Methods for differentiating autoimmune hepatitis, differential antigen proteins, antibody test agents, and antibody test kits.

Abstract

The objectives are to identify disease-specific autoantibodies involved in the diagnosis or pathogenesis of autoimmune hepatitis, and to provide a diagnostic method for AIH using these antibodies. [Solution] A method for assisting in the differentiation of autoimmune hepatitis in a subject is provided, comprising the step of detecting an autoantibody derived from the subject against a differential antigen protein, wherein the differential antigen protein is the DOK2 protein.

Description

Technical Field

[0001] The present invention relates to a method for differentiating autoimmune hepatitis, an antigen protein for differentiation, an antibody test agent, and an antibody test kit.

Background Art

[0002] Autoantibodies such as antinuclear antibody and anti-smooth muscle antibody, which are detected in many cases of autoimmune hepatitis (AIH), lack disease specificity, and often cause difficulties in diagnosis in atypical cases and acute onset cases. The survival rate of AIH in non-transplanted cases of fulminant hepatitis is extremely poor, and one of the factors is the existence of difficult-to-diagnose cases. On the other hand, the etiology of AIH is considered to be mainly an autoimmune response by cytotoxic lymphocytes, and the identification of the corresponding antigen of autoantibodies is also important for elucidating the pathogenesis of this disease.

[0003] So far overseas, anti-liver kidney microsome-1 (LKM-1) antibody and anti-SLA / LP antibody have been reported as candidate autoantibodies involved in the etiology of AIH (Non-Patent Documents 1 and 2). Patent Document 1 also describes identifying a protein recognized by SLA / LP autoantibody and using the protein as a diagnostic or therapeutic agent for AIH. However, in AIH cases in Japan, the positive cases of anti-LKM-1 antibody (10%) and anti-SLA / LP antibody (15 - 20%) are few, and the search for disease-specific autoantibodies involved in the diagnosis or pathology of AIH has been an issue. Patent Document 2 also discloses using miR-21-5p, which is a miRNA, as a biomarker other than autoantibodies of AIH. However, the problems are that the cut-off value and positive rate of miR-21-5p are not shown, the expression in liver tissue has not been proven, and the measurement method of miRNA is complicated.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

[0005] [Non-Patent Document 1] Manns MP, Johnson EF, Griffin KJ, Tan EM, Sullivan KF. Major antigen of liver kidney microsomal autoantibodies in idiopathic autoimmune hepatitis is cytochrome P450db1. J Clin Invest. 1989 83:1066-72. [Non-Patent Document 2] Ballot E, Homberg JC, Johanet C. Antibodies to soluble liver antigen: an additional marker in type 1 auto-immune hepatitis. J Hepatol. 2000 33:208-15. [Overview of the project] [Problems that the invention aims to solve]

[0006] The present invention aims to identify a novel autoantibody that can be used as a marker for the differentiation of artificial intestinal hemorrhage (AIH). Autoantibody detection is advantageous compared to miRNA in that it has a clear cutoff value, a high positive rate, proven expression in liver tissue, and a simple measurement method. Furthermore, the present invention aims to provide a method for differentiating AIH using this autoantibody. [Means for solving the problem]

[0007] The inventors comprehensively detected autoantibodies from AIH patient serum using antibody profiling with a human protein microarray developed in the Fukushima Pharmaceutical and Pharmaceutical Industry Support Hub Project, and searched for disease-specific autoantibodies that contribute to diagnosis. This microarray, synthesized in a wheat germ cell-free system, carries over 16,000 types of antigen proteins, including cell membrane, cytoplasmic, and nuclear proteins, and had the potential to detect novel autoantibodies that had not been previously discovered. Using this protein microarray system, the inventors obtained and analyzed blood antibody profiles from samples of patients diagnosed with AIH and healthy individuals. As a result, they identified autoantibodies against antigens significantly present in the AIH group and used them as AIH differential markers. The present invention was completed based on the above findings and includes the following embodiments: One aspect of the present invention is, [1] A method to assist in the differential diagnosis of autoimmune hepatitis in a subject, The process includes detecting autoantibodies derived from the subject against a differential antigen protein, This invention relates to a method for assisting differentiation, wherein the differential antigen protein is the DOK2 protein. In one embodiment, the method for assisting identification according to the present invention is as follows: [2] A method to assist in the identification described in [1] above, The detection step is characterized by measuring the antibody response level of the autoantibody derived from the subject to the differential antigen protein. Furthermore, in one embodiment, the method for assisting identification according to the present invention is as follows: [3] A method to assist in the identification described in [1] or [2] above, The method further includes a step of comparing the antibody response level obtained in the measurement step with the antibody response level to the antigen protein used to differentiate autoantibodies from patients with autoimmune hepatitis or healthy individuals. Furthermore, in one embodiment, the method for assisting identification according to the present invention is as follows: [4] A method to assist in the identification described in any of [1] to [3] above, The method further includes a step of comparing the antibody response level obtained in the measurement step with the antibody response level to the antigen protein used to differentiate autoantibodies from autoimmune hepatitis patients or healthy individuals by cluster analysis. Furthermore, in one embodiment, the method for assisting identification according to the present invention is as follows: [5] A method to assist in the identification described in any of [1] to [4] above, A step of comparing the antibody response level obtained in the measurement step with a predetermined threshold. It is characterized by further including the following. Furthermore, in one embodiment, the method for assisting identification according to the present invention is as follows: [6] A method to assist in the identification described in any of [1] to [5] above, The autoantibodies derived from the subject are characterized in that they originate from the subject's bodily fluids. Furthermore, in one embodiment, the method for assisting identification according to the present invention is as follows: [7] A method to assist in the identification described in any of [1] to [6] above, In the measurement step, the antibody response level of the autoantibody against the differential antigen protein is measured by ELISA or protein microarray.

[0008] Furthermore, in another aspect, the present invention is: [8] This relates to a differential antigen protein, which is DOK2, used for the differential diagnosis of autoimmune hepatitis in a subject.

[0009] Furthermore, the present invention has manifested itself in another aspect. [9] An antibody test kit for use in the differential diagnosis of autoimmune hepatitis in a subject, The antibody test agent comprises the differential antigen protein described in claim 8, This invention relates to an antibody test kit that differentiates whether or not a person has autoimmune hepatitis by detecting autoantibodies derived from the subject against the differential antigen protein. In one embodiment, the antibody test agent of the present invention is 〔10〕The antibody test agent according to the above 〔9〕, characterized by comprising a substrate having the discrimination antigen protein immobilized on the surface thereof.

[0010] In another aspect, the present invention 〔11〕An antibody test kit for use in discriminating autoimmune hepatitis in a subject, comprising the antibody test agent according to the above 〔9〕 or 〔10〕, and at least one component selected from the group consisting of a reference antibody, a buffer solution, a preservative, a diluent, a blocking solution, a washing solution, and an instruction manual.

Advantages of the Invention

[0011] According to the present invention, compared with the conventional method for discriminating AIH, it is possible to more simply and with higher accuracy discriminate AIH, which brings great benefits to the clinical management of AIH.

Brief Description of the Drawings

[0012] [Figure 1] The antibody response levels of autoantibodies against the discrimination antigen protein of the AIH patient group and the healthy subject group measured in Example 1 below are shown as a heat map. [Figure 2] The figure shows a group scatter plot of the antibody response scores of each test sample belonging to the AIH patient group, the drug-induced liver injury (DILI) patient group, and the healthy subject group measured in Example 2 below. [Figure 3] The figure shows a ROC curve created based on the antibody response scores of each test sample belonging to the AIH patient group, the DILI patient group, or the healthy subject group measured in Example 2 below. [Figure 4] The figure is an image showing the results of immunohistochemical staining of DOK2 and CD68 in liver tissues derived from AIH patients and healthy subjects performed in Example 3 below. [Figure 5]The figure below shows a scatter plot of the antibody response scores of each test sample belonging to the AIH patient group and the healthy control group, as measured in Example 4. [Modes for carrying out the invention]

[0013] 1. Methods for differentiating autoimmune hepatitis (AIH) in test subjects 1-1. Overview A first aspect of the present invention is a method for differentiating whether or not a subject has AIH using the antibody response level of an autoantibody against a specific antigen as an indicator. Therefore, in the present invention, the autoantibody can be rephrased as an AIH differential marker. The differential method according to the first aspect of the present invention does not include the act of diagnosing AIH in humans. Therefore, the first aspect of the present invention can also be rephrased as a method to assist in differentiating whether or not a subject has AIH, or a method for testing for AIH using a sample derived from the subject.

[0014] The AIH differential marker of the present invention is an autoantibody characteristic of AIH patients that recognizes a specific protein as an antigen. In this specification, the protein recognized by the AIH differential marker is referred to as the "differential antigen protein." A sample derived from a subject contains autoantibodies, and these autoantibodies can produce an antigen-antibody reaction with the differential antigen protein. By measuring the antibody response level of the autoantibody to the differential antigen protein, it is possible to differentiate (or assist in differentiation) whether or not a subject has AIH.

[0015] 1-2.Definition In this specification, "autoimmune hepatitis" (AIH) refers to hepatitis that follows a chronic or acute hepatitis-like course, in which an autoimmune mechanism is thought to be involved in the development of liver cell damage. It has been reported to be more common in women of middle age and older.

[0016] In this specification, "AIH differential marker" refers to an autoantibody that recognizes a differential antigen protein contained in a biological sample, such as a test subject's sample, and can be used as a biomarker to differentiate whether or not a person has AIH.

[0017] In this specification, "drug-induced liver injury" (DILI) refers to liver damage caused by metabolites resulting from the metabolism of drugs in the liver. Drug-induced liver injury includes hepatocellular injury type, cholestatic type, mixed type, acute hepatic failure type, and other type liver diseases caused by drugs.

[0018] In this specification, the protein that can be used as a "differential antigen protein" is the DOK2 protein. The "differential antigen protein" may also be used in combination with other proteins useful for differentiating AIH.

[0019] The "differentiation antigen protein" is not particularly limited as long as it reacts immunologically with autoantibodies present in body fluids, and may be from humans, non-human animals, etc. From the viewpoint of improving the accuracy of detection or measurement of autoantibodies, it is preferable that the origin of the differentiation antigen protein is the same as that of the sample. For example, when using human body fluids as a sample, it is preferable that the antigen is also of human origin. The differentiation antigen protein may be recovered and purified from humans or non-human animals, or a recombinant produced by genetic engineering techniques may be used. The amino acid sequence of the differentiation antigen protein and the nucleotide sequence encoding it are publicly known. Those skilled in the art can obtain amino acid sequence or nucleotide sequence information from databases such as NCBI using the Gene ID information described in the examples, and recombinant human-derived differentiation antigen proteins can be produced by known genetic engineering techniques.

[0020] The "differential antigen protein" may be a natural-type protein, a mutant or modified protein having amino acid mutations or other modifications from the natural-type protein, or a part (fragment) of the above, as long as it can react with autoantibodies derived from the subject.

[0021] In this specification, a variant means a protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, or added to the amino acid sequence constituting the differential antigen protein, and which can react with autoantibodies derived from the test subject. Here, "several" refers to 1 to 200, 1 to 190, 1 to 150, 1 to 10, 1 to 50, 1 to 40, 1 to 30, 1 to 20, or 1 to 10 amino acid residues. A modified protein means a protein in which a chemical structure other than that of the differential antigen protein is added to the amino acid sequence of the differential antigen protein by chemical modification of a part of the amino acid sequence of the protein, or a protein in which a part of the chemical structure in the differential antigen protein is removed, and which can react with autoantibodies derived from the test subject.

[0022] In one embodiment, the DOK2 protein can be identified as the following polypeptides (a) to (c): (a) Polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 (b) Polypeptides consisting of amino acid sequences in which one or more amino acids are substituted, added, or deleted from the amino acid sequence shown in Sequence ID No. 1. (c) A polypeptide having 90% or more identity (more preferably 95%, 96%, 97%, 98%, or 99% or more) with respect to the amino acid sequence shown in SEQ ID NO: 1. Furthermore, the polypeptide identified by (b) or (c) above is a polypeptide that can react with an antigen-antibody reaction with an autoantibody derived from the test subject that specifically binds to the polypeptide in (a) above.

[0023] In this specification, "identity" refers to the percentage (%) of identical amino acids in the amino acid sequence of the polypeptide being compared relative to the total number of amino acid residues, after the two amino acid sequences have been aligned and gaps introduced as necessary to maximize the degree of agreement between the two amino acid sequences. The identity of amino acid sequences can be determined, for example, using the sequence analysis tool FASTA and its default parameters.

[0024] The differential antigen protein may be a fusion protein formed by fusing it with another protein to facilitate immobilization on the solid phase in immunological measurements. For example, it can be a fusion protein of the differential antigen protein with other proteins such as GST (glutathione-S-transferase) or streptavidin-binding peptides. The differential antigen protein may also be tagged or labeled to enable detection or measurement of antigen-antibody reactions with autoantibodies. Known methods can be used for immobilizing the protein on the solid phase and for tagging or labeling to detect or measure the antigen-antibody reaction.

[0025] "Autoantibodies" refer to antibodies present in the body of a test subject that recognize components of their own cells or tissues as antigens. In this specification, "autoantibodies" refer to antibodies present in the body of a test subject that recognize AIH differential markers as antigens.

[0026] In this specification, "antibody response score" is a score determined by the antibody response level of an autoantibody, which is an "AIH differential marker," to a differential antigen protein. The type of score and the method of calculation are not particularly limited. In one embodiment, the antibody response score can be the antibody response level of the autoantibody to the differential antigen protein used directly as the score. In another embodiment, a cutoff value for the antibody response level of the AIH differential marker can be set, and the score can be determined by comparing it with the cutoff value (for example, if the antibody response level to the differential antigen protein is equal to or greater than the cutoff value, the antibody response score is set to "1", and if it is less than the cutoff value, the antibody response score is set to "0" or "-1").

[0027] In this specification, "sample score" refers to a score determined by the antibody response level of autoantibodies against the AIH differential marker given for each test sample. There are no particular restrictions on the type of score or the calculation method. The "sample score" may be the same as or different from the "antibody response score". When antigen proteins other than DOK2 are used in combination as a "differentiation antigen protein group", for example, the "sample score" can be treated as the sum of the antibody response levels of autoantibodies against the "differentiation antigen protein group" in each test sample, or the sum of the antibody response scores.

[0028] In this specification, "measured value" refers to a value obtained by a method for measuring the antibody response level of autoantibodies against a differential antigen protein. The measured value may be treated as a quantitative value or a qualitative value. The measured value may be an absolute value expressed in weight, such as ng (nanograms) or μg (micrograms), representing the amount of antibody in the sample, or a relative value expressed as absorbance or fluorescence intensity from a labeled molecule relative to a control value.

[0029] The measured antibody response level of autoantibodies can be calculated, depending on the measurement method, as a relative ratio (antibody response ratio) to a common sample (hereinafter referred to as the "common reference"). The common reference used to calculate the antibody response ratio can be any sample, as long as the measurement conditions are the same for both samples being compared. For example, commercially available antibody mixtures such as Goat Reference Antibody Mixture I (Fukushima Protein Factory) can be used as the common reference.

[0030] In this specification, "antibody response level" includes the binding strength (affinity) of an autoantibody to the differential antigen protein, or the antibody concentration. The antibody response level as used herein can be expressed as the antibody response level to the wild-type protein of the differential antigen protein. However, it is not limited to this and may also include the antibody response level to proteins encoded by mutant genes such as point mutation genes.

[0031] 1-3.Measurement method The method for differentiating AIH in a subject according to the present invention includes the step of detecting an autoantibody derived from the subject against a differential antigen protein. More specifically, the differential antigen protein is the DOK2 protein.

[0032] In one embodiment of the method for differentiating AIH in a subject according to the present invention, the step of detecting autoantibodies derived from the subject against a differential antigen protein is a step of measuring the antibody response level of the autoantibodies derived from the subject against the differential antigen protein.

[0033] The detection and measurement methods will be explained in detail below. The "detection step" is the process of detecting the presence or absence of autoantibodies derived from the subject that recognize the differential antigen protein as an antigen (show an antibody reaction). The detection step may include a measurement step simultaneously with or after detection. Detection can be performed by ELISA, chemiluminescence, indirect immunofluorescence, or immunohistochemical staining, but is not limited to these methods. The "measurement step" is a step of measuring the antibody response level of autoantibodies derived from the test sample to the differential antigen protein and obtaining the measured value. In one embodiment, it is preferable to measure the antibody response level of the autoantibodies by measuring the antibody response level per unit amount for each differential antigen protein.

[0034] The differential antigen protein includes the DOK2 protein. Using a combination of antigen proteins other than the DOK2 protein is preferable because it improves the accuracy of differentiating the presence or absence of AIH. When selecting two or more proteins, the combination of these proteins is not limited.

[0035] In one embodiment, the combination of differential antigen proteins can be selected such that cutoff values ​​can be set for sensitivity and specificity of 70% or more, 75% or more, 80% or more, 85% or more, and 90% or more when ROC curves are drawn for the AIH patient group and the healthy control group. In one embodiment, the combination of differential antigen proteins can be selected such that cutoff values ​​can be set for sensitivity and specificity of 70% or higher, 75% or higher, 80% or higher, 85% or higher, and 90% or higher when ROC curves are drawn for the AIH patient group and the DILI group.

[0036] In this specification, "test sample" refers to a sample taken from a subject. "Subject" is not particularly limited, but may be an individual suspected of having AIH, or an individual with a history of AIH. "Individual with a history of AIH" includes patients currently suffering from AIH, and individuals with a history of AIH. Furthermore, in this specification, "subject" refers to a human individual who provides a sample and is subjected to testing.

[0037] In this specification, there are no particular restrictions on the physical conditions of "subjects," such as age, height, and weight, nor on the number of subjects. Subjects are preferably female. On the other hand, healthy individuals may be male or female, but female is more preferable. This is preferable because comparing the antibody response levels of a healthy group consisting only of women with the antibody response levels of the subjects provides a higher degree of accuracy compared to when the healthy group includes men. Furthermore, subjects are preferably Japanese (Yamato, Ryukyuan, or Ainu people), and they may reside outside of Japan if they belong to one of these ethnic groups.

[0038] The diagnostic method of the present invention can differentiate AIH even if the subject is suffering from a disease other than AIH. Examples of other diseases include, but are not limited to, hypertension, diabetes, rheumatoid arthritis, heart disease (ventricular septal defect, etc.), kidney disease (cystic kidney disease, etc.), dyslipidemia (hypercholesterolemia, etc.), appendicitis, and cerebrovascular disease (moyamoya disease, etc.). On the other hand, in one embodiment of the present invention, the diagnostic method can be used for subjects who do not suffer from any other diseases besides AIH.

[0039] In this specification, "sample" refers to a sample taken from the subject and used in the identification method of this embodiment, and includes, for example, body fluids, tissues, or cells. Here, "body fluid" refers to a liquid biological sample taken from the subject. Examples include blood (including serum, plasma, and interstitial fluid), cerebrospinal fluid, urine, lymph, saliva, sweat, mucus, tears, nasal secretions, digestive fluids, ascites, pleural fluid, periradicular fluid, and extracts of various tissues or cells. Blood, interstitial fluid, and lymph are preferred. Here, "tissue" and "cells" may originate from any part of the subject, but are preferably specimens taken by biopsy or surgical excision. Examples of biopsy tissues include, but are not limited to, biopsy tissues from the labial glands, lacrimal glands, submandibular glands, and kidneys.

[0040] If the sample is a bodily fluid, it should be collected according to known collection methods in the relevant field. For example, blood or lymph fluid should be collected according to known blood collection methods. If the sample is a tissue or cell, it should be obtained by biopsy or surgical excision.

[0041] The amount of sample required in the detection method of this embodiment is not particularly limited. If it is a body fluid such as blood or lymph, a volume of at least 0.1 mL, preferably at least 1 mL, and more preferably at least 10 mL is sufficient. If it is a tissue or cell, at least 10 μg, preferably at least 0.1 mg is desirable. Biopsy material may also be used. The sample to be tested can be prepared and processed as necessary so that the antibody reaction of autoantibodies against the differential antigen protein can be detected or measured. For example, if it is blood, serum can be recovered by centrifugation after blood collection. The method for preparing serum can be carried out in accordance with known methods. If the sample is tissue or cell, examples of processing include homogenization, cell lysis, removal of impurities by centrifugation or filtration, and addition of protease inhibitors. Details of these processing methods are described in detail in Green & Sambrook, Molecular Cloning, 2012, Fourth Ed., Cold Spring Harbor Laboratory Press, which can be used as a reference.

[0042] In this specification, "unit amount" refers to an arbitrarily defined amount of sample. For example, this could be volume (expressed as μL or mL) or weight (expressed as μg, mg, or g). While there is no specific definition of unit amount, it is preferable that the unit amount measured by a series of detection or measurement methods be constant. For example, when comparing a test sample from a healthy individual with a test sample from an AIH patient, keeping the unit amount constant allows for more accurate detection. In particular, when measuring the antibody response level of autoantibodies as an absolute value, it is necessary to keep the unit amount constant.

[0043] The following provides a detailed explanation of methods for detecting AIH differential markers and measuring antibody response levels. Methods for detecting antibodies that specifically recognize antigens, measuring binding strength (affinity) in antigen-antibody reactions, and measuring antibody concentrations of antibodies that specifically recognize antigens are well-known. While representative antibody detection and measurement methods are described below, the method is not limited to these, and known detection and measurement methods can be used.

[0044] The detection or measurement of AIH differential markers is not limited to methods that can detect the binding of subject-derived autoantibodies to differential antigen proteins or measure the antibody response level, and known antibody detection or measurement methods can be employed. Such methods are not limited to the following, but include, enzyme immunosorbent assays (ELISA, EIA), fluorescence immunoassays (FIA), radioimmunoassays (RIA), luminescence immunoassays (LIA), enzyme immunoassays, fluorescence immunoassays, immunochromatography, immunoturbidimetry, latex turbidimetry, latex agglutination assays, hemagglutination assays, particle agglutination assays, etc.

[0045] When the measurement method of the present invention is implemented using a method based on a labeling immunoassay such as enzyme immunoassay, the present invention can be applied to any method such as an indirect method, a sandwich method, or a competitive method. In the measurement method of the present invention, the measurement may be performed manually or using an analytical instrument or other device. When detecting the binding of autoantibodies to a differential antigen protein or measuring the antibody response level using an indirect method, for example, the differential antigen protein can be immobilized on a solid phase, the autoantibodies contained in the subject's sample can be reacted with the differential antigen protein, and the autoantibodies can be detected or the antibody response level measured using a labeled anti-human IgG antibody that recognizes the autoantibodies after the reaction.

[0046] 1-4.Identification method The method for assisting the differentiation of AIH in a subject according to the present invention assists in differentiating whether or not a subject has AIH based on the presence or absence of autoantibodies or antibody response levels against the differentiation antigen protein detected or measured as described above.

[0047] Herein, one embodiment of the identification method of the present invention includes a step of comparing the antibody response level of an autoantibody against a differentiation antigen protein measured in the measurement step with the antibody response level against the corresponding differentiation antigen protein in a sample derived from an AIH patient, a healthy person, or a patient with drug-induced liver injury, thereby differentiating whether or not the patient has AIH. Herein, the comparison of antibody response levels against differentiation antigen proteins includes not only the comparison of antibody response levels against a single differentiation antigen protein, but also the comparison of antibody response profiles obtained from antibody response levels against two or more differentiation antigen proteins. Herein, an antibody response profile includes multiple antibody response levels and also refers to information that aggregates multiple antibody response levels.

[0048] For comparison with the test sample, the antibody response levels or antibody response profiles of autoantibodies derived from AIH patients, healthy individuals, or patients with drug-induced liver injury may be those measured in advance, or newly detected or measured antibody response levels of autoantibodies against differential antigen proteins in samples derived from AIH patients, healthy individuals, or patients with drug-induced liver injury may be used. Therefore, in one embodiment, the differentiation method according to the present invention includes a step of detecting autoantibodies from AIH patients, healthy individuals, or patients with drug-induced liver injury against a differentiation antigen protein. Furthermore, in one embodiment, the differentiation method according to the present invention further includes a step of measuring the antibody response level between the autoantibodies from AIH patients, healthy individuals, or patients with drug-induced liver injury and the differentiation antigen protein. The antibody response level or antibody response profile (hereinafter referred to as "antibody response level, etc.") obtained by this step can be compared with the antibody response level, etc. of the test sample. The sample from AIH patients, healthy individuals, or patients with drug-induced liver injury may be a sample from a single individual or may contain samples from two or more individuals. It is preferable that the number of individuals from which the sample originates can be averaged out, thereby increasing the accuracy of detection.

[0049] In this step, comparing the antibody response level of autoantibodies against the differential antigen protein obtained in the measurement step with the antibody response level of autoantibodies against the corresponding differential antigen protein in test samples from AIH patients, healthy individuals, or patients with drug-induced liver injury, for example, if the test sample has an antibody response level equivalent to that of autoantibodies against the corresponding differential antigen protein in samples from AIH patients, the patient can be evaluated as having AIH, or if the test sample has an antibody response level different from that of autoantibodies against the corresponding differential antigen protein in samples from healthy individuals or patients with drug-induced liver injury, the patient can be evaluated as having AIH (or having a high probability of having AIH). Furthermore, if the test sample has an expression level equivalent to that of autoantibodies against the corresponding differential antigen protein in samples from healthy individuals or patients with drug-induced liver injury, it can be evaluated as not having AIH. Alternatively, if the test sample has an antibody response level different from that of autoantibodies against the corresponding differential antigen protein in samples from patients with AIH, it can be evaluated as not having AIH (or having a high probability of not having AIH).

[0050] Here, "having equivalent antibody response levels, etc." means that the antibody response levels, etc., of the autoantibodies against each protein included in the differential antigen protein group are the same or similar. Conversely, "having different antibody response levels, etc." means that the antibody response levels, etc., of the autoantibodies against each protein included in the differential antigen protein group are not similar.

[0051] Specific methods for determining whether the antibody response levels of autoantibodies against each protein in the differential antigen protein group are equivalent or different can be known methods. These methods, though not limited to the following, include, for example: (i) a method of classifying the test sample into the AIH group, the healthy control group, or the drug-induced liver injury group based on hierarchical clustering analysis; (ii) a method of evaluation by comparing antibody response levels; and (iii) a method of evaluating whether the test sample is suffering from AIH by setting a threshold.

[0052] (i) Hierarchical clustering analysis In one embodiment of the present invention, the identification method allows for the classification of test samples by performing cluster analysis on the antibody response levels of autoantibodies against the identification antigen proteins in the test samples. The data used for cluster analysis may be antibody response profiles of autoantibodies against multiple proteins included in the group of identification antigen proteins. More specifically, hierarchical cluster analysis can be performed by comparing the antibody response levels measured in the test sample with the antibody response levels to the corresponding differential antigen proteins in samples derived from AIH patients. When differentiating whether a subject providing a test sample has AIH using hierarchical clustering analysis, the following are required to create hierarchical clusters: (i) the antibody response levels to the differential antigen proteins in the test sample, and (ii) the antibody response levels to the differential antigen proteins in samples derived from AIH patients, healthy individuals, or patients with drug-induced liver injury. Hierarchical cluster analysis can employ known methods. In particular, a preferred embodiment of the present invention is cluster analysis using the group average method with Euclidean distance. Known software can be used for cluster analysis, and is not limited to the following, but commercially available software such as ExpressionView Pro software (MicroDiagnostic, Tokyo, Japan) can be used. Hierarchical cluster analysis allows us to create a hierarchical structure (tree diagram) consisting of clusters belonging to patients with AIH and clusters belonging to healthy individuals or those with drug-induced liver injury. This makes it possible to differentiate whether or not the subjects providing the test samples have AIH.

[0053] (ii) Method of evaluation by comparing antibody response levels In one embodiment, the presence or absence of AIH in a subject can be differentiated by comparing the antibody response level to a differential antigen protein in the test sample with the antibody response level to the corresponding differential antigen protein in samples from AIH patients, healthy individuals, or patients with drug-induced liver injury. While not limited to the following, one embodiment is described as follows: The antibody response level to a differential antigen protein is measured, and the antibody response levels to the differential antigen protein in AIH patients, healthy individuals, or patients with drug-induced liver injury are plotted as a group scatter plot to confirm where the antibody response level of the autoantibody (AIH differential marker) in the test sample is plotted. Based on the plotted position, it is possible to evaluate whether the subject providing the test sample is likely to have AIH. In one embodiment, if the plotted value does not have a significant difference from the antibody response level of the comparison target (or the sum of the antibody response levels) or the mean value in their group scatter plots, it can be determined that they have "equivalent antibody response levels, etc.", and if there is a significant difference, it can be determined that they have "different antibody response levels, etc."

[0054] (iii) Method of differentiation by setting a threshold In one embodiment, it is possible to differentiate whether or not a person has AIH by comparing the antibody response level to an AIH differentiation marker in the test sample with a predetermined threshold. Here, "predetermined threshold" refers to a predetermined cutoff value based on the antibody response level to the differential antigen protein in samples from AIH patients, healthy individuals, or patients with drug-induced liver injury. The cutoff value can be set, for example, as follows, but is not limited to this. That is, the antibody response level of autoantibodies to the differential antigen protein is measured in samples from AIH patients, healthy individuals, or patients with drug-induced liver injury, and the antibody response level is calculated for each sample. Then, the predetermined cutoff value can be derived by creating an ROC curve from the obtained antibody response levels. The ROC curve may be created, for example, based on the antibody response level in AIH patients and the antibody response level in healthy individuals, or based on the antibody response level in AIH patients and the antibody response level in patients with drug-induced liver injury. By setting a cutoff value, it is possible to differentiate whether or not a person has AIH, or the possibility thereof, based on whether or not the antibody response level to the AIH differential marker obtained from the test sample exceeds the cutoff value.

[0055] The ROC curve (Receiver Operating Characteristic curve) is created by plotting the true position fraction (TPF), or sensitivity, on the vertical axis and the false position fraction (FPF), or (1-specificity), on the horizontal axis, while varying the cutoff point, which is the threshold value for determining whether a test result is positive, as a mediating variable. Specificity is the rate at which negative individuals are accurately identified as negative. The method for setting the cutoff value from the created ROC curve can basically be set to increase both sensitivity and specificity (to approach 1). To achieve this, the cutoff value should be set to the value that gives the point closest to (0,1) on the ROC curve. In the most preferred embodiment, the cutoff value should be set to one that can clearly distinguish between samples from AIH patients and samples from healthy individuals or drug-induced liver injury patients.

[0056] 1-5. Effects According to the method for differentiating AIH in a subject of this embodiment, it is possible to determine whether or not a subject has AIH by examining the presence of autoantibodies against a differential antigen protein in a sample derived from the subject, or the antibody response level of said autoantibodies.

[0057] The present invention's method for assisting in the differentiation of autoimmune hepatitis can be used in combination with known diagnostic methods or criteria for differentiating autoimmune hepatitis. Examples of such diagnostic methods or criteria include detection of antinuclear antibodies or anti-smooth muscle antibodies, IgG levels, confirmation of histological interface hepatitis or plasma cell infiltration, and confirmation of the therapeutic effect of corticosteroids. Furthermore, the method for assisting in the differential diagnosis of autoimmune hepatitis according to the present invention can be carried out in combination with known diagnostic methods or diagnostic criteria for differentiating other related diseases. Other related diseases include primary biliary cholangitis, viral hepatitis, alcoholic liver disease, and drug-induced liver injury.

[0058] 2. Antigen proteins for differentiation Another aspect of the present invention provides a differential antigen protein for use in the differentiation of AIH. The differential antigen protein is as described in the first aspect.

[0059] 3. Antibody test kit Another aspect of the present invention provides an antibody test for differentiating AIH in a subject. The antibody test of the present invention comprises a differential antigen protein. In one embodiment, the antibody test of the present invention may, in addition to the differential antigen protein, include a substrate on which the differential antigen protein is immobilized.

[0060] The substrates that can be used in the present invention are not limited as long as they can have a differential antigen protein immobilized on their surface so that autoantibodies derived from the subject can recognize them. Substrates made of inert materials that do not affect biological analysis using antibodies are preferred. Examples of such substrates include inorganic materials such as metals, metal oxides, glass, quartz, silicon, and ceramics; synthetic polymers such as elastomers, plastics, polyester resins, polyethylene resins, polypropylene resins, ABS resins, nylon, acrylic resins, fluororesins, polycarbonate resins, polyurethane resins, methylpentene resins, phenolic resins, melamine resins, epoxy resins, and vinyl chloride resins; and natural polymers such as chitin, chitosan, and cellulose. The shape of the substrate is not limited and can include flat shapes such as plates, flat membranes, films, and porous membranes, or three-dimensional shapes such as cylinders, stamps, multiwell plates, microchannels, fine particles, porous carriers, and microchips. A substrate made of glass, quartz, or silicon is more preferred, and glass is even more preferred.

[0061] Techniques for immobilizing proteins onto a solid phase substrate are well known and are not limited to those that allow for antigen-antibody reactions between the differential antigen protein and the autoantibody. These techniques can be carried out by known methods such as physical adsorption, chemical binding, or combinations thereof.

[0062] By contacting a sample derived from a subject (e.g., serum) with the antibody test agent of the present invention, autoantibodies contained in the sample bind to the differential antigen protein, allowing for the detection of autoantibodies against the differential antigen protein or the measurement of the antibody response level of said autoantibodies.

[0063] 4. Antibody test kit Another aspect of the present invention provides an antibody test kit for differentiating AIH in a subject. The antibody test kit of the present invention appropriately includes, in addition to an antibody test agent, an optional component such as a reference antibody, a buffer, a preservative, a diluent, a blocking solution, a washing solution, and instructions for use.

[0064] The present invention will be described in detail below using the following examples, but the present invention is not limited to the following examples. [Examples]

[0065] (Example 1. Protein microarray) In this example, autoantibodies were comprehensively detected from the serum of AIH patients using antibody profiling with human protein microarrays, and disease-specific autoantibodies were searched for.

[0066] 1-1. Reagents, etc. (Protein microarray) As the human protein microarray, a human protein microarray containing 18,429 spots of human protein (wheat germ synthesis system) was used. P-DE1 (Fukushima Protein Factory, Cat:HC4-01-DE) was used as the protein microarray cassette.

[0067] (antibody) ·Goat Reference Antibody Mixture II (Fukushima Protein Factory,Cat:PA5011) ·Alexa Fluor 647 labeled anti-Human IgG antibody ·Cy3 labeled anti-Goat IgG antibody

[0068] (reagent) Blocking One (Nacalai tesque, Cat: 03953-95) • Solution A (10X) for protein microarrays (Fukushima Protein Factory, Cat: PA1010) • Solution B (10X) for protein microarrays (Fukushima Protein Factory, Cat: PA1110) • Final washing solution for protein microarrays (10X) (Fukushima Protein Factory, Cat: PA1210) • Primary antibody diluent (2X) for protein microarrays (Fukushima Protein Factory, Cat: PA1302) • Secondary antibody diluent (2X) for protein microarrays (Fukushima Protein Factory, Cat: PA1402)

[0069] 1-2. Preparation of serum samples Serum samples (100 μl each) were used from eight patients with autoimmune hepatitis (AIH) and ten healthy individuals. Serum samples were collected from each subject by blood sampling, separated, and frozen at -80°C.

[0070] 1-3. Protein microarrays All subsequent antibody reactions and washing / solution replacement treatments were carried out at 26°C (37°C for the primary antibody reaction) with shaking. The protein microarray was treated with ethanol and then washed with ultrapure water. To replace the solution, the protein microarray was treated with protein microarray-specific solution A. Blocking was performed for 1 hour using Blocking One. To replace the solution, the microarray was treated with protein microarray-specific solution A. 1 / 2,000 volume of Goat Reference Antibody Mixture II was added to the protein microarray-specific primary antibody diluent, and each test sample was diluted to 1 / 2,000th with this solution (primary antibody solution). 2 mL of the primary antibody solution was added to the protein microarray cassette P-DE1. The array was placed in the cassette, the lid was closed, and it was sealed with 8 dedicated clips and allowed to react for 17 hours (primary antibody reaction). After the primary antibody reaction, it was washed with protein microarray-specific solution A. To the secondary antibody diluent for protein microarrays, 1 / 500 volume of Cy3-labeled anti-Goat IgG antibody and 1 / 500 volume of Alexa Fluor 647-labeled anti-Human IgG antibody were added, and 2 mL of this solution was prepared per array (secondary antibody solution). 2 mL of the secondary antibody solution was added to the protein microarray cassette P-DE1. The array was placed in the cassette, the lid was closed, and it was sealed with 8 dedicated clips and allowed to react for 1 hour (secondary antibody reaction). After the secondary antibody reaction, the array was washed with protein microarray-specific solution A. After washing with protein microarray-specific solution B, it was washed with protein microarray-specific final washing solution. The array was centrifuged (250 G, 1,500 rpm, 1 minute), dried, and scanned with a GenePix 4000B scanner.

[0071] 1-4. Analysis and Results By combining the blood autoantibody profiles of five test samples and five control samples from healthy individuals, and extracting spots with strong autoantibody reactions, a total of 695 spots (652 genes) were identified. Next, by extracting spots that met the conditions of having zero samples with a value of 3.0 or higher in the healthy group and two or more samples with a value of 3.0 or higher in the AIH group, the DOK2 gene was successfully identified (Figure 1). Furthermore, as a verification, the three test samples were compared with five additional control samples from healthy individuals, showing lower values ​​in the healthy group and higher values ​​in the AIH group. Information on the DOK2 gene is shown in the table below. It was confirmed that the Alexa Fluor 647-labeled anti-Human IgG antibody was bound to the positive control human IgG loaded onto the protein microarray. Therefore, it was confirmed that there were no defects in the protein microarray or the test procedure.

[0072] [Table 1]

[0073] (Example 2. ELISA method using recombinant protein as antigen 1) In this example, the amount of autoantibodies was evaluated using an ELISA method with recombinant human DOK2 protein as the antigen, and autoantibodies derived from AIH patients, drug-induced liver injury (DILI) patients, and healthy controls (HC).

[0074] 2-1. Preparation of serum samples Serum samples were obtained from 25 AIH patients, 26 drug-induced liver injury patients, and 67 healthy individuals (100 μl each). Serum samples were collected from each subject by blood collection, separated, and frozen at -80°C.

[0075] 2-2.ELISA method Recombinant human DOK2 (Abnova, Taipei, Taiwan) was immobilized on a 96-well microtiter plate (F96 CERT MAXISORP, NUNC-IMMUNOPlate, Thermo Fisher Scientific, Roskilde, Denmark). Each well was coated with 0.125 μg of recombinant DOK2 in 50 μL / well of 0.1 M carbonic acid-bicarbonate buffer (pH 9.8) overnight at 4°C. Control wells without DOK2 were also assayed to measure nonspecific binding of serum samples to the wells. All procedures were performed at room temperature, except for the antigen coating step. After each step, the wells were washed four times with PBS-T. The ELISA plate was incubated for 2 hours in 200 μL / well of blocking buffer (PBS-T containing 0.05% skimmed dried milk). Serum samples (serum samples diluted 1 / 50 with blocking buffer) were added to each well in duplicate. After a 2-hour incubation, bound IgG was detected with biotin-conjugated goat F(ab′)2 anti-human IgG (diluted to 1 / 1000 with blocking buffer), followed by the addition of VECTASTAIN ABC reagent (Vector Laboratories, Burlingame, CA, USA) and incubation for 30 minutes. After further washing, 3,3′,5,5′-tetramethylbenzidine + substrate chromogen was added. 0.5M sulfuric acid was added to stop the color development, and the optical density (OD) at 450 nm was measured using a microplate reader (Benchmark Plus Microplate Spectrophotometer; Bio-Rad Laboratories, Hercules, CA, USA).

[0076] In each serum sample, specific binding of the anti-DOK2 antibody was evaluated by subtracting the mean OD value of the control well from the mean OD value of the DOK2 coat well. The obtained antibody titers were expressed in arbitrary units (AU) based on positive and negative reference serum samples set to 100 OD units and 0 OD units, respectively. The cutoff value was set at or above the mean AU value of serum samples from healthy individuals + 2 standard deviations.

[0077] 2-3.Results Figure 2 shows a scatter plot of anti-DOK2 antibody titers in each serum sample. As shown in Figure 2, the mean anti-DOK2 antibody titer in the AIH group was significantly higher than the mean anti-DOK2 antibody titers in either the HC group or the DILI group (P < 0.0001).

[0078] Furthermore, the ROC curves obtained from the AIH group and HC group are shown in Figure 3, and the ROC curves obtained from the AIH group and DILI group are also shown in Figure 3. As shown in Figure 3, the ROC curves obtained from the AIH group and HC group could be set to a cutoff value of 7.949, at which point the sensitivity was 83.98% and the specificity was 80%. The ROC curves obtained from the AIH group and DILI group could be set to a cutoff value of 7.491, at which point the sensitivity was 85.07% and the specificity was 69.23%. These results indicate that detecting anti-DOK2 antibodies in serum samples derived from the subject is useful in differentiating AIH.

[0079] (Example 3. Confirmation of DOK2 localization in liver tissue) In this example, immunohistochemical staining of DOK2 was performed on liver tissue from AIH patients and healthy individuals to confirm its localization.

[0080] 3-1.Intercept The procedure was performed on 4 μm sections obtained from formalin-fixed, paraffin-containing tissue samples.

[0081] 3-2. Antibody Reagents The following antibodies were used as primary antibodies: Dok-2 mouse monoclonal antibody (SANTA CRUZ BIOTECHNOLOGY, INK.) (Clone name: E-10, Catalog number: sc-17830, Dilution ratio: 1:25) • CD68 mouse monoclonal antibody (Nichirei Bioscience) (Clone name: PG-M1, Catalog number: AT1379-1, Dilution ratio: RTU (Ready to use))

[0082] 3-3. Immunohistochemical staining Immunohistochemical staining was performed using Nichirei's automated immunohistochemical staining system (HISTOSTAINER AT). Reagents other than the primary antibody were Nichirei's HISTOSTAINER AT-specific reagents. For the control, FFPE (Formalin Fixed Paraffin Embedded) sections of normal appendiceal tissue were used. Deparaffinization of the sections was performed using Dewax-1 for 5 minutes and Dewax-2 for 4 minutes and 45 seconds. Antigen retrieval was performed by treating with TR-pH9 at 101°C for 27 minutes. The primary antibody was reacted at 25°C for 20 minutes. Antibody detection was performed using the 3-step polymer method, each step lasting 9 minutes. DAB staining was performed for 7 minutes, followed by nuclear staining with Meyer's hematoxylin solution for 9 minutes.

[0083] 3-4.Results The results are shown in Figure 4. As shown in Figure 4, immunohistochemical staining for DOK2 revealed positive cells in the hepatic lobules of AIH patients. Furthermore, the DOK2-positive cells were found to be consistent with CD68-positive cells and were confirmed to be expressed in the cytoplasm of Kupffer cells.

[0084] (Example 4. ELISA method using recombinant protein as antigen 2) In this example, the antibody levels were evaluated using the same method as in Example 2 for autoantibodies derived from different AIH patients and HC than those in Example 2.

[0085] 4-1. Preparation of serum samples Serum samples were obtained from 80 AIH patients and 120 HC-derived serum samples (100 μl each). Serum samples were collected from each subject by blood collection, separated, and frozen at -80°C.

[0086] 4-2.ELISA method The ELISA method was performed in the same manner as in Example 2.

[0087] 4-3.Results Figure 5 shows a scatter plot of anti-DOK2 antibody titers in each serum sample. The mean anti-DOK2 antibody titer in the AIH group was 32 AU. In contrast, the mean anti-DOK2 antibody titer in the HC group was 0.6 AU. The mean anti-DOK2 antibody titer in the AIH group was significantly higher than that of the HC group (P < 0.0001).

[0088] These results were consistent with those shown in Example 2. Thus, it was confirmed that the detection of anti-DOK2 antibodies in serum samples derived from the subject is useful in differentiating AIH.

Claims

1. A method to assist in the differential diagnosis of autoimmune hepatitis in a subject, The process includes detecting autoantibodies derived from the subject against a differential antigen protein, A method to assist in differentiation, wherein the differential antigen protein is the DOK2 protein.

2. A method for assisting identification as described in claim 1, A method for assisting differentiation, wherein the detection step is a step of measuring the antibody response level of the autoantibody derived from the subject to the differential antigen protein.

3. A method for assisting identification as described in claim 2, A step of comparing the antibody response level obtained in the measurement step with the antibody response level of autoantibodies from autoimmune hepatitis patients or healthy individuals against the corresponding differential antigen protein. Methods to aid in differentiation, including further methods.

4. A method for assisting identification according to claim 2 or 3, A step of comparing the antibody response level obtained in the measurement step with the antibody response level of autoantibodies from autoimmune hepatitis patients or healthy individuals against the corresponding differential antigen protein by cluster analysis. Methods to aid in differentiation, including further methods.

5. A method for assisting identification according to claim 2 or 3, A step of comparing the antibody response level obtained in the measurement step with a predetermined threshold. Methods to aid in differentiation, including further methods.

6. A method for assisting identification as described in claim 1, A method to assist in the differentiation of autoantibodies derived from the subject as originating from the subject's bodily fluids.

7. A method for assisting identification as described in claim 1, A method for assisting differentiation, wherein in the measurement step, the antibody response level of an autoantibody against the differential antigen protein is measured by ELISA or a protein microarray.

8. A differential antigen protein, which is DOK2, used for the differential diagnosis of autoimmune hepatitis in a subject.

9. An antibody test kit for use in differentiating autoimmune hepatitis in a subject, The antibody test agent comprises the differential antigen protein described in claim 8. An antibody test kit for differentiating whether or not a person has autoimmune hepatitis by detecting autoantibodies derived from the subject against the differential antigen protein.

10. An antibody test agent according to claim 9, An antibody test agent comprising a substrate on which the aforementioned differential antigen protein is immobilized on its surface.

11. An antibody test kit for use in differentiating autoimmune hepatitis in a subject, An antibody test kit comprising the antibody test agent according to claim 9 or 10, and at least one component selected from the group consisting of a reference antibody, a buffer, a preservative, a diluent, a blocking solution, a washing solution, and instructions for use.

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