JPH1082788A - Diagnosis medicine for autoimmune disease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a diagnosis medicine for autoimmune disease and a kit thereof, and to detect an antibody thereof, by using polypeptide selected from the HGM-1 family, or the HGM-2 family, or an antigen including a kind of a piece of the polypeptide that can react with the antibody of a patient having an autoimmune disease. SOLUTION: This medicine can be applied to an autoimmune disease such as chronic joint rheumatism, systemic erythematodes and primary biliary cirrhosis. Peptide is selected from HMG-1 or HMG-2 of a man, a cow, a pig, chicken, a mouse or a rat. Polypeptide is one with deficiency, conversion or addition of one or more amino-acid and includes polypeptide or a piece thereof that can react with an antigen of a patient having the autoimmune disease. The polypeptide is made form a tissue or a cultured cell or made by installing a vector into which a gene coding polypeptide is incorporated to a host cell to create the polypeptide.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a high mobility group protein-1 (HMG) to which an antibody of an autoimmune disease patient reacts.
-1) A diagnostic agent or a diagnostic kit for an autoimmune disease using high mobility group protein-2 (HMG-2) or a fragment thereof, and a method for detecting an antibody in an autoimmune disease patient Things.
In particular, rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, Behcet's disease, scleroderma, primary biliary cirrhosis, microscopic polyangiitis / polyarteritis nodosa,
Antibody reacts to patients with ulcerative colitis and Crohn's disease
Rheumatoid arthritis, systemic lupus erythematosus using HMG-1, HMG-2 or fragments of their polypeptides,
Diagnostic agent or kit for Sjogren's syndrome, Behcet's disease, scleroderma, primary biliary cirrhosis, microscopic polyangitis / polyarteritis nodosa, ulcerative colitis, and Crohn's disease, and the patient's The present invention relates to a method for detecting an antibody.

[0002]

2. Description of the Related Art It has been reported that various anti-neutrophil cytoplasmic antibodies (ANCA) exist in autoimmune diseases and inflammatory diseases. ANCA is an autoantibody detected by indirect immunofluorescence (IIF), and cytoplasmic-ANCA (cANCA) and perinuclear-ANCA (pANCA)
Is distinguished. cANCA is detected as frequently as 80% in Wegener's granulomatosis, and more than 90% of its antigens are proteinases.
-3 (proteinase-3: PR-3). On the other hand, pANCA is microscopic polyarteritis, pauci-imm
une-type necrotizing crescent-forming nephritis
c gromerulonephritis (NCGN) with a high frequency of 80%, and its antigen is 80% myelopoxidase (myerop
eroxidase (MPO-ANCA). Thus, early diagnosis and differential diagnosis of vasculitis syndrome can be made by measuring antibodies having high disease specificity.

Recently, chronic inflammatory bowel disease (IBD) such as ulcerative colitis (UC), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE),
PANCA has been recognized in patients with various inflammatory diseases such as autoimmune hepatitis (AIH), malignant tumor, amoeba abscess, and Sweet's disease. Lactoferrin, cathepsin G, elastase, lysozyme, etc. have been identified as antigens of pANCA, and their relation to etiology and pathological conditions has been studied.
The specificity for A is low, suggesting the presence of other antigens.

[0004] In the inflammatory bowel diseases ulcerative colitis (UC) and Crohn's disease (CD), the ratio (positive rate) of detection of these anti-neutrophil cytoplasmic antibodies (ANCA) by the indirect fluorescent antibody method is as follows. They are 40-87% and 6-27%, respectively. The staining pattern is pANCA 80-9 for ulcerative colitis
PANCA and cA in Crohn's disease, compared to 5%
NCA is evenly detected. On the other hand, in rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome
Mostly pANCA, 33% (4%), 43% (2%), 50
% (8%) (the percentage in parentheses is the positive rate of cANCA).

