Fig.8D .exist Figures 8A to 8D In the table, the vertical axis represents the relative value of the absorbance at 450 nm, which is proportional to the amount of the detection antibody on the solid phase. "Ulcerative colitis" represents serum samples from patients with ulcerative colitis (n=47), "Crohn's disease" represents serum samples from patients with Crohn's disease (n=8), and "other intestinal diseases" represents serum samples from patients with the aforementioned other intestinal diseases (n=8). Figures 8A to 8D In the figure, the cutoff value indicated by the dotted line is a value obtained by adding 3 times the standard deviation (SD) to the mean value of the relative absorbance values ​​of the serum samples of the other intestinal disease patients.

[0399] Fig. 8A The following results are shown: For IgG1 binding to human integrin αVβ6, 45 out of 47 serum samples from patients with ulcerative colitis were positive (sensitivity 96%), 8 out of 8 serum samples from patients with Crohn's disease were negative, and 8 out of 8 serum samples from patients with other intestinal diseases were negative.

[0400] Figure 8B The following results are shown: IgG2 binding to human integrin αVβ6 was positive in 29 of 47 serum samples from patients with ulcerative colitis (sensitivity 62%), negative in 7 of 8 serum samples from patients with Crohn's disease, and negative in 8 of 8 serum samples from patients with other intestinal diseases.

[0401] Figure 8C The following results are shown: IgG3 binding to human integrin αVβ6 was positive in 10 of 47 serum samples from patients with ulcerative colitis (sensitivity 21%), negative in 6 of 8 serum samples from patients with Crohn's disease, and negative in 8 of 8 serum samples from patients with other intestinal diseases.

[0402] Fig.8D The following results are shown: IgG4 binding to human integrin αVβ6 was positive in 19 of 47 serum samples from patients with ulcerative colitis (sensitivity 40%), negative in 8 of 8 serum samples from patients with Crohn's disease, and negative in 8 of 8 serum samples from patients with other intestinal diseases.

[0403] These experimental results confirmed that IgG1 is dominant among the IgG subtypes of anti-human integrin αVβ6 autoantibodies in patients with ulcerative colitis.

[0404] <Experiment 9: Isotypes of anti-human integrin αVβ6 autoantibodies in patients with ulcerative colitis>

[0405] In this experiment, isotypes of IgA, IgM, and IgE other than IgG, which are anti-human integrin αVβ6 autoantibodies, were detected in the sera of patients with ulcerative colitis.

[0406] In this experiment, the number of samples used was n=47, including serum samples from patients with ulcerative colitis, n=8, and serum samples from patients with Crohn's disease, n=8, and serum samples from patients with other intestinal diseases (infectious enteritis, ischemic enteritis, Behcet's disease, eosinophilic gastroenteritis, and polyposis-pigmentation-alopecia-onychia dystrophy syndrome or IBDU).

[0407] In this experiment, patients with ulcerative colitis but not complicated with primary sclerosing cholangitis were selected.

[0408] In order to detect IgA, IgM, and IgE that specifically bind to human integrin αVβ6, the ELISA method described in Experiment 1 using human integrin αVβ6 as an antigen was used, except that HRP-conjugated anti-human IgA antibody (Goat anti-Human IgA Antibody HRP Conjugated, A80-102P, BETHYL), HRP-conjugated anti-human IgM antibody (Goat anti-Human IgM Antibody HRP Conjugated, A80-100P, BETHYL), or HRP-conjugated anti-human IgE antibody (Goat anti-Human IgE Cross-Adsorbed Secondary Antibody, HRP, A18799, Invitrogen) was used as the detection antibody and CaCl was added to each of the two assays. 2 and MgCl 2 ELISA was performed in the same manner as in Experiment 1 except that the ELISA washing solution, ELISA blocking buffer, and conjugate diluent were added to a final concentration of 1 mM.

[0409] The results of IgA detection are shown in Fig.9A The results of IgM detection are shown in Fig. 9B The IgE test results are shown in Fig. 9C . Figures 9A to 9C In the table, the vertical axis represents the relative value of the absorbance at 450 nm, which is proportional to the amount of the detection antibody on the solid phase. "Ulcerative colitis" represents serum samples from patients with ulcerative colitis (n=47), "Crohn's disease" represents serum samples from patients with Crohn's disease (n=8), and "other intestinal diseases" represents serum samples from patients with the aforementioned other intestinal diseases (n=8). Figures 9A to 9CIn the figure, the cutoff value indicated by the dotted line is a value obtained by adding 3 times the standard deviation (SD) to the mean value of the relative absorbance values ​​of the serum samples of the other intestinal disease patients.

[0410] Fig.9A The following results are shown: Regarding IgA binding to human integrin αVβ6, 35 of 47 serum samples from patients with ulcerative colitis were positive (sensitivity 74%), 8 of 8 serum samples from patients with Crohn's disease were negative, and 8 of 8 serum samples from patients with other intestinal diseases were negative.

[0411] Fig. 9B The following results are shown: For IgM binding to human integrin αVβ6, 11 of 47 serum samples from patients with ulcerative colitis were positive (sensitivity 23%), 8 of 8 serum samples from patients with Crohn's disease were negative, and 8 of 8 serum samples from patients with other intestinal diseases were negative.

[0412] Fig. 9C The following results are shown: Regarding IgE binding to human integrin αVβ6, 10 of 47 serum samples from patients with ulcerative colitis were positive (sensitivity 21%), 7 of 8 serum samples from patients with Crohn's disease were negative, and 8 of 8 serum samples from patients with other intestinal diseases were negative.

[0413] These experimental results confirmed that anti-human integrin αVβ6 autoantibodies in patients with ulcerative colitis include IgA, IgM, and IgE in addition to IgG, with IgA being predominant.

[0414] <Experiment 10: Correlation between the severity of ulcerative colitis and the titer of anti-human integrin αVβ6 autoantibodies>

[0415] The correlation between the severity of ulcerative colitis and the antibody titer of anti-human integrin αVβ6 autoantibody (IgG) was verified.

[0416] The partial Mayo score obtained by excluding the mucosal observation results (endoscopic observation results) from the Mayo score was used for the evaluation of the condition.

[0417] The antibody titer of anti-human integrin αVβ6 autoantibody (IgG) was measured by the same procedure as in Experiment 2 above.

[0418] Fig. 10A and Fig. 10B Partial Mayo scores (left vertical axis) and antibody titers of anti-human integrin αVβ6 autoantibodies (IgG) (right vertical axis) of two representative patients with ulcerative colitis at various time points from November 2017 to November 2018 are shown.

[0419] In both patients, antibody titers changed similarly along with changes in partial Mayo scores.

[0420] <Experiment 11: Diagnosis of ulcerative colitis based on anti-human integrin αVβ3 antibody>

[0421] In this experiment, it was confirmed that the method for diagnosing ulcerative colitis based on the serum concentration of anti-human integrin αVβ3 antibodies can specifically diagnose ulcerative colitis as positive. In this experiment, serum samples of patients with ulcerative colitis were n=66, serum samples of patients with Crohn's disease were n=47, and serum samples of patients with other intestinal diseases (infectious enteritis, ischemic enteritis, Behcet's disease, eosinophilic gastroenteritis, polyposis-pigmentation-alopecia-nail dystrophy syndrome or IBDU) were n=9.

[0422] In this experiment, patients with ulcerative colitis but not complicated with primary sclerosing cholangitis were selected.

[0423] The concentration of anti-human integrin αVβ3 antibody in each serum sample was measured by the ELISA method in the same manner as described in Experiment 4.

[0424] The results are shown in Fig.11 . Fig.11 The middle vertical axis represents the relative value of absorbance at 450 nm, which is proportional to the amount of detection antibody on the solid phase. "Ulcerative colitis" represents serum samples of patients with ulcerative colitis, "Crohn's disease" represents serum samples of patients with Crohn's disease, and "Other intestinal diseases" represents serum samples of patients with other intestinal diseases. The cutoff value represented by the dotted line is the value obtained by adding 3 times the standard deviation (SD) to the average value of the relative absorbance values ​​of serum samples of patients with other intestinal diseases.

[0425] In terms of anti-human integrin αVβ3 antibodies, 60 out of 66 serum samples from patients with ulcerative colitis were positive, with a sensitivity of 91%. On the other hand, in terms of anti-human integrin αVβ3 antibodies, 38 out of 47 serum samples from patients with Crohn's disease and 9 out of 9 serum samples from patients with other intestinal diseases were negative, with a specificity of 84%. These results show that the blood concentration of autoantibodies to human integrin αVβ3 is specifically high in patients with ulcerative colitis.

[0426] <Experiment 12: Diagnosis of ulcerative colitis based on anti-human integrin αVβ3 antibody>

[0427] In this experiment, it was confirmed that the method for diagnosing ulcerative colitis based on the concentration of anti-human integrin αVβ3 antibodies in serum can specifically diagnose ulcerative colitis as positive. In this experiment, the number of samples used was n=51 serum samples from patients with ulcerative colitis, n=26 serum samples from patients with Crohn's disease, n=24 serum samples from patients with other intestinal diseases (infectious enteritis, ischemic enteritis, Behcet's disease, eosinophilic gastroenteritis, polyposis-pigmentation-alopecia-nail dystrophy syndrome or IBDU), n=27 serum samples from patients with collagen diseases, and n=22 serum samples from normal persons.

[0428] In this experiment, patients with ulcerative colitis but not complicated with primary sclerosing cholangitis were selected.

[0429] The concentration of anti-human integrin αVβ3 antibody in each serum sample was measured by the ELISA method in the same manner as described in Experiment 4.

[0430] The results are shown in Fig.12 . Fig.12 The middle vertical axis represents the relative value of absorbance at 450 nm, which is proportional to the amount of detection antibody on the solid phase. "UC" represents serum samples of patients with ulcerative colitis, "CD" represents serum samples of patients with Crohn's disease, "other intestinal diseases" represents serum samples of patients with other intestinal diseases, "collagen disease" represents serum samples of patients with collagen disease, and "normal person" represents serum samples of normal people. The cutoff value represented by the dotted line is the value obtained by adding 3 times the standard deviation (SD) to the average value of the relative absorbance values ​​of normal human serum samples.

[0431] For anti-human integrin αVβ3 antibodies, 42 out of 51 serum samples from patients with ulcerative colitis were positive, with a sensitivity of 82%. On the other hand, for anti-human integrin αVβ3 antibodies, 21 out of 26 serum samples from patients with Crohn's disease were negative, 21 out of 24 serum samples from patients with other intestinal diseases were negative, 27 out of 27 serum samples from patients with collagen diseases were negative, and 22 out of 22 serum samples from normal people were negative, with a specificity of 92%. This result also shows that the blood concentration of autoantibodies to human integrin αVβ3 is specifically high in patients with ulcerative colitis, as in Experiment 11.

[0432] <Experiment 13: IgG subtypes of anti-human integrin αVβ3 autoantibodies in patients with ulcerative colitis>

[0433] In this experiment, the IgG subtype of anti-human integrin αVβ3 autoantibodies in the sera of patients with ulcerative colitis was confirmed.

[0434] In this experiment, the number of samples used was n=47, including serum samples from patients with ulcerative colitis, n=8, and serum samples from patients with Crohn's disease, n=8, and serum samples from patients with other intestinal diseases (infectious enteritis, ischemic enteritis, Behcet's disease, eosinophilic gastroenteritis, and polyposis-pigmentation-alopecia-onychia dystrophy syndrome or IBDU).

[0435] In this experiment, patients with ulcerative colitis but not complicated with primary sclerosing cholangitis were selected.

[0436] In order to distinguish and detect IgG1, IgG2, IgG3, and IgG4 that specifically bind to human integrin αVβ3, the ELISA method described in Experiment 1 using human integrin αVβ3 as an antigen was used, except that HRP-conjugated anti-human IgG1 antibody (Anti-IgG1-HRP, AP006, Binding Site), HRP-conjugated anti-human IgG2 antibody (Anti-IgG2-HRP, AP007, Binding Site), HRP-conjugated anti-human IgG3 antibody (Anti-IgG3-HRP, AP008, Binding Site), or HRP-conjugated anti-human IgG4 antibody (Anti-IgG4-HRP, AP009, Binding Site) was used as the detection antibody, and CaCl was added to each of them. 2 and MgCl 2 ELISA was performed in the same manner as in Experiment 1 except that the ELISA washing solution, ELISA blocking buffer, and conjugate diluent were added to a final concentration of 1 mM.

[0437] The detection results of IgG1 are shown in Fig.13A The results of IgG2 detection are shown in Fig. 13B The results of IgG3 are shown in Fig. 13C The results of IgG4 are shown in Fig.13D .exist FIG. 13A to FIG. 13D In the table, the vertical axis represents the relative value of the absorbance at 450 nm, which is proportional to the amount of the detection antibody on the solid phase. "Ulcerative colitis" represents serum samples from patients with ulcerative colitis (n=47), "Crohn's disease" represents serum samples from patients with Crohn's disease (n=8), and "other intestinal diseases" represents serum samples from patients with the aforementioned other intestinal diseases (n=8). FIG. 13A to FIG. 13D In the figure, the cutoff value indicated by the dotted line is a value obtained by adding 3 times the standard deviation (SD) to the mean value of the relative absorbance values ​​of the serum samples of the other intestinal disease patients.

[0438] Fig.13AThe following results are shown: IgG1 binding to human integrin αVβ3 was positive in 39 of 47 serum samples from patients with ulcerative colitis (sensitivity 83%), negative in 8 of 8 serum samples from patients with Crohn's disease, and negative in 8 of 8 serum samples from patients with other intestinal diseases.

[0439] Fig. 13B The following results are shown: IgG2 binding to human integrin αVβ3 was positive in 14 of 47 serum samples from patients with ulcerative colitis (sensitivity 30%), negative in 8 of 8 serum samples from patients with Crohn's disease, and negative in 8 of 8 serum samples from patients with other intestinal diseases.

[0440] Fig. 13C The following results are shown: IgG3 binding to human integrin αVβ3 was positive in 16 of 47 serum samples from patients with ulcerative colitis (sensitivity 34%), negative in 8 of 8 serum samples from patients with Crohn's disease, and negative in 8 of 8 serum samples from patients with other intestinal diseases.

[0441] Fig.13D The following results are shown: IgG4 binding to human integrin αVβ3 was positive in 3 of 47 serum samples from patients with ulcerative colitis (sensitivity 6%), negative in 8 of 8 serum samples from patients with Crohn's disease, and negative in 8 of 8 serum samples from patients with other intestinal diseases.

[0442] These experimental results confirmed that IgG1 is dominant among the IgG subtypes of anti-human integrin αVβ3 autoantibodies in patients with ulcerative colitis.

[0443] <Experiment 14: Isotypes of anti-human integrin αVβ3 autoantibodies in patients with ulcerative colitis>

[0444] In this experiment, isotypes of IgA, IgM, and IgE other than IgG, which are anti-human integrin αVβ3 autoantibodies, were detected in the sera of patients with ulcerative colitis.

[0445] In this experiment, the number of samples used was n=47, including serum samples from patients with ulcerative colitis, n=8, and serum samples from patients with Crohn's disease, n=8, and serum samples from patients with other intestinal diseases (infectious enteritis, ischemic enteritis, Behcet's disease, eosinophilic gastroenteritis, and polyposis-pigmentation-alopecia-onychia dystrophy syndrome or IBDU).

[0446] In this experiment, patients with ulcerative colitis but not complicated with primary sclerosing cholangitis were selected.

[0447] In order to detect IgA, IgM, and IgE that specifically bind to human integrin αVβ3, the ELISA method described in Experiment 1 using human integrin αVβ3 as an antigen was used, except that HRP-conjugated anti-human IgA antibody (Goat anti-Human IgA Antibody HRP Conjugated, A80-102P, BETHYL), HRP-conjugated anti-human IgM antibody (Goat anti-Human IgM Antibody HRP Conjugated, A80-100P, BETHYL), or HRP-conjugated anti-human IgE antibody (Goat anti-Human IgE Cross-Adsorbed Secondary Antibody, HRP, A18799, Invitrogen) was used as the detection antibody and CaCl was added to each of the two assays. 2 and MgCl 2 ELISA was performed in the same manner as in Experiment 1 except that the ELISA washing solution, ELISA blocking buffer, and conjugate diluent were added to a final concentration of 1 mM.

[0448] The results of IgA detection are shown in Fig.14A The results of IgM detection are shown in Fig. 14B The IgE test results are shown in Fig. 14C .exist Figures 14A to 14C In the table, the vertical axis represents the relative value of the absorbance at 450 nm, which is proportional to the amount of the detection antibody on the solid phase. "Ulcerative colitis" represents serum samples from patients with ulcerative colitis (n=47), "Crohn's disease" represents serum samples from patients with Crohn's disease (n=8), and "other intestinal diseases" represents serum samples from patients with the aforementioned other intestinal diseases (n=8). Figures 14A to 14C In the figure, the cutoff value indicated by the dotted line is a value obtained by adding 3 times the standard deviation (SD) to the mean value of the relative absorbance values ​​of the serum samples of the other intestinal disease patients.

[0449] Fig.14A The following results are shown: For IgA binding to human integrin αVβ3, 5 of 47 serum samples from patients with ulcerative colitis were positive (sensitivity 11%), 8 of 8 serum samples from patients with Crohn's disease were negative, and 8 of 8 serum samples from patients with other intestinal diseases were negative.

[0450] Fig. 14BThe following results are shown: For IgM binding to human integrin αVβ3, 10 of 47 serum samples from patients with ulcerative colitis were positive (sensitivity 21%), 8 of 8 serum samples from patients with Crohn's disease were negative, and 8 of 8 serum samples from patients with other intestinal diseases were negative.

[0451] Fig. 14C The following results were shown: 0 of 47 serum samples from patients with ulcerative colitis were positive for IgE binding to human integrin αVβ3 (sensitivity 0%), 8 of 8 serum samples from patients with Crohn's disease were negative, and 8 of 8 serum samples from patients with other intestinal diseases were negative.

[0452] These experimental results confirmed that the isotypes of anti-human integrin αVβ3 autoantibodies in patients with ulcerative colitis were low in IgA, IgM, and IgE.

[0453] All publications, patents and patent applications cited in this specification are incorporated into this specification by reference in their original form. Sequence Listing <110> National University Corporation Kyoto University <120> Methods of examination for ulcerative colitis and primary sclerosing cholangitis <130> PH-8191-PCT <150> JP 2019-000060 <151> 2019-01-04 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 1048 <212> PRT <213> Homo sapiens <400> 1 Met Ala Phe Pro Pro Arg Arg Arg Leu Arg Leu Gly Pro Arg Gly Leu 1 5 10 15 Pro Leu Leu Leu Ser Gly Leu Leu Leu Pro Leu Cys Arg Ala Phe Asn 20 25 30 Leu Asp Val Asp Ser Pro Ala Glu Tyr Ser Gly Pro Glu Gly Ser Tyr 35 40 45 Phe Gly Phe Ala Val Asp Phe Phe Val Pro Ser Ala Ser Ser Arg Met 50 55 60 Phe Leu Leu Val Gly Ala Pro Lys Ala Asn Thr Thr Gln Pro Gly Ile 65 70 75 80 Val Glu Gly Gly Gln Val Leu Lys Cys Asp Trp Ser Ser Thr Arg Arg 85 90 95 Cys Gln Pro Ile Glu Phe Asp Ala Thr Gly Asn Arg Asp Tyr Ala Lys 100 105 110 Asp Asp Pro Leu Glu Phe Lys Ser His Gln Trp Phe Gly Ala Ser Val 115 120 125 Arg Ser Lys Gln Asp Lys Ile Leu Ala Cys Ala Pro Leu Tyr His Trp 130 135 140 Arg Thr Glu Met Lys Gln Glu Arg Glu Pro Val Gly Thr Cys Phe Leu 145 150 155 160 Gln Asp Gly Thr Lys Thr Val Glu Tyr Ala Pro Cys Arg Ser Gln Asp 165 170 175 Ile Asp Ala Asp Gly Gln Gly Phe Cys Gln Gly Gly Phe Ser Ile Asp 180 185 190 Phe Thr Lys Ala Asp Arg Val Leu Leu Gly Gly Pro Gly Ser Phe Tyr 195 200 205 Trp Gln Gly Gln Leu Ile Ser Asp Gln Val Ala Glu Ile Val Ser Lys 210 215 220 Tyr Asp Pro Asn Val Tyr Ser Ile Lys Tyr Asn Asn Gln Leu Ala Thr 225 230 235 240 Arg Thr Ala Gln Ala Ile Phe Asp Asp Ser Tyr Leu Gly Tyr Ser Val 245 250 255 Ala Val Gly Asp Phe Asn Gly Asp Gly Ile Asp Asp Phe Val Ser Gly 260 265 270 Val Pro Arg Ala Ala Arg Thr Leu Gly Met Val Tyr Ile Tyr Asp Gly 275 280 285 Lys Asn Met Ser Ser Leu Tyr Asn Phe Thr Gly Glu Gln Met Ala Ala 290 295 300 Tyr Phe Gly Phe Ser Val Ala Ala Thr Asp Ile Asn Gly Asp Asp Tyr 305 310 315 320 Ala Asp Val Phe Ile Gly Ala Pro Leu Phe Met Asp Arg Gly Ser Asp 325 330 335 Gly Lys Leu Gln Glu Val Gly Gln Val Ser Val Ser Leu Gln Arg Ala 340 345 350 Ser Gly Asp Phe Gln Thr Thr Lys Leu Asn Gly Phe Glu Val Phe Ala 355 360 365 Arg Phe Gly Ser Ala Ile Ala Pro Leu Gly Asp Leu Asp Gln Asp Gly 370 375 380 Phe Asn Asp Ile Ala Ile Ala Ala Pro Tyr Gly Gly Glu Asp Lys Lys 385 390 395 400 Gly Ile Val Tyr Ile Phe Asn Gly Arg Ser Thr Gly Leu Asn Ala Val 405 410 415 Pro Ser Gln Ile Leu Glu Gly Gln Trp Ala Ala Arg Ser Met Pro Pro 420 425 430 Ser Phe Gly Tyr Ser Met Lys Gly Ala Thr Asp Ile Asp Lys Asn Gly 435 440 445 Tyr Pro Asp Leu Ile Val Gly Ala Phe Gly Val Asp Arg Ala Ile Leu 450 455 460 Tyr Arg Ala Arg Pro Val Ile Thr Val Asn Ala Gly Leu Glu Val Tyr 465 470 475 480 Pro Ser Ile Leu Asn Gln Asp Asn Lys Thr Cys Ser Leu Pro Gly Thr 485 490 495 Ala Leu Lys Val Ser Cys Phe Asn Val Arg Phe Cys Leu Lys Ala Asp 500 505 510 Gly Lys Gly Val Leu Pro Arg Lys Leu Asn Phe Gln Val Glu Leu Leu 515 520 525 Leu Asp Lys Leu Lys Gln Lys Gly Ala Ile Arg Arg Ala Leu Phe Leu 530 535 540 Tyr Ser Arg Ser Pro Ser His Ser Lys Asn Met Thr Ile Ser Arg Gly 545 550 555 560 Gly Leu Met Gln Cys Glu Glu Leu Ile Ala Tyr Leu Arg Asp Glu Ser 565 570 575 Glu Phe Arg Asp Lys Leu Thr Pro Ile Thr Ile Phe Met Glu Tyr Arg 580 585 590 Leu Asp Tyr Arg Thr Ala Ala Asp Thr Thr Gly Leu Gln Pro Ile Leu 595 600 605 Asn Gln Phe Thr Pro Ala Asn Ile Ser Arg Gln Ala His Ile Leu Leu 610 615 620 Asp Cys Gly Glu Asp Asn Val Cys Lys Pro Lys Leu Glu Val Ser Val 625 630 635 640 Asp Ser Asp Gln Lys Lys Ile Tyr Ile Gly Asp Asp Asn Pro Leu Thr 645 650 655 Leu Ile Val Lys Ala Gln Asn Gln Gly Glu Gly Ala Tyr Glu Ala Glu 660 665 670 Leu Ile Val Ser Ile Pro Leu Gln Ala Asp Phe Ile Gly Val Val Arg 675 680 685 Asn Asn Glu Ala Leu Ala Arg Leu Ser Cys Ala Phe Lys Thr Glu Asn 690 695 700 Gln Thr Arg Gln Val Val Cys Asp Leu Gly Asn Pro Met Lys Ala Gly 705 710 715 720 Thr Gln Leu Leu Ala Gly Leu Arg Phe Ser Val His Gln Gln Ser Glu 725 730 735 Met Asp Thr Ser Val Lys Phe Asp Leu Gln Ile Gln Ser Ser Asn Leu 740 745 750 Phe Asp Lys Val Ser Pro Val Val Ser His Lys Val Asp Leu Ala Val 755 760 765 Leu Ala Ala Val Glu Ile Arg Gly Val Ser Ser Pro Asp His Val Phe 770 775 780 Leu Pro Ile Pro Asn Trp Glu His Lys Glu Asn Pro Glu Thr Glu Glu 785 790 795 800 Asp Val Gly Pro Val Val Gln His Ile Tyr Glu Leu Arg Asn Asn Gly 805 810 815 Pro Ser Ser Phe Ser Lys Ala Met Leu His Leu Gln Trp Pro Tyr Lys 820 825 830 Tyr Asn Asn Asn Thr Leu Leu Tyr Ile Leu His Tyr Asp Ile Asp Gly 835 840 845 Pro Met Asn Cys Thr Ser Asp Met Glu Ile Asn Pro Leu Arg Ile Lys 850 855 860 Ile Ser Ser Leu Gln Thr Thr Glu Lys Asn Asp Thr Val Ala Gly Gln 865 870 875 880 Gly Glu Arg Asp His Leu Ile Thr Lys Arg Asp Leu Ala Leu Ser Glu 885 890 895 Gly Asp Ile His Thr Leu Gly Cys Gly Val Ala Gln Cys Leu Lys Ile 900 905 910 Val Cys Gln Val Gly Arg Leu Asp Arg Gly Lys Ser Ala Ile Leu Tyr 915 920 925 Val Lys Ser Leu Leu Trp Thr Glu Thr Phe Met Asn Lys Glu Asn Gln 930 935 940 Asn His Ser Tyr Ser Leu Lys Ser Ser Ala Ser Phe Asn Val Ile Glu 945 950 955 960 Phe Pro Tyr Lys Asn Leu Pro Ile Glu Asp Ile Thr Asn Ser Thr Leu 965 970 975 Val Thr Thr Asn Val Thr Trp Gly Ile Gln Pro Ala Pro Met Pro Val 980 985 990 Pro Val Trp Val Ile Ile Leu Ala Val Leu Ala Gly Leu Leu Leu Leu 995 1000 1005 Ala Val Leu Val Phe Val Met Tyr Arg Met Gly Phe Phe Lys Arg 1010 1015 1020 Val Arg Pro Pro Gln Glu Glu Gln Glu Arg Glu Gln Leu Gln Pro 1025 1030 1035 His Glu Asn Gly Glu Gly Asn Ser Glu Thr 1040 1045 <210> 2 <211> 788 <212> PRT <213> Homo sapiens <400> 2 Met Gly Ile Glu Leu Leu Cys Leu Phe Phe Leu Phe Leu Gly Arg Asn 1 5 10 15 Asp His Val Gln Gly Gly Cys Ala Leu Gly Gly Ala Glu Thr Cys Glu 20 25 30 Asp Cys Leu Leu Ile Gly Pro Gln Cys Ala Trp Cys Ala Gln Glu Asn 35 40 45 Phe Thr His Pro Ser Gly Val Gly Glu Arg Cys Asp Thr Pro Ala Asn 50 55 60 Leu Leu Ala Lys Gly Cys Gln Leu Asn Phe Ile Glu Asn Pro Val Ser 65 70 75 80 Gln Val Glu Ile Leu Lys Asn Lys Pro Leu Ser Val Gly Arg Gln Lys 85 90 95 Asn Ser Ser Asp Ile Val Gln Ile Ala Pro Gln Ser Leu Ile Leu Lys 100 105 110 Leu Arg Pro Gly Gly Ala Gln Thr Leu Gln Val His Val Arg Gln Thr 115 120 125 Glu Asp Tyr Pro Val Asp Leu Tyr Tyr Leu Met Asp Leu Ser Ala Ser 130 135 140 Met Asp Asp Asp Leu Asn Thr Ile Lys Glu Leu Gly Ser Arg Leu Ser 145 150 155 160 Lys Glu Met Ser Lys Leu Thr Ser Asn Phe Arg Leu Gly Phe Gly Ser 165 170 175 Phe Val Glu Lys Pro Val Ser Pro Phe Val Lys Thr Thr Pro Glu Glu 180 185 190 Ile Ala Asn Pro Cys Ser Ser Ile Pro Tyr Phe Cys Leu Pro Thr Phe 195 200 205 Gly Phe Lys His Ile Leu Pro Leu Thr Asn Asp Ala Glu Arg Phe Asn 210 215 220 Glu Ile Val Lys Asn Gln Lys Ile Ser Ala Asn Ile Asp Thr Pro Glu 225 230 235 240 Gly Gly Phe Asp Ala Ile Met Gln Ala Ala Val Cys Lys Glu Lys Ile 245 250 255 Gly Trp Arg Asn Asp Ser Leu His Leu Leu Val Phe Val Ser Asp Ala 260 265 270 Asp Ser His Phe Gly Met Asp Ser Lys Leu Ala Gly Ile Val Ile Pro 275 280 285 Asn Asp Gly Leu Cys His Leu Asp Ser Lys Asn Glu Tyr Ser Met Ser 290 295 300 Thr Val Leu Glu Tyr Pro Thr Ile Gly Gln Leu Ile Asp Lys Leu Val 305 310 315 320 Gln Asn Asn Val Leu Leu Ile Phe Ala Val Thr Gln Glu Gln Val His 325 330 335 Leu Tyr Glu Asn Tyr Ala Lys Leu Ile Pro Gly Ala Thr Val Gly Leu 340 345 350 Leu Gln Lys Asp Ser Gly Asn Ile Leu Gln Leu Ile Ile Ser Ala Tyr 355 360 365 Glu Glu Leu Arg Ser Glu Val Glu Leu Glu Val Leu Gly Asp Thr Glu 370 375 380 Gly Leu Asn Leu Ser Phe Thr Ala Ile Cys Asn Asn Gly Thr Leu Phe 385 390 395 400 Gln His Gln Lys Lys Cys Ser His Met Lys Val Gly Asp Thr Ala Ser 405 410 415 Phe Ser Val Thr Val Asn Ile Pro His Cys Glu Arg Arg Ser Arg His 420 425 430 Ile Ile Ile Lys Pro Val Gly Leu Gly Asp Ala Leu Glu Leu Leu Val 435 440 445 Ser Pro Glu Cys Asn Cys Asp Cys Gln Lys Glu Val Glu Val Asn Ser 450 455 460 Ser Lys Cys His His Gly Asn Gly Ser Phe Gln Cys Gly Val Cys Ala 465 470 475 480 Cys His Pro Gly His Met Gly Pro Arg Cys Glu Cys Gly Glu Asp Met 485 490 495 Leu Ser Thr Asp Ser Cys Lys Glu Ala Pro Asp His Pro Ser Cys Ser 500 505 510 Gly Arg Gly Asp Cys Tyr Cys Gly Gln Cys Ile Cys His Leu Ser Pro 515 520 525 Tyr Gly Asn Ile Tyr Gly Pro Tyr Cys Gln Cys Asp Asn Phe Ser Cys 530 535 540 Val Arg His Lys Gly Leu Leu Cys Gly Gly Asn Gly Asp Cys Asp Cys 545 550 555 560 Gly Glu Cys Val Cys Arg Ser Gly Trp Thr Gly Glu Tyr Cys Asn Cys 565 570 575 Thr Thr Ser Thr Asp Ser Cys Val Ser Glu Asp Gly Val Leu Cys Ser 580 585 590 Gly Arg Gly Asp Cys Val Cys Gly Lys Cys Val Cys Thr Asn Pro Gly 595 600 605 Ala Ser Gly Pro Thr Cys Glu Arg Cys Pro Thr Cys Gly Asp Pro Cys 610 615 620 Asn Ser Lys Arg Ser Cys Ile Glu Cys His Leu Ser Ala Ala Gly Gln 625 630 635 640 Ala Arg Glu Glu Cys Val Asp Lys Cys Lys Leu Ala Gly Ala Thr Ile 645 650 655 Ser Glu Glu Glu Asp Phe Ser Lys Asp Gly Ser Val Ser Cys Ser Leu 660 665 670 Gln Gly Glu Asn Glu Cys Leu Ile Thr Phe Leu Ile Thr Thr Asp Asn 675 680 685 Glu Gly Lys Thr Ile Ile His Ser Ile Asn Glu Lys Asp Cys Pro Lys 690 695 700 Pro Pro Asn Ile Pro Met Ile Met Leu Gly Val Ser Leu Ala Ile Leu 705 710 715 720 Leu Ile Gly Val Val Leu Leu Cys Ile Trp Lys Leu Leu Val Ser Phe 725 730 735 His Asp Arg Lys Glu Val Ala Lys Phe Glu Ala Glu Arg Ser Lys Ala 740 745 750 Lys Trp Gln Thr Gly Thr Asn Pro Leu Tyr Arg Gly Ser Thr Ser Thr 755 760 765 Phe Lys Asn Val Thr Tyr Lys His Arg Glu Lys Gln Lys Val Asp Leu 770 775 780 Ser Thr Asp Cys 785 <210> 3 <211> 788 <212> PRT <213> Homo sapiens <400> 3 Met Arg Ala Arg Pro Arg Pro Arg Pro Leu Trp Val Thr Val Leu Ala 1 5 10 15 Leu Gly Ala Leu Ala Gly Val Gly Val Gly Gly Pro Asn Ile Cys Thr 20 25 30 Thr Arg Gly Val Ser Ser Cys Gln Gln Cys Leu Ala Val Ser Pro Met 35 40 45 Cys Ala Trp Cys Ser Asp Glu Ala Leu Pro Leu Gly Ser Pro Arg Cys 50 55 60 Asp Leu Lys Glu Asn Leu Leu Lys Asp Asn Cys Ala Pro Glu Ser Ile 65 70 75 80 Glu Phe Pro Val Ser Glu Ala Arg Val Leu Glu Asp Arg Pro Leu Ser 85 90 95 Asp Lys Gly Ser Gly Asp Ser Ser Gln Val Thr Gln Val Ser Pro Gln 100 105 110 Arg Ile Ala Leu Arg Leu Arg Pro Asp Asp Ser Lys Asn Phe Ser Ile 115 120 125 Gln Val Arg Gln Val Glu Asp Tyr Pro Val Asp Ile Tyr Tyr Leu Met 130 135 140 Asp Leu Ser Tyr Ser Met Lys Asp Asp Leu Trp Ser Ile Gln Asn Leu 145 150 155 160 Gly Thr Lys Leu Ala Thr Gln Met Arg Lys Leu Thr Ser Asn Leu Arg 165 170 175 Ile Gly Phe Gly Ala Phe Val Asp Lys Pro Val Ser Pro Tyr Met Tyr 180 185 190 Ile Ser Pro Pro Glu Ala Leu Glu Asn Pro Cys Tyr Asp Met Lys Thr 195 200 205 Thr Cys Leu Pro Met Phe Gly Tyr Lys His Val Leu Thr Leu Thr Asp 210 215 220 Gln Val Thr Arg Phe Asn Glu Glu Val Lys Lys Gln Ser Val Ser Arg 225 230 235 240 Asn Arg Asp Ala Pro Glu Gly Gly Phe Asp Ala Ile Met Gln Ala Thr 245 250 255 Val Cys Asp Glu Lys Ile Gly Trp Arg Asn Asp Ala Ser His Leu Leu 260 265 270 Val Phe Thr Thr Asp Ala Lys Thr His Ile Ala Leu Asp Gly Arg Leu 275 280 285 Ala Gly Ile Val Gln Pro Asn Asp Gly Gln Cys His Val Gly Ser Asp 290 295 300 Asn His Tyr Ser Ala Ser Thr Thr Met Asp Tyr Pro Ser Leu Gly Leu 305 310 315 320 Met Thr Glu Lys Leu Ser Gln Lys Asn Ile Asn Leu Ile Phe Ala Val 325 330 335 Thr Glu Asn Val Val Asn Leu Tyr Gln Asn Tyr Ser Glu Leu Ile Pro 340 345 350 Gly Thr Thr Val Gly Val Leu Ser Met Asp Ser Ser Asn Val Leu Gln 355 360 365 Leu Ile Val Asp Ala Tyr Gly Lys Ile Arg Ser Lys Val Glu Leu Glu 370 375 380 Val Arg Asp Leu Pro Glu Glu Leu Ser Leu Ser Phe Asn Ala Thr Cys 385 390 395 400 Leu Asn Asn Glu Val Ile Pro Gly Leu Lys Ser Cys Met Gly Leu Lys 405 410 415 Ile Gly Asp Thr Val Ser Phe Ser Ile Glu Ala Lys Val Arg Gly Cys 420 425 430 Pro Gln Glu Lys Glu Lys Ser Phe Thr Ile Lys Pro Val Gly Phe Lys 435 440 445 Asp Ser Leu Ile Val Gln Val Thr Phe Asp Cys Asp Cys Ala Cys Gln 450 455 460 Ala Gln Ala Glu Pro Asn Ser His Arg Cys Asn Asn Gly Asn Gly Thr 465 470 475 480 Phe Glu Cys Gly Val Cys Arg Cys Gly Pro Gly Trp Leu Gly Ser Gln 485 490 495 Cys Glu Cys Ser Glu Glu Asp Tyr Arg Pro Ser Gln Gln Asp Glu Cys 500 505 510 Ser Pro Arg Glu Gly Gln Pro Val Cys Ser Gln Arg Gly Glu Cys Leu 515 520 525 Cys Gly Gln Cys Val Cys His Ser Ser Asp Phe Gly Lys Ile Thr Gly 530 535 540 Lys Tyr Cys Glu Cys Asp Asp Phe Ser Cys Val Arg Tyr Lys Gly Glu 545 550 555 560 Met Cys Ser Gly His Gly Gln Cys Ser Cys Gly Asp Cys Leu Cys Asp 565 570 575 Ser Asp Trp Thr Gly Tyr Tyr Cys Asn Cys Thr Thr Arg Thr Asp Thr 580 585 590 Cys Met Ser Ser Asn Gly Leu Leu Cys Ser Gly Arg Gly Lys Cys Glu 595 600 605 Cys Gly Ser Cys Val Cys Ile Gln Pro Gly Ser Tyr Gly Asp Thr Cys 610 615 620 Glu Lys Cys Pro Thr Cys Pro Asp Ala Cys Thr Phe Lys Lys Glu Cys 625 630 635 640 Val Glu Cys Lys Lys Phe Asp Arg Glu Pro Tyr Met Thr Glu Asn Thr 645 650 655 Cys Asn Arg Tyr Cys Arg Asp Glu Ile Glu Ser Val Lys Glu Leu Lys 660 665 670 Asp Thr Gly Lys Asp Ala Val Asn Cys Thr Tyr Lys Asn Glu Asp Asp 675 680 685 Cys Val Val Arg Phe Gln Tyr Tyr Glu Asp Ser Ser Gly Lys Ser Ile 690 695 700 Leu Tyr Val Val Glu Glu Pro Glu Cys Pro Lys Gly Pro Asp Ile Leu 705 710 715 720 Val Val Leu Leu Ser Val Met Gly Ala Ile Leu Leu Ile Gly Leu Ala 725 730 735 Ala Leu Leu Ile Trp Lys Leu Leu Ile Thr Ile His Asp Arg Lys Glu 740 745 750 Phe Ala Lys Phe Glu Glu Glu Arg Ala Arg Ala Lys Trp Asp Thr Ala 755 760 765 Asn Asn Pro Leu Tyr Lys Glu Ala Thr Ser Thr Phe Thr Asn Ile Thr 770 775 780 Tyr Arg Gly Thr 785

Claims

1. Use of a fragment or whole of integrin αVβ6 and / or a fragment or whole of integrin αVβ3 for detecting autoantibodies in the manufacture of a test kit or test reagent for detecting ulcerative colitis, The test kit or test reagent is a test kit or test reagent for detecting autoantibodies in a sample as an indicator of ulcerative colitis, wherein the autoantibodies are antibodies that immunologically react with a fragment or the whole of integrin αVβ6 and / or antibodies that immunologically react with a fragment or the whole of integrin αVβ3, The fragment of integrin αVβ6 is a dimer comprising the following αV chain and / or β6 chain, wherein as the αV chain, an αV chain comprising a partial sequence from Phe at position 31 to Val at position 992 in the amino acid sequence of the αV chain shown in SEQ ID NO: 1; and as the β6 chain, a β6 chain comprising a partial sequence from Gly at position 22 to Asn at position 707 in the amino acid sequence of the β6 chain shown in SEQ ID NO: 2, The fragment of integrin αVβ3 is a dimer comprising the following αV chain and / or β3 chain, where, as the αV chain, it comprises a partial sequence of Phe at position 31 to Val at position 992 in the amino acid sequence of the αV chain shown in sequence number 1; and as the β3 chain, it comprises a partial sequence of Gly at position 27 to Asp at position 718 in the amino acid sequence of the β3 chain shown in sequence number 3.

2. The use according to claim 1, wherein the fragment or the whole of integrin αVβ6 and / or the fragment or the whole of integrin αVβ3 are in the form of being fixed to a solid phase.

3. Use of an antibody that immunoreacts with a fragment or the whole of integrin αVβ6 and / or an antibody that immunoreacts with a fragment or the whole of integrin αVβ3 in the manufacture of a reagent for preparing a non-human model animal of ulcerative colitis, wherein the antibody that immunoreacts with a fragment or the whole of integrin αVβ6 and / or the antibody that immunoreacts with a fragment or the whole of integrin αVβ3 are autoantibodies, and the autoantibodies are used as indicators of ulcerative colitis. The fragment of integrin αVβ6 is a dimer comprising the following αV chain and / or β6 chain, wherein as the αV chain, an αV chain comprising a partial sequence from Phe at position 31 to Val at position 992 in the amino acid sequence of the αV chain shown in SEQ ID NO: 1; and as the β6 chain, a β6 chain comprising a partial sequence from Gly at position 22 to Asn at position 707 in the amino acid sequence of the β6 chain shown in SEQ ID NO: 2, The fragment of integrin αVβ3 is a dimer comprising the following αV chain and / or β3 chain, where, as the αV chain, it comprises a partial sequence of Phe at position 31 to Val at position 992 in the amino acid sequence of the αV chain shown in sequence number 1; and as the β3 chain, it comprises a partial sequence of Gly at position 27 to Asp at position 718 in the amino acid sequence of the β3 chain shown in sequence number 3.

4. Use of a fragment or the whole of integrin αVβ6 and / or a fragment or the whole of integrin αVβ3 in the manufacture of a reagent for preparing a non-human model animal of ulcerative colitis; a fragment or the whole of integrin αVβ6 and / or a fragment or the whole of integrin αVβ3 is used to detect autoantibodies, wherein the autoantibodies are antibodies that immunologically react with a fragment or the whole of integrin αVβ6 and / or antibodies that immunologically react with a fragment or the whole of integrin αVβ3; The fragment of integrin αVβ6 is a dimer comprising the following αV chain and / or β6 chain, wherein as the αV chain, an αV chain comprising a partial sequence from Phe at position 31 to Val at position 992 in the amino acid sequence of the αV chain shown in SEQ ID NO: 1; and as the β6 chain, a β6 chain comprising a partial sequence from Gly at position 22 to Asn at position 707 in the amino acid sequence of the β6 chain shown in SEQ ID NO: 2, The fragment of integrin αVβ3 is a dimer comprising the following αV chain and / or β3 chain, where, as the αV chain, it comprises a partial sequence of Phe at position 31 to Val at position 992 in the amino acid sequence of the αV chain shown in sequence number 1; and as the β3 chain, it comprises a partial sequence of Gly at position 27 to Asp at position 718 in the amino acid sequence of the β3 chain shown in sequence number 3.

5. Use of a fragment or the whole of integrin αVβ6 for detecting autoantibodies in the manufacture of a test kit or a test reagent for detecting primary sclerosing cholangitis, wherein the test kit or the test reagent is a test kit or a test reagent for detecting autoantibodies in a sample as an indicator of primary sclerosing cholangitis, wherein the autoantibodies are antibodies that immunologically react with a fragment or the whole of integrin αVβ6, The fragment of integrin αVβ6 is a dimer comprising the following αV chain and / or β6 chain, wherein as the αV chain, it is an αV chain comprising a partial sequence from Phe at position 31 to Val at position 992 in the amino acid sequence of the αV chain shown in sequence number 1; and as the β6 chain, it is a β6 chain comprising a partial sequence from Gly at position 22 to Asn at position 707 in the amino acid sequence of the β6 chain shown in sequence number 2.

6. The use according to claim 5, wherein the fragment or the whole of integrin αVβ6 is in a form fixed to a solid phase.

7. Use of an antibody that immunoreacts with a fragment or the whole of integrin αVβ6 in the manufacture of a reagent for preparing a non-human model animal for primary sclerosing cholangitis, wherein the antibody that immunoreacts with a fragment or the whole of integrin αVβ6 is an autoantibody, and the autoantibody is used as an indicator of primary sclerosing cholangitis. The fragment of integrin αVβ6 is a dimer comprising the following αV chain and / or β6 chain, wherein as the αV chain, it is an αV chain comprising a partial sequence from Phe at position 31 to Val at position 992 in the amino acid sequence of the αV chain shown in sequence number 1; and as the β6 chain, it is a β6 chain comprising a partial sequence from Gly at position 22 to Asn at position 707 in the amino acid sequence of the β6 chain shown in sequence number 2.

8. Use of a fragment or the whole of integrin αVβ6 in the manufacture of a reagent for preparing a non-human model animal for primary sclerosing cholangitis, wherein the fragment or the whole of integrin αVβ6 is used to detect autoantibodies, wherein the autoantibodies are antibodies that react immunologically with the fragment or the whole of integrin αVβ6, The fragment of integrin αVβ6 is a dimer comprising the following αV chain and / or β6 chain, wherein as the αV chain, it is an αV chain comprising a partial sequence from Phe at position 31 to Val at position 992 in the amino acid sequence of the αV chain shown in sequence number 1; and as the β6 chain, it is a β6 chain comprising a partial sequence from Gly at position 22 to Asn at position 707 in the amino acid sequence of the β6 chain shown in sequence number 2.

Images