Method for diagnosing inflammatory bowel disease, diagnostic probe, and diagnostic kit

Abstract

Provided are a novel inflammatory bowel disease determination method, a diagnosis probe and a diagnosis kit. This method for determining whether inflammatory bowel disease is present includes the measurement of anti-endothelial cell protein C receptor antibodies (anti-EPCR antibodies) in a specimen collected from a test subject.

Description

[Technical field]

[0001] The present disclosure relates to methods, diagnostic probes and diagnostic kits for diagnosing inflammatory bowel disease. [Background technology]

[0002] Inflammatory bowel disease (IBD), represented by ulcerative colitis and Crohn's disease, is a disease in which inflammation occurs in the large intestine. IBD was common in Europe and the United States, but has been increasing in Japan in recent years. The number of Japanese patients with ulcerative colitis is 170,000, and the number of new patients exceeds 10,000 per year, and the incidence rate of ulcerative colitis in Japan is increasing at the same rate as in Europe and the United States. The number of Crohn's disease patients is over 40,000, and although the number of patients is not as high as ulcerative colitis, it is increasing compared to the past. Both ulcerative colitis and Crohn's disease are chronic inflammation of the intestine that occurs in relatively young people, and are intractable diseases that repeatedly relapse and go into remission.

[0003] Ulcerative colitis is a diffuse non-specific inflammation of the large intestine that mainly attacks the mucosa and causes erosions and ulcers, and it is known that immunological mechanisms form the pathology. Serum markers in the clinical setting of ulcerative colitis are non-specific inflammatory findings. It has also been reported that autoantibodies such as antineutrophil cytoplasmic antibodies and anti-smooth muscle cell antibodies are present in ulcerative colitis (Non-Patent Document 1), and although efforts have been made to identify antigens corresponding to such antibodies, the positive rates for each are low at 50% or less. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Zhou G et al., Digestive Diseases 2016;34(1-2): 90-7 Summary of the Invention [Problem to be solved by the invention]

[0005] There is a need for a marker that can specifically detect or diagnose inflammatory bowel disease and a method for detecting inflammatory bowel disease using the same.

[0006] An object of the present disclosure is to provide a method, a diagnostic probe, and a diagnostic kit that can specifically diagnose inflammatory bowel disease. [Means for solving the problem]

[0007] This disclosure includes subject matter described in the following sections.

[0008] [1] 1. A method for providing data for determining inflammatory bowel disease, comprising: Measure anti-endothelial cell protein C receptor antibodies (anti-EPCR antibodies) in samples collected from subjects Process, and comparing said measured value with a reference value; A method for diagnosing inflammatory bowel disease, comprising:

[0009] [2] contacting a sample collected from a subject with endothelial cell protein C receptor (EPCR); and The amount of anti-endothelial cell protein C receptor antibody (anti-EPCR antibody) in the sample is measured. process The method according to claim 1, comprising:

[0010] [3] The method according to [1] or [2], wherein the determination is determination of the presence or absence of inflammatory bowel disease, determination of the risk of developing inflammatory bowel disease, determination of the severity of inflammatory bowel disease, determination of the preventive effect of inflammatory bowel disease, determination of the therapeutic effect in patients suffering from inflammatory bowel disease, determination of the presence or absence of recurrence of inflammatory bowel disease, determination of the risk of recurrence of inflammatory bowel disease, determination of the presence or absence of relapse of inflammatory bowel disease, or determination of the presence or absence and risk of relapse of inflammatory bowel disease. [4] The method according to any one of [1] to [3], wherein the sample is blood, serum or plasma. [5] The method according to any of [1] to [4], wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease. [6] The method according to [1] or [2], wherein the reference value is an average value calculated from the amounts of anti-EPCR antibodies in multiple patients suffering from inflammatory bowel disease or a value greater than the average value.

[0011] [7] The determination is a determination of the presence or absence of inflammatory bowel disease, A higher value than the reference value indicates that the subject is suffering from inflammatory bowel disease. The method according to [1] or [2]. [8] The assessment is an assessment of the risk of developing inflammatory bowel disease, A higher value than the reference value indicates that the subject is at high risk of developing inflammatory bowel disease. The method according to [1] or [2].

[0012] [ 9 ] The assessment is an assessment of risk of recurrence of inflammatory bowel disease, The subject is a subject who has recovered from inflammatory bowel disease, The method according to [1] or [2], wherein a measurement value higher than the reference value indicates a high risk of recurrence of inflammatory bowel disease in the subject. [ 10 ] 1. A method for providing data for determining efficacy of treatment in a patient suffering from inflammatory bowel disease, comprising: Measuring the amount of anti-endothelial cell protein C receptor antibodies (anti-EPCR antibodies) in a sample taken from the subject prior to undergoing treatment for treating inflammatory bowel disease; measuring the amount of anti-endothelial cell protein C receptor antibodies (anti-EPCR antibodies) in a sample taken from the subject after the subject has undergone treatment for treating inflammatory bowel disease; comparing the measured values ​​of anti-endothelial cell protein C receptor antibodies (anti-EPCR antibodies) in the two samples; and The method, wherein the amount of anti-EPCR antibody in a sample taken from the subject after undergoing treatment for treating inflammatory bowel disease is lower than the amount of anti-EPCR antibody in a sample taken from the subject before undergoing treatment for treating inflammatory bowel disease, indicating that treatment of inflammatory bowel disease in the subject is effective.

[0013] [ 11 ]A diagnostic probe for inflammatory bowel disease consisting of endothelial cell protein C receptor (EPCR).

[0014] [ 12 ]A diagnostic kit for inflammatory bowel disease with endothelial cell protein C receptor (EPCR). Effect of the Invention

[0015] According to the present disclosure, inflammatory bowel disease can be specifically diagnosed. [Brief description of the drawings]

[0016] [Figure 1] The frequency and titer of anti-EPCR antibody are shown. Control: control, UC: ulcerative colitis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] 1. Subjects The subjects include not only patients suffering from inflammatory bowel disease, but also patients suspected of suffering from inflammatory bowel disease. A "subject suspected of suffering from inflammatory bowel disease" may be a person who is subjectively suspected by the subject himself (not limited to a person who has some subjective symptoms, but including a person who simply wishes to undergo a preventive medical examination), or a person based on some objective grounds (for example, a person who a doctor judges to have a reasonable possibility of suffering from inflammatory bowel disease as a result of a conventionally known clinical test (e.g., heart rate, blood pressure, blood or urine test) and / or a medical examination).

[0018] The inflammatory bowel disease is not particularly limited as long as it is a disease in which inflammation occurs in the large intestine, and is preferably ulcerative colitis or Crohn's disease, more preferably ulcerative colitis.

[0019] 2. Sample The sample collected from the subject is not particularly limited, and examples of the sample include body fluids such as blood (whole blood), serum, plasma, lymph, etc. From the viewpoint of detection accuracy, blood, serum, or plasma is preferable.

[0020] 3. Antigen EPCR Prior to the filing of the present application, disease-specific antigens corresponding to autoantibodies in inflammatory bowel disease had not been identified, and diagnosis of the disease currently relies on screening tests for inflammatory reactions, etc. Identification of autoantibodies and their corresponding antigens is useful for (1) use as diagnostic probes in clinical practice, (2) diagnosis and classification of diseases, (3) elucidation of pathology, and (4) selection of intervention targets.

[0021] Incidentally, it has been difficult to identify membrane protein autoantigens using conventional techniques for identifying proteins, such as Western blotting, two-dimensional electrophoresis, mass spectrometry, and expression libraries. However, the inventors previously constructed a Serological identification system for Autoantigens using a Retroviral vector and Flow cytometry (SARF) that enables the identification of membrane protein autoantigens (Shirai T et al., Clin Dev Immunol. 2013;2013:453058. doi: 10.1155 / 2013 / 453058).

[0022] In this study, the inventors attempted to identify the antigens corresponding to autoantibodies in vascular endothelial cells using SARF, and identified Endothelial cell Protein C Receptor (EPCR) and Scavenger Receptor Class B member I (SCARBI) as two new membrane protein autoantigens in vasculitis syndrome. Using EPCR and SCARBI as detection or diagnostic probes, the inventors measured the levels of anti-EPCR and anti-SCARBI antibodies in the serum of patients with ulcerative colitis and healthy subjects, and surprisingly found that anti-EPCR antibody activity was significantly increased in patients with ulcerative colitis compared to that in healthy subjects.

[0023] Endothelial cell protein C receptor (EPCR) is a specific receptor for protein C, which is one of the blood coagulation factors, and is a transmembrane protein. Human-derived EPCR has the amino acid sequence shown in SEQ ID NO: 1 (NP#006395.2), and the EPCR of the present disclosure can be a protein having the amino acid sequence shown in SEQ ID NO: 1 or a protein having an amino acid sequence substantially identical to the amino acid sequence shown in SEQ ID NO: 1. An amino acid sequence substantially identical to the amino acid sequence shown in SEQ ID NO: 1 refers to an amino acid sequence having about 90% or more, preferably about 95% or more, more preferably about 98% or more identity to the amino acid sequence shown in SEQ ID NO: 1. Preferably, the EPCR of the present disclosure is a protein having the amino acid sequence shown in SEQ ID NO: 1.

[0024] Furthermore, the EPCR of the present disclosure can also be a protein having an amino acid sequence in which one or more amino acids (preferably 1 to 10, more preferably 1 to 5, and even more preferably 1, 2 or 3) have been deleted, added or substituted in the amino acid sequence represented by SEQ ID NO: 1. When an amino acid sequence has been deleted, added or substituted, the position of the deletion, addition or substitution is not particularly limited as long as the activity of the protein is maintained, but is preferably a position other than the amino acid sequence corresponding to the epitope.

[0025] In addition, the "identity" of a sequence refers to the percentage of identical bases out of the total overlapping bases in the optimal alignment when two base sequences are aligned using a mathematical algorithm known in the art (preferably, the algorithm may take into account the introduction of gaps into one or both of the sequences for optimal alignment).

[0026] Other algorithms for determining the identity of base sequences include, but are not limited to, the algorithm described in Karlin S et al., Proc. Natl. Acad. Sci. USA, 90:5873-5877(1993), Needleman SB et al., J. Mol. Biol., 48-444-453(1970), Myers EW et al., CABIOS, 4:11-17(1988), and Pearson WR et al., Proc. Natl. Acad. Sci. USA, 85:2444-2448(1988).

[0027] EPCR can be prepared, for example, from cells or tissues of a subject using a known method or a method equivalent thereto, or can be chemically synthesized by a known peptide synthesis method using a peptide synthesizer or the like, or can be produced by culturing a transformant containing DNA encoding EPCR to express the protein, or can be biochemically synthesized using a cell-free transcription / translation system with a nucleic acid encoding EPCR as a template. EPCR may be modified in various ways, such as glycosylation.

[0028] 4. Anti-EPCR antibody Anti-EPCR antibodies can be identified using all or part of the amino acid sequence represented by SEQ ID NO: 1 as an antigen (preferably 4 or more consecutive amino acids, more preferably 5 or more consecutive amino acids, even more preferably 6 or more consecutive amino acids, and preferably 20 or less consecutive amino acids, more preferably 18 or less consecutive amino acids, even more preferably 15 or less consecutive amino acids, of the amino acid sequence represented by SEQ ID NO: 1).

[0029] The anti-EPCR antibody may be either a polyclonal antibody or a monoclonal antibody, and may include not only an intact antibody molecule, but also a fragment thereof, such as Fab, F(ab')2, ScFv, and minibody.

[0030] 5. Method for measuring the amount of anti-EPCR antibodies In the present disclosure, examples of immunoassays using the above-mentioned EPCR protein as an antigen include Western blot using the antigen, immunoassays such as enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay, and fluorescent immunoassay, immunoprecipitation, immunoturbidimetry, flow cytometry, and fluorescent staining, etc. These can be performed by detecting a band or spot or a peak that matches the molecular weight of the antigen, but are not limited to these.

[0031] In the case of Western blotting, proteins can be separated based on the molecular weight and isoelectric point of the antigen, so the presence or absence of an antibody can be easily determined from the position of the band size. In the case of immunoassay, cultured cells having an antigen are fixed on a solid support (e.g., a slide glass) with an appropriate solvent, and a sample from a subject is contacted to measure the amount of labeled anti-human immunoglobulin antibody, thereby quantifying the antibody of the present disclosure. In the flow cytometry method, a sample from a subject is contacted with cells in which an antigen is overexpressed, and then fluorescently labeled anti-human IgG cells are reacted to quantify the antibody of the present disclosure. In the flow cytometry method, cells are used without being fixed, so that antibody activity against the extracellular domain of an autoantigen and its higher-order structure can be quantified.

[0032] However, the method for measuring the amount of anti-EPCR antibody should not be limited to the above-mentioned method, and any method may be used as long as it detects the amount of antigen, antibody corresponding to the amount of antigen, or antibody-antigen complex in a sample by chemical or physical means, and calculates the amount of anti-EPCR antibody. In the measurement, the antigen or antibody may be bound to a labeling agent such as a radioisotope, enzyme, fluorescent substance, or luminescent substance. Examples of radioisotopes include, 125 I], [ 131 I], [ 3 H], [ 14C], etc. Examples of the enzyme include alkaline phosphatase, peroxidase, malate dehydrogenase, etc. Examples of fluorescent substances include fluorescamine, fluorescein isothiocyanate, etc. Examples of luminescent substances include luminol, luminol derivatives, luciferin, lucigenin, etc. Furthermore, a biotin-(strept)avidin system can also be used to bind an antibody to a labeling agent.

[0033] In measuring the amount of antibody, a secondary antibody against the antibody specifically bound to the antigen on the transfer membrane or solid phase can be used to enhance the reaction, and the secondary antibody is preferably labeled. As the secondary antibody, as long as it recognizes human immunoglobulin, it includes, but is not limited to, natural antibodies such as polyclonal antibodies and monoclonal antibodies (mAbs), chimeric antibodies that can be produced using gene recombination techniques, humanized antibodies, single-chain antibodies, and binding fragments thereof. Such secondary antibodies are well known in the art.

[0034] 6. Method for diagnosing inflammatory bowel disease The present disclosure encompasses a method for determining inflammatory bowel disease, comprising measuring anti-endothelial cell protein C receptor antibody (anti-EPCR antibody) in a sample collected from a subject.

[0035] Since a high proportion of patients with inflammatory bowel disease are positive for anti-EPCR antibodies, inflammatory bowel disease can be specifically determined or diagnosed in vitro by measuring the amount of anti-EPCR antibodies in a sample collected from a subject.

[0036] The assessment includes assessment of the presence or absence of inflammatory bowel disease, assessment of the risk of developing inflammatory bowel disease, assessment of the severity of inflammatory bowel disease, assessment of the preventive effect of inflammatory bowel disease, assessment of the effectiveness of treatment in patients with inflammatory bowel disease, assessment of the presence or absence of recurrence of inflammatory bowel disease, assessment of the risk of recurrence of inflammatory bowel disease, assessment of the presence or absence of relapse of inflammatory bowel disease, and assessment of the risk of relapse of inflammatory bowel disease.

[0037] When comparing measured values ​​of anti-EPCR antibodies, the comparison is made based on the presence or absence of statistically significant differences.

[0038] In one embodiment, the method for determining inflammatory bowel disease of the present invention comprises: contacting a sample obtained from a subject with endothelial cell protein C receptor (EPCR); Measuring the amount of anti-endothelial cell protein C receptor antibody (anti-EPCR antibody) in the sample; and determining that the subject is suffering from inflammatory bowel disease when the measured value is higher than a reference value; Includes.

[0039] The reference value is preferably a known amount of anti-EPCR antibody, for example, an average value calculated from the amounts of anti-EPCR antibodies in a number of patients suffering from inflammatory bowel disease, or a value larger than the average value.

[0040] If the amount of anti-EPCR antibody in the sample collected from the subject is higher than the reference value, the subject can be determined to have inflammatory bowel disease. If the amount of anti-EPCR antibody in the sample collected from the subject is the same as or lower than the reference value, the subject can be determined to not have inflammatory bowel disease. By such a determination, the presence or absence of inflammatory bowel disease can be determined.

[0041] In another embodiment, the method for determining inflammatory bowel disease of the present invention comprises: contacting a sample obtained from a subject with endothelial cell protein C receptor (EPCR); Measuring the amount of anti-endothelial cell protein C receptor antibody (anti-EPCR antibody) in the sample; and determining that the subject is at high risk of developing inflammatory bowel disease when the measured value is higher than a reference value; Includes.

[0042] The above-mentioned standard value is an average value calculated from the amounts of anti-EPCR antibodies in a group of multiple healthy subjects, or a certain value that is higher than the average value calculated from the amounts of anti-EPCR antibodies in a group of multiple healthy subjects and lower than the average value calculated from the amounts of anti-EPCR antibodies in a group of multiple patients suffering from inflammatory bowel disease.

[0043] When the amount of anti-EPCR antibody in the sample collected from the subject is higher than the reference value, the subject can be determined to have a high risk of developing inflammatory bowel disease. When the amount of anti-EPCR antibody in the sample collected from the subject is the same as or lower than the reference value, the subject can be determined to have a low risk of developing inflammatory bowel disease. By such a determination, the risk of developing inflammatory bowel disease can be determined.

[0044] In another embodiment, the method for determining inflammatory bowel disease of the present invention comprises: contacting a sample obtained from a subject with endothelial cell protein C receptor (EPCR); Measuring the amount of anti-endothelial cell protein C receptor antibody (anti-EPCR antibody) in the sample; and A step of comparing the measured value with a reference value to determine the severity of the subject's inflammatory bowel disease. Includes.

[0045] The above-mentioned reference value may be an average value calculated from the amounts of anti-EPCR antibodies in multiple patients suffering from inflammatory bowel disease of a certain severity.

[0046] When the measured value of the subject is higher than the above average value, it can be determined that the subject's inflammatory bowel disease is highly likely to fall under the severity level. When the measured value of the subject is equal to or lower than the above average value, it can be determined that the subject's inflammatory bowel disease is low likely to fall under the severity level. By such a determination, the severity of the inflammatory bowel disease can be determined.

[0047] In another embodiment, the method for determining inflammatory bowel disease of the present invention comprises: contacting a sample taken from the subject before receiving a treatment for preventing inflammatory bowel disease and a sample taken from the subject after receiving a treatment for preventing inflammatory bowel disease with endothelial cell protein C receptor (EPCR); Measuring the amount of anti-endothelial cell protein C receptor antibody (anti-EPCR antibody) in the two samples; and A step of comparing the measured values ​​of the two samples to determine the preventive effect of inflammatory bowel disease in the subject. Includes.

[0048] When the amount of anti-EPCR antibody in a sample collected from a subject after undergoing measures to prevent inflammatory bowel disease is lower than the amount of anti-EPCR antibody in a sample collected from a subject before undergoing measures to prevent inflammatory bowel disease, it can be determined that there is a preventive effect against inflammatory bowel disease in the subject. When the amount of anti-EPCR antibody in a sample collected from a subject after undergoing measures to prevent inflammatory bowel disease is the same as or higher than the amount of anti-EPCR antibody in a sample collected from a subject before undergoing measures to prevent inflammatory bowel disease, it can be determined that there is no preventive effect against inflammatory bowel disease in the subject. Measures to prevent inflammatory bowel disease include administration of anti-inflammatory drugs such as 5-aminosalicylic acid preparations and steroid preparations, immunosuppressants, anti-TNFα antibodies, exercise therapy, diet therapy, etc. By such a judgment, the preventive effect against inflammatory bowel disease can be determined.

[0049] In another embodiment, the method for determining inflammatory bowel disease of the present invention comprises: contacting a sample taken from the subject prior to undergoing treatment for treating inflammatory bowel disease and a sample taken from the subject after undergoing treatment for treating inflammatory bowel disease with endothelial cell protein C receptor (EPCR); Measuring the amount of anti-endothelial cell protein C receptor antibody (anti-EPCR antibody) in the two samples; and A step of comparing the measured values ​​of the two samples to determine the efficacy of treatment of inflammatory bowel disease in the subject. Includes.

[0050] When the amount of anti-EPCR antibody in a sample collected from a subject after undergoing treatment for treating inflammatory bowel disease is lower than the amount of anti-EPCR antibody in a sample collected from a subject before undergoing treatment for treating inflammatory bowel disease, it can be determined that the treatment of inflammatory bowel disease in the subject is effective. When the amount of anti-EPCR antibody in a sample collected from a subject after undergoing treatment for treating inflammatory bowel disease is the same as or higher than the amount of anti-EPCR antibody in a sample collected from a subject before undergoing treatment for treating inflammatory bowel disease, it can be determined that the treatment of inflammatory bowel disease in the subject is ineffective. Measures for treating inflammatory bowel disease include surgical operations such as vascular bypass surgery, administration of anti-inflammatory drugs such as 5-aminosalicylic acid preparations and steroid preparations, immunosuppressants, and drugs such as anti-TNFα antibodies, exercise therapy, diet therapy, etc. By such a judgment, the treatment effect of inflammatory bowel disease can be determined.

[0051] In another embodiment, the method for determining inflammatory bowel disease of the present invention comprises: contacting a sample obtained from a subject who has recovered from inflammatory bowel disease with an endothelial cell protein C receptor (EPCR); Measuring the amount of anti-endothelial cell protein C receptor antibody (anti-EPCR antibody) in the sample; and determining that the subject is experiencing a recurrence of inflammatory bowel disease when the measured value is higher than a reference value; Includes.

[0052] The reference value is preferably a known amount of anti-EPCR antibody, for example, an average value calculated from the amounts of anti-EPCR antibodies in a number of patients suffering from inflammatory bowel disease, or a value larger than the average value.

[0053] When the amount of anti-EPCR antibody in a sample collected from a subject who has been cured of inflammatory bowel disease is higher than the reference value, it can be determined that the inflammatory bowel disease in the subject has recurred. When the amount of anti-EPCR antibody in a sample collected from a subject who has been cured of inflammatory bowel disease is equal to or lower than the reference value, it can be determined that the inflammatory bowel disease in the subject has not recurred. Measures for treating inflammatory bowel disease include surgical operations such as vascular bypass surgery, administration of anti-inflammatory drugs such as 5-aminosalicylic acid preparations and steroid preparations, immunosuppressants, anti-TNFα antibodies, exercise therapy, diet therapy, etc. By such a determination, the presence or absence of recurrence of inflammatory bowel disease can be determined.

[0054] In another embodiment, the method for determining inflammatory bowel disease of the present invention comprises: contacting a sample obtained from a subject who has recovered from inflammatory bowel disease with an endothelial cell protein C receptor (EPCR); Measuring the amount of anti-endothelial cell protein C receptor antibody (anti-EPCR antibody) in the sample; and A step of comparing the measured value of the sample with a reference value to determine the risk of recurrence of inflammatory bowel disease in the subject. Includes.

[0055] The above-mentioned reference value includes a known amount of anti-EPCR antibody, for example, an average value calculated from the amount of anti-EPCR antibody in multiple patients suffering from inflammatory bowel disease or a greater value thereof, a value calculated from the amount of anti-EPCR antibody at the time of complete recovery in a subject who has been cured of the above-mentioned inflammatory bowel disease or a greater value thereof, or an average value calculated from the amount of anti-EPCR antibody at the time of complete recovery in multiple patients who have been cured of the above-mentioned inflammatory bowel disease or a greater value thereof.

[0056] When the amount of anti-EPCR antibody in a sample collected from a subject who has been cured of inflammatory bowel disease is higher than the reference value, it can be determined that the subject has a high risk of recurrence of inflammatory bowel disease. When the amount of anti-EPCR antibody in a sample collected from a subject who has been cured of inflammatory bowel disease is equal to or lower than the reference value, it can be determined that the subject has a low risk of recurrence of inflammatory bowel disease. Measures for treating inflammatory bowel disease include surgical operations such as vascular bypass surgery, administration of anti-inflammatory drugs such as 5-aminosalicylic acid preparations and steroid preparations, immunosuppressants, anti-TNFα antibodies, exercise therapy, diet therapy, etc. By such a determination, the risk of recurrence of inflammatory bowel disease can be determined.

[0057] In another embodiment, the method for determining inflammatory bowel disease of the present invention comprises: contacting each sample taken from the subject after discontinuing a treatment for treating inflammatory bowel disease with an endothelial cell protein C receptor (EPCR); Measuring the amount of anti-endothelial cell protein C receptor antibody (anti-EPCR antibody) in the sample; and A step of comparing the measured value of the sample with a reference value to determine whether or not the subject has relapsed in inflammatory bowel disease. Includes.

[0058] The above-mentioned standard value includes a certain value higher than the average value calculated from the amounts of anti-EPCR antibodies in a group of multiple healthy individuals, or the average value calculated from the amounts of anti-EPCR antibodies in multiple patients suffering from inflammatory bowel disease.

[0059] If the amount of anti-EPCR antibody in the sample collected from the subject after the treatment for inflammatory bowel disease is discontinued is higher than the amount of anti-EPCR antibody in the sample collected from the subject after the treatment for inflammatory bowel disease is discontinued, it can be determined that the subject has relapsed. If the amount of anti-EPCR antibody in the sample collected from the subject after the treatment for inflammatory bowel disease is the same as or lower than the reference value, it can be determined that the subject has not relapsed. Measures for treating inflammatory bowel disease include surgical operations such as vascular bypass surgery, administration of anti-inflammatory drugs such as 5-aminosalicylic acid preparations and steroid preparations, immunosuppressants, anti-TNFα antibodies, exercise therapy, diet therapy, etc. By such a judgment, the presence or absence of relapse of inflammatory bowel disease can be determined.

[0060] In another embodiment, the method for determining inflammatory bowel disease of the present invention comprises: contacting a sample taken from a subject undergoing treatment for treating inflammatory bowel disease with endothelial cell protein C receptor (EPCR), and a sample taken from the subject after the treatment for treating inflammatory bowel disease has been discontinued; Measuring the amount of anti-endothelial cell protein C receptor antibody (anti-EPCR antibody) in the two samples; and comparing the measurements from the two samples to determine the risk of relapse of inflammatory bowel disease in the subject. Includes.

[0061] When the amount of anti-EPCR antibody in a sample collected from a subject after the treatment for inflammatory bowel disease is discontinued is higher than the reference value, it can be determined that the subject has a high risk of relapse of inflammatory bowel disease. When the amount of anti-EPCR antibody in a sample collected from a subject after the treatment for inflammatory bowel disease is discontinued is equal to or lower than the reference value, it can be determined that the subject has a low risk of relapse of inflammatory bowel disease. Measures for treating inflammatory bowel disease include surgical procedures such as vascular bypass surgery, administration of anti-inflammatory drugs such as 5-aminosalicylic acid preparations and steroid preparations, immunosuppressants, anti-TNFα antibodies, exercise therapy, diet therapy, etc. By such a determination, the risk of relapse of inflammatory bowel disease can be determined.

[0062] 7. Diagnostic Probes The present disclosure also includes a diagnostic probe for inflammatory bowel disease that consists of endothelial cell protein C receptor (EPCR).

[0063] The inflammatory bowel disease is not particularly limited as long as it is a disease in which inflammation occurs in the large intestine, and is preferably ulcerative colitis or Crohn's disease, more preferably ulcerative colitis.

[0064] EPCR can be a protein having the amino acid sequence shown in SEQ ID NO: 1 or a protein having an amino acid sequence substantially identical to the amino acid sequence shown in SEQ ID NO: 1. An amino acid sequence substantially identical to the amino acid sequence shown in SEQ ID NO: 1 refers to an amino acid sequence having about 90% or more, preferably about 95% or more, and more preferably about 98% or more identity to the amino acid sequence shown in SEQ ID NO: 1. Preferably, the EPCR of the present disclosure is a protein having the amino acid sequence shown in SEQ ID NO: 1.

[0065] EPCR can also be a protein having an amino acid sequence in which one or more amino acids (preferably 1 to 10, more preferably 1 to 5, and even more preferably 1, 2 or 3) have been deleted, added or substituted in the amino acid sequence represented by SEQ ID NO: 1. When an amino acid sequence has been deleted, added or substituted, the position of the deletion, addition or substitution is not particularly limited as long as the activity of the protein is maintained, but is preferably a position other than the amino acid sequence corresponding to the epitope.

[0066] 8. Diagnostic kits The present disclosure also includes a diagnostic kit for inflammatory bowel disease with endothelial cell protein C receptor (EPCR).

[0067] The inflammatory bowel disease is not particularly limited as long as it is a disease in which inflammation occurs in the large intestine, and is preferably ulcerative colitis or Crohn's disease, more preferably ulcerative colitis.

[0068] EPCR can be a protein having the amino acid sequence shown in SEQ ID NO: 1 or a protein having an amino acid sequence substantially identical to the amino acid sequence shown in SEQ ID NO: 1. An amino acid sequence substantially identical to the amino acid sequence shown in SEQ ID NO: 1 refers to an amino acid sequence having about 90% or more, preferably about 95% or more, and more preferably about 98% or more identity to the amino acid sequence shown in SEQ ID NO: 1. Preferably, the EPCR of the present disclosure is a protein having the amino acid sequence shown in SEQ ID NO: 1.

[0069] EPCR can also be a protein having an amino acid sequence in which one or more amino acids (preferably 1 to 10, more preferably 1 to 5, and even more preferably 1, 2 or 3) have been deleted, added or substituted in the amino acid sequence represented by SEQ ID NO: 1. When an amino acid sequence has been deleted, added or substituted, the position of the deletion, addition or substitution is not particularly limited as long as the activity of the protein is maintained, but is preferably a position other than the amino acid sequence corresponding to the epitope.

[0070] In addition to EPCR, the diagnostic kit may contain other substances necessary for a reaction to detect the amount of antibody, which do not adversely affect the reaction when stored in the presence of the other substances, such as a color-developing reagent, a labeled secondary antibody, and various reagents such as a blocking agent. The diagnostic kit may further contain a buffer solution, a washing solution, an instruction manual, and the like.

[0071] The antigen protein, EPCR, is preferably provided in a state of being immobilized on a suitable solid support. Examples of the solid support include various carriers made of insoluble polysaccharides (e.g., agarose, dextran, cellulose, etc.), synthetic resins, glass, metals, etc., which are used in conventional antigen-antibody reactions, such as microplates, tubes, membranes, columns, beads, sensor chips, etc. Immobilization may be performed by physical adsorption, or by chemical bonding, which is usually used for insolubilizing and immobilizing proteins.

[0072] The diagnostic kit of the present disclosure can be applied to clinical tests such as the diagnosis or monitoring of inflammatory bowel disease, and can be used in drug discovery research targeting signal transduction by EPCR.

[0073] The disclosures of all patent applications and publications cited herein are hereby incorporated by reference in their entireties.

[0074] Although the embodiments of the present disclosure have been specifically described above, the present disclosure is not limited to the above-described embodiments, and various modifications based on the technical ideas of the present disclosure are possible.

[0075] The present disclosure will be described more specifically below with reference to examples, but the present invention is not limited to these. EXAMPLES

[0076] 1. Subjects At Tohoku University Hospital, blood samples were collected from 36 patients diagnosed with ulcerative colitis according to the diagnostic criteria for ulcerative colitis (revised on January 21, 2017) described in the "Survey on Refractory Inflammatory Bowel Disorders" policy research project for intractable diseases funded by the Ministry of Health, Labor and Welfare Science Research Grant, and from 79 healthy controls. Blood was drawn from the subjects' veins and centrifuged in the usual way to obtain serum, which was then stored in a freezer until use.

[0077] 2. Flow Cytometry and Antibody Binding Experiments The DNA sequence of EPCR (SEQ ID NO: 2) was amplified by PCR and inserted into the pMX-GFP vector. The retrovirus vectors with EPCR-GFP inserted were transfected into Plat E cells using Fugene to create retroviruses with EPCR-GFP inserted sequences. The retroviruses were then infected into rat myeloma cells to establish cells that forcibly express EPCR protein. Each EPCR protein-expressing cell expressed GFP, and after mixing with GFP-negative cells, it was reacted with serum-derived IgG as the primary antibody and PE (phycoerythrin)-anti-human IgG antibody as the secondary antibody. Next, anti-EPCR antibody activity was measured by comparing the fluorescence intensity of PE between the GFP-positive and negative groups using FACS.

[0078] Anti-EPCR antibody titers were measured in ulcerative colitis patients and healthy subjects (Figure 1). The vertical axis represents the mean fluorescence intensity (MFI) ratio of serum IgG binding to EPCR-expressing cells relative to binding to EPCR non-expressing cells. Anti-EPCR antibodies were detected in 25 of 36 ulcerative colitis patients, or approximately 70%, but not in healthy subjects. The amount of anti-EPCR antibodies in ulcerative colitis patients was significantly higher than that in healthy subjects (Mann-Whitenny test).

[0079] The expression of anti-SCARBI antibodies in subjects was also examined, but no significant difference was found between ulcerative colitis patients and healthy subjects (data not shown).

Claims

1. 1. A method for providing data for determining inflammatory bowel disease, comprising: Measuring anti-endothelial cell protein C receptor antibodies (anti-EPCR antibodies) in a sample collected from a subject; and comparing said measured value with a reference value; The method includes:

2. contacting a sample obtained from a subject with an endothelial cell protein C receptor (EPCR); The method of claim 1, further comprising a step of measuring the amount of anti-endothelial cell protein C receptor antibody (anti-EPCR antibody) in the sample.

3. The method according to claim 1 or 2, wherein the determination is determination of the presence or absence of inflammatory bowel disease, determination of the risk of developing inflammatory bowel disease, determination of the severity of inflammatory bowel disease, determination of the preventive effect of inflammatory bowel disease, determination of the therapeutic effect in patients suffering from inflammatory bowel disease, determination of the presence or absence of recurrence of inflammatory bowel disease, determination of the risk of recurrence of inflammatory bowel disease, determination of the presence or absence of relapse of inflammatory bowel disease, or determination of the presence or absence and risk of relapse of inflammatory bowel disease.

4. The method according to any one of claims 1 to 3, wherein the sample is blood, serum or plasma.

5. The method according to any one of claims 1 to 4, wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease.

6. The method according to claim 1 or 2, wherein the reference value is an average value calculated from the amounts of anti-EPCR antibodies in a plurality of patients suffering from inflammatory bowel disease or a value greater than the average value.

7. The determination is a determination of the presence or absence of inflammatory bowel disease, A higher value than the reference value indicates that the subject is suffering from inflammatory bowel disease. The method according to claim 1 or 2.

8. The assessment is an assessment of the risk of developing inflammatory bowel disease, A higher value than the reference value indicates that the subject is at high risk of developing inflammatory bowel disease. The method according to claim 1 or 2.

9. The assessment is an assessment of risk of recurrence of inflammatory bowel disease, The subject is a subject who has recovered from inflammatory bowel disease, The method of claim 1 or 2, wherein the measured value being higher than the reference value indicates an increased risk of recurrence of inflammatory bowel disease in the subject.

10. 1. A method for providing data for determining efficacy of treatment in a patient suffering from inflammatory bowel disease, comprising: measuring the amount of anti-endothelial cell protein C receptor antibodies (anti-EPCR antibodies) in a sample taken from the subject prior to undergoing treatment for treating inflammatory bowel disease; measuring the amount of anti-endothelial cell protein C receptor antibodies (anti-EPCR antibodies) in a sample taken from the subject after the subject has undergone treatment for treating inflammatory bowel disease; comparing the measured values ​​of anti-endothelial cell protein C receptor antibodies (anti-EPCR antibodies) in the two samples; and The method, wherein the amount of anti-EPCR antibody in a sample taken from the subject after undergoing treatment for treating inflammatory bowel disease is lower than the amount of anti-EPCR antibody in a sample taken from the subject before undergoing treatment for treating inflammatory bowel disease, indicating that treatment of inflammatory bowel disease in the subject is effective.

11. A diagnostic probe for inflammatory bowel disease consisting of endothelial cell protein C receptor (EPCR).

12. Diagnostic kit for inflammatory bowel disease with endothelial cell protein C receptor (EPCR).

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