[0005] Lactoferrin (Lactoferin) is an antigen corresponding to ANCA detected in ulcerative colitis and Crohn's disease.
errin), cathepsin G, myeloperoxidase and myeloperoxidase + elastase, myeloperoxidase + elastase + cathepsin G, and various reports have been reported. At present, the corresponding antigen has not yet been determined. Antigens corresponding to other pANCA-positive diseases have not been identified as well.

The standard clinical diagnosis of ulcerative colitis and Crohn's disease includes endoscopy and X-ray examination. These tests have the disadvantage of being expensive, painful, and long detained for the patient. On the other hand, detection of pANCA by indirect fluorescent antibody method has recently been reported as a serodiagnosis of ulcerative colitis. However, this method does not have high sensitivity and tends to have a high background.Furthermore, since neutrophils or other cells are immobilized on a plate with ethanol, the results may vary depending on the state of the cells and immobilization techniques. It has the disadvantage of being unreliable and is not yet commonly used. No autoantibodies have been found for Crohn's disease. As described above, at present, no specific and simple method for diagnosing ulcerative colitis or Crohn's disease using patient serum has been developed.

Various autoantibodies such as rheumatoid factor and antinuclear antibody are produced in rheumatoid arthritis and systemic lupus erythematosus, but are detected in scleroderma, polymyositis, Behcet's disease, periarteritis nodosa and the like. There are few autoantibodies, and clinical symptoms dominate the diagnosis. For these diseases, diagnose the disease at an early stage,
It has been pointed out that the prognosis is important to give sufficient treatment to prevent progression to a mature disease, but a simple serological diagnosis method for early diagnosis has not been developed.

In general, when an autoimmune disease is suspected, a test for antinuclear antibodies is used as a diagnostic method in primary screening. Antinuclear antibodies are a general term for autoantibodies that use cell nucleus nucleic acids and various nucleoprotein components as antigens, and their types are diverse. As a method for detecting an antinuclear antibody, an indirect fluorescent antibody method is mainly used. As antinuclear antibodies,
Anti-DNA antibody, anti-histone antibody, anti-E
An NA antibody, an anti-centromere antibody, an anti-nucleolar antibody, etc. are presumed. However, it has been pointed out that the measurement of the antinuclear antibody by the indirect fluorescent antibody method has many problems in keeping the accuracy constant. For example, nuclear materials (cells) vary from facility to facility, calibration curves cannot be drawn, and autoantibodies are heterogeneous. The biggest disadvantage is that this test method is determined by visual observation. Therefore, the judgment criterion and the judgment technique are non-objective.

However, the measurement of antinuclear antibodies by the indirect fluorescent antibody method is indispensable for diagnosing various clinical diseases, such as systemic lupus erythematosus, and for understanding the clinical picture, despite these disadvantages. . On the other hand, autoantibodies (self-antigens) which are replaced by antinuclear antibodies for the reasons described above
It is also a fact that a simple primary screening method for autoimmune diseases, which is simple and uses no difference between facilities, is desired.

[0010]

Therefore, identifying an autoantigen in a patient with an autoimmune disease and detecting an autoantibody using the autoantigen requires confirmation of diagnosis as an autoimmune disease and appropriate treatment. It paves the way for establishing a policy.
Therefore, identification and isolation of a common self-antigen appearing in an autoimmune disease, and development of a simple antibody detection method using this antigen are desired.

[0011]

Means for Solving the Problems The present inventors have conducted intensive studies in order to overcome the above-mentioned conventional problems, and as a result, have found that antibodies in the serum of patients with autoimmune diseases, particularly pANCA-positive ulcerative colitis, have been obtained. Using this protein, high mobility group protein-1 (H
MG-1) and high mobility group protein-2 (HMG-2)
For the first time. By constructing an ELISA system using this HMG-1 and HMG-2, this antigen, rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, Behcet's disease, scleroderma, primary biliary cirrhosis, microscopy Polyclonal arthritis / polyarteritis nodosa, ulcerative colitis, and Crohn's disease show high rates of antibodies, and this ELISA system compares to the indirect immunofluorescence assay for antinuclear antibodies Showing that it is relatively sensitive, simple, reliable and objective, and that the detection of antibodies against the antigen can be used to detect rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, Behcet's disease, scleroderma, primary Biliary cirrhosis, microscopic polyangitis / polyarteritis nodosa, ulcerative colitis,
The inventors have found that the marker can be used as a diagnostic marker for Crohn's disease patients and the like, and have completed the present invention.

The HMG antigen has not yet been measured as an anti-nuclear antibody in autoimmune diseases but not as an antigen of ANCA. Dennis JS and colleagues report 10.3% anti-HMG-1 antibody and 6.9% anti-HMG-2 antibody in patients with systemic lupus erythematosus, and 0% for both antibodies in mixed connective tissue disease and rheumatoid arthritis. Science 215, 1245-1247, 1982). In addition, Briolay J. et al. Tested immunoblotting for systemic lupus erythematosus, rheumatoid arthritis, and scleroderma.
As a result of detecting HMG-1 antibody and anti-HMG-2 antibody, both antibodies are reported to have no diagnostic value (Autoimmunity
2, 165-176, 1989). There may be problems with HMG antigen purity, ELISA, immunoblotting, and other techniques, as well as the condition of the patient and the number of samples measured.At this point, HMG-1 and HMG-1
The invention of a diagnostic drug using -2 has not been completed. But anti
HMG-1 antibody and anti-HMG-2 antibody-positive cases are reported to be positive for anti-HMG-1 and / or anti-HMG-2 antibodies in 39% of antinuclear antibody-positive juvenile rheumatic patients (Wittemann B et al.) ,
Arthritis and Rheumatism 33, 1378-1383, 1990). In the present invention, an ELISA system was constructed using high-purity HMG-1 and HMG-2, and a positive rate was measured in various diseases. As a result, a statistically significant difference was observed in the above nine diseases as compared with a healthy person. As a result, the present invention has been completed.

[0013] The present invention includes at least one kind of a polypeptide selected from the HMG-1 family, a polypeptide selected from the HMG-2 family, or a fragment thereof, which can react with an antibody of an autoimmune disease patient. The present invention relates to a diagnostic agent for an autoimmune disease, a kit for diagnosing these diseases, and a method for detecting an antibody in a patient with an autoimmune disease.

[0014] In a preferred embodiment, the autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus,
Sjogren's syndrome, Behcet's disease, scleroderma, primary biliary cirrhosis, microscopic polyangitis / polyarteritis nodosa, ulcerative colitis, and Crohn's disease.

In a preferred embodiment, the polypeptide is selected from human, bovine, porcine, chicken, mouse, and rat HMG-1 or HMG-2.

This achieves the object of the present invention.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION HMG (high mobility group prote
in) was discovered in 1964 as a large amount of non-histone proteins contained in the chromatin structure, and is a protein that is universally contained in all higher plants and animals. In addition, it is known that abundance exists not only in the nucleus but also in the cytoplasm. Although the physiological effect is not clearly understood, HMG binds to DNA, but is not specific to the base sequence when binding to DNA, but relaxes the double helix structure.
It is thought to function as an extremely wide range of transcription promoters and nucleosome relaxing factors, which increase the transcriptional activity by changing the higher order structure of E. coli to an optimal structure. There are several types of HMG.

The polypeptide used in the present invention is HM
It is selected from polypeptides contained in the G-1 family or HMG-2 family, or fragments thereof.

HMG-1 (high mobility group protein-
1) The family is the human HMG-1 shown in SEQ ID NO: 1.
And 90% or more amino acid homology, for example, bovine HMG-1 (SEQ ID NO: 3),
HMG- such as pig (SEQ ID NO: 4) and rat (SEQ ID NO: 5)
Including 1. Preferably, human HMG-1 is used, but pigs, cows, and rats can be used because of their high homology. these
FIG. 13 shows a comparison of the amino acid sequence of HMG-1.

On the other hand, HMG-2 (high mobility group prote
in-2) Family is the human HMG shown in SEQ ID NO: 2.
Refers to a polypeptide having 80% or more amino acid homology with HB-2, porcine HMG-2 (SEQ ID NO: 6), bovine HMG
HMG-2 such as chicken HMG-2 (SEQ ID NO: 8), chicken HMG-2a (SEQ ID NO: 9), and mouse HMG-2 (SEQ ID NO: 10). Preferred is human, porcine, or bovine HMG-2. A comparison of the amino acid sequences of these HMG-2 is shown in FIG.

The polypeptide belonging to the HMG-1 or HMG-2 family includes a polypeptide in which one or more amino acids are deleted, substituted, or added, or
Also included are polypeptides that are fragments thereof and that can react with antibodies of an autoimmune disease patient.

These fragments refer to fragments of a polypeptide belonging to the HMG-1 family or the HMG-2 family which can react with an antibody of an autoimmune disease patient.
Fragments can be chemically synthesized or made using appropriate proteolytic enzymes. Whether or not the produced fragment reacts with the antibody can be determined by reacting with a serum obtained from an autoimmune disease patient. This method is well known to those skilled in the art, and the same method as the following antibody detection method can be used.

Since HMG-1 and HMG-2 are universal proteins possessed by all cells, they can be prepared by extracting from any organ, tissue or cell. For example, human thymus, porcine thymus, bovine thymus, human placenta, neutrophils, HL-60 cell line and the like. Extraction and purification methods are known and described, for example, in GH Goodwin et al. (Biochemica et Bio
phisica Acta, 405, 280-291, 1975) and M. Yoshida and K. Shimura (J. Biochem. Tokyo, 95, 117-124, 19).
80), Y. Adachi et al. (J. Chromatogr, 530, 39-46,199
It can be prepared by the method of 2). Also, a mixture of bovine HMG-1 and HMG-2 is sold by Wako Pure Chemical Industries.

The above-mentioned polypeptide belonging to the HMG-1 family or HMG-2 family can be obtained by introducing a vector into which a gene encoding the polypeptide has been introduced into a host cell from the above-mentioned tissue or cultured cell producing the same. It can be produced by expression. Polypeptides in which one or more amino acids have been deleted, substituted or added can be obtained by known methods, for example, site-directed mutation, M13 based on the gene sequence of HMG-1 or HMG-2. It can be produced by modifying a gene sequence by a method such as deletion mutation using a phage and expressing it.
Both prokaryote and eukaryote can be used as host cells. For example, bacteria such as Escherichia coli and Bacillus, yeast, mold, insect cells, mammalian cells and the like can be mentioned.
For purification of the polypeptide, known methods, for example, chromatography such as gel filtration chromatography, ion exchange chromatography, affinity chromatography, and reversed-phase liquid chromatography can be used alone or in combination. Preferably, reverse phase HPLC or ion exchange chromatography can be used. Reverse phase HP
As the LC column, various commercially available columns can be used, and preferably, a protein separation column, for example, a YMC-protein RP column (manufactured by YMC) can be used.
Also, as ion exchange chromatography, for example, po
lybuffer-exchanger PBE94 column chromatography,
Alternatively, a monoQ column (manufactured by Pharmacia) or the like can be used.

In the present invention, as autoimmune diseases, rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, Behcet's disease, scleroderma, polymyositis / dermatomyositis, primary biliary cirrhosis, microscopic polyangiitis / Polyarteritis nodosa, ulcerative colitis, and Crohn's disease.

The ulcerative colitis referred to in the present invention refers to those having the following symptoms. First, ulcerative colitis refers to an idiopathic, nonspecific inflammatory disease of the large intestine, especially the rectum, which mainly affects the mucous membrane and submucosa. The disease is more common in adults younger than 30 years, but also in children and those older than 50. The cause is unknown, and it is thought that immune mechanisms, genetic factors, and psychological factors are involved. He usually shows bloody diarrhea and various systemic symptoms. When the whole colon is destroyed for a long period of time, there is a tendency for malignancy. What is intractable ulcerative colitis?
Cases in which the ulcerative colitis is under strict medical treatment, is chronically persistent, is still active for more than 6 months after relapse, and meets any of the following conditions: Say.

[0027] Crohn's disease is an inflammatory bowel disease of unknown cause that can occur in the entire digestive tract, including the small and large intestines.
It is characterized by discontinuous or segmental lesions, longitudinal ulcers, full-thickness inflammatory lesions (mass or stenosis), noncaseating granulomas, and is often associated with young (15-24 years). No autoantibodies have been discovered and there is no simple serological diagnosis.

[0028] Rheumatoid arthritis (RA) is a chronic progressive incurable disease of unknown cause. The nature of RA is in chronic synovitis that does not show a tendency to spontaneously heal, and synovial cell proliferation is seen along with lymphocyte infiltration, angiogenesis, and synovial cell stratification. The small joints of the limbs and the large joints such as the knees, elbows, shoulders and hips are placed symmetrically. The persistence of synovial inflammation and proliferation of inflamed tissue in such joints eventually destroys cartilage and bone, resulting in joint deformation and disability.

[0029] Systemic lupus erythematosus is a multi-organ disease and accompanies various diseases. Non-infectious inflammatory lesions occur in almost all tissues and organs including skin symptoms such as butterfly erythema, oral ulcers, arthritis, lupus nephritis, and central nervous system lesions. Although various autoantibodies are produced, disease-specific autoantibodies have not been identified. Diagnostically, the appearance rate of antinuclear antibodies by indirect fluorescent antibody method is almost 100%, but the specificity is not high. It is an overwhelmingly common disease among young women. In recent years, the 5-year survival rate exceeds 95%, but the course is long-term, and remissions are often repeated.

Sjogren's syndrome is a chronic inflammatory disease of the exocrine glands, mainly lacrimal glands and salivary glands, or of the water secreting tissue, and is a syndrome of dry mouth and eyes due to decreased tear and salivary secretion. Most cases are only dry syndrome, but some may be accompanied by disorders such as thyroid, lung, gastrointestinal, liver, and kidneys. About 30 to 40% of patients have rheumatoid arthritis, systemic lupus erythematosus, and scleroderma. May overlap and present a variety of clinical conditions. Increased erythrocyte sedimentation, hypergammaglobulinemia, and various autoantibodies are observed. On immunological tests, the autoantibodies were diversified and had rheumatoid factor (80
%), Antinuclear antibodies are found in the majority.

Scleroderma (systemic sclerosis) is a connective tissue disease accompanied by hardening of the skin and organ damage. There are various types of the disease, and the disease is not always systemic or progressive. Extensive skin sclerosis (diffuse sclerosis) throughout the body and the esophagus, intestines, lungs,
The prognosis varies from those with poor prognosis, which is associated with organ disorders such as kidney and thyroid gland, and whose progress is fast, to those with skin hardening confined to the face and fingers, and visceral lesions that gradually appear with a good prognosis called CREST syndrome . Among the antinuclear antibodies, the anti-Sc1-70 antibody, which is an autoantibody to topoisomerase 1, has high specificity for a widespread disease (positive rate: 30%). Anti-Centromere antibodies have high diagnostic value in CREST syndrome among this disease, and in the overlapping syndrome of this disease and polymyositis.

[0032] Primary biliary cirrhosis is a chronic disease (symptomatic) characterized by pruritus cutaneous and jaundice, which occurs frequently in middle-aged women and later, but is asymptomatically discovered without these symptoms There is also. Beginning with the destruction of the interlobular bile duct, the fibrous tissue grows gradually and progresses to a histological image of cirrhosis. These biliary changes are reflected in the clinical picture characterized by persistent jaundice. The etiology has not been elucidated. Autoimmune diseases (Sjogren's syndrome, rheumatoid arthritis, etc.) in about 30% of both asymptomatic and symptomatic patients
It is known to merge.

[0033] Polymyositis (PM) is a disease in which skeletal muscles systematically cause non-suppurative inflammation, exhibiting muscle pain and weakness. Red-purple erythema (heliotrope) on the eyelids even in the same PM
Those with keratotic erythema on the back of the finger joints (Gottron's sign) are called dermatomyositis (DM). These striated muscles first impede the proximal muscles, making them unable to stand up or raise their hands. In severe cases, the neck and head cannot be supported. Since some of these diseases are accompanied by cancer or malignant tumors, treatment for cancer is essential, and Behcet's disease is a pre-determined disease. %), Skin symptoms (84%)
Erythema nodosum, folliculitis, acne-like eruption, subcutaneous thrombophlebitis, and skin irritation with razor loss and needle reaction, and ocular symptoms (90%) for iridocyclitis and retinochoroiditis Intractable disease of unknown cause. Other secondary symptoms include arthritis and gastrointestinal symptoms.

Polyarteritis nodosa is a typical necrotizing vasculitis with systematic inflammation in the arteries. Although this disease is rare and difficult to diagnose, it is also a disease that requires early and accurate treatment.
A variety of lesions occur throughout the body in arteritis, and particularly important are skin lesions, kidney lesions, gastrointestinal tract lesions, and central nervous system lesions. It is common in men and no antinuclear antibodies are found. An antibody refers to a component contained in a body fluid that is present in a body fluid of an autoimmune disease and is induced by a specific antigenic substance. For example, when referring to an antibody of a patient with ulcerative colitis or an antibody of a patient with rheumatoid arthritis, it refers to a body fluid component contained in a body fluid such as serum of a patient diagnosed with ulcerative colitis or rheumatoid arthritis, respectively. Antibodies for autoimmune diseases include IgM, IgG, IgE, IgD, IgA and the like.

The diagnostic agent of the present invention includes the above-mentioned polypeptides belonging to the HMG-1 family or HMG-2 family, or fragments thereof. Diagnostic drugs include HMG-1
It is sufficient that at least one family polypeptide, HMG-2 family polypeptide or a fragment thereof is included. Preference is given to using a mixture of HMG-1 and HMG-2.

The diagnostic agent of the present invention reacts with an antibody of an autoimmune disease patient to form an antigen-antibody complex. Thus, it may contain additional components that can detect the formed antigen-antibody complex. These components can be used, for example, in precipitation reactions, ELIS
It is a component compatible with methods such as Method A, RIA, and Western blotting.

The polypeptide contained in the HMG-1 family or HMG-2 family, or a fragment thereof can be used as a diagnostic kit. The diagnostic kit, for example, detects an antigen-antibody complex bound to an antibody of an autoimmune disease patient and an ELISA plate on which a polypeptide contained in the HMG-1 family or the HMG-2 family or a fragment thereof is immobilized. And a reagent for performing This reagent contains a component compatible with a method such as a precipitation reaction method, an ELISA method, an RIA method, and a western blotting method. In the ELISA method, for example, a reagent for detection includes a secondary antibody reagent. Secondary antibody reagents are goat or mouse anti-human IgG or anti-human (IgA + IgG + IgM)
Reacts with IgG, IgM, and IgA. These secondary antibodies may be labeled with a labeling agent generally used in an immunoassay. Such labeling agents include radioisotopes (eg, ³² P, ³ H, ¹²⁵ I, etc.), enzymes (eg, β-galactosidase, peroxidase, alkaline phosphatase, glucose oxidase, lactate oxidase,
Alcohol oxidase, monoamine oxidase, etc.), coenzyme / prosthetic group (for example, FAD, FMN, ATP,
Biotin, heme, etc.), fluorescein derivatives (eg, fluorescein isothiocyanate, fluorescein thiol bamil, etc.), rhodamine derivatives (eg,
Tetramethylrhodamine B isothiocyanate, etc.),
Umbelliferone and 1-anilino-8-naphthalenesulfonic acid, luminol derivatives (eg, luminol, isoluminol, etc.) and the like can be used. Preferably, it is alkaline phosphatase or peroxidase. In the former case, the substrate is paranitrophenyl phosphate, and in the latter case, it is tetramethylbenzidine (TMBZ). The binding between the antibody and the labeling agent can be determined by a known method as described in a compendium (for example, “Seizai Kagaku Kenkyusho 5 Immunobiochemical Research Method” (Tokyo Kagaku Dojin, 1986, p102-112)). Can be appropriately selected from the methods described above. Many of the labeled secondary antibodies are commercially available and can be used. For example, alkaline phosphatase-labeled goat anti-human IgG F (ab ') ₂ polyclonal antibody can be obtained from Immunotech SA (France).

The form of the kit includes a form in which the antigen is contained in an appropriate container, a carrier such as a resin, a membrane, and a film.
Alternatively, a form in which the antigen is immobilized on a carrier such as a container, a resin, a membrane, or a film may be used. As the carrier, polyvinyl chloride, polystyrene, styrene-divinylbenzene copolymer, styrene-maleic anhydride copolymer, nylon, polyvinyl alcohol, polyacrylamide,
Synthetic organic polymer compounds such as polyacrylonitrile, polypropylene, and polymethylene methacrylate; dextran derivatives (such as Sephadex); agarose gels (such as sepharose and biogel); polysaccharides such as cellulose (paper desk and filter paper); glass; silica gel; An inorganic polymer compound such as silicone can be exemplified. These may be those into which a functional group such as an amino group, a carboxyl group, a carbonyl group, a hydroxyl group or a sulfhydryl group has been introduced. Preferred examples are polystyrene,
Polyvinyl chloride.

The carrier may be in the form of a flat plate (microtiter plate, disk, etc.), particulate (beads, etc.), tubular (test tube, etc.), fibrous, membrane, fine particles (latex particles, etc.), capsule, The carrier may be in any form such as an endoplasmic reticulum, and a carrier having a suitable shape may be appropriately selected according to the measurement method. Preferably, it is a 96-well microtiter plate capable of processing a large amount of sample at a time in an ELISA system. For example, an EB plate (manufactured by Lab Systems), H
Type plate, C type plate (Sumitomo Bakelite), maxi soap plate (Nunc) and EI
A./RIA plate (manufactured by Coaster) can be exemplified.

The binding between the carrier and the antigen can be performed by a known method such as a physical adsorption method, an ion bonding method, a covalent bonding method, and an entrapment method (for example, "Immobilized enzyme" edited by Ichiro Chibatake, March 20, 1975
JP, published by Kodansha Co., Ltd.) may be employed, and the physical adsorption method is particularly preferred because it is simple. The antigen and the carrier can be bound directly or between the antigen and the carrier via another substance (spacer) or the like. The immobilized antigen may be subjected to a blocking treatment with a blocking agent such as gelatin or BSA to suppress non-specific binding.

The method for detecting an antibody of an autoimmune disease according to the present invention comprises the steps of: (a) selecting a polypeptide selected from the HMG-1 family;
This is a method including a step of reacting a polypeptide selected from the MG-2 family or a fragment (antigen) thereof with a body fluid component of an autoimmune disease. Conditions well known to those skilled in the art are applied as conditions for reacting the antigen with the antibody. For the detection of the antigen-antibody reactant, a method known to those skilled in the art can be applied.
Examples of the detection method include a sedimentation reaction method, an ELISA method, an RIA method, and a western blotting method. For example, an appropriately diluted patient serum is allowed to react with an antigen.
Secondary antibody, alkaline phosphatase-labeled anti-human
An IgG antibody was added to react, and then an alkaline phosphatase substrate, p-nitrophenyl phosphate, was added to develop a color, and the anti-HMG- was determined by measuring the absorbance at 405 nm.
1 and anti-HMG-2 antibodies can be measured.

In the kit, in addition to the HMG antigen, if necessary, appropriate reagents according to the measurement method can be appropriately selected from various reagents such as a coloring reagent, a reaction stopping reagent, a standard antigen reagent, and a sample pretreatment reagent. It can be attached to the kit of the present invention.

In the following, taking an example of a patient with ulcerative colitis, an antigen reacting with the antibody is screened, and
Method for identifying G-1 and HMG-2, and HMG-
A method for measuring anti-HMG-1 antibody and anti-HMG-2 antibody by an ELISA system using HMG-2 and HMG-2 antigens will be described.

First, blood is collected from a patient with ulcerative colitis to obtain a serum component. Next, the presence of an anti-neutrophil cytoplasmic antibody (ANCA) in the serum component is measured by a fluorescent antibody method. On the other hand, a neutrophil fraction is separated from peripheral blood of a healthy person by specific gravity centrifugation. Next, this neutrophil fraction is processed,
Obtain neutrophil lysate and perform Western blotting. For example, 10 ⁶ neutrophils are dissolved in a sample buffer containing 2-mercaptoethanol and SDS, boiled for 10 minutes, quenched with ice cold, and subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Do. After the electrophoresis, the protein band is transferred to a nylon membrane according to a conventional method, and nonspecific binding is blocked using skim milk or the like.

On the other hand, the serum of an ANCA-positive patient was
Pass through the column and purify the IgG antibody fraction. The purified IgG antibody fraction is reacted with the neutrophil lysate transferred to the nylon membrane. After washing the nylon membrane, for example,
Chemiluminescence is performed with a detection agent such as an ECL kit (manufactured by Amersham), bands are detected, and the presence of the antigen is determined.

The polypeptide having antigenicity (antigen peptide) can be confirmed by the above method. Such an antigen polypeptide can be purified by applying a known protein purification method.

Alternatively, a cell line that produces an antigenic peptide can be identified using the above antibody. An antigenic peptide can be produced using such a cell line and purified by applying a known protein purification method.

By using the above method, 28 kDa
An antigen against anti-neutrophil cytoplasmic antibody (ANCA) could be identified. As will be shown later in the examples, 10 out of 24 ANCA-positive patients
At least one antigen of 28 / 39.5 / 44/47/58 kDa was detected in humans, of which 7 had 28 kDa antigen, and 5 out of 7 were diagnosed with refractory ulcerative colitis It had been. On the other hand, no positive band was detected in the serum of a patient with ulcerative colitis that was negative for ANCA by indirect immunofluorescence. The molecular weight of the antigen in the present invention is 10% SDS-PAGE
Means the one determined in

When the cells producing the 28 kDa antigen were screened by the above screening method, the HL-60 cell line (ATCC CCL-), a neutrophil cell derived from promyelocytic leukemia, was screened.
240) has a 28kDa antigen, and the results of Western blotting show that the
It was suggested that the antigen was present. From this HL-60 cell line, the 28 kDa and 29 kDa antigens can be purified. 28kDa
For the purification of the antigen and the 29 kDa antigen, known protein purification methods can be applied. For example, the HL-60 cell line is
The cells are cultured in RPMI1640 medium supplemented with S, the cells are dissolved in 6M guanidine hydrochloride, and sonication is performed to inactivate the protease. The protein is completely dissolved, concentrated by dialysis, eg, ultrafiltration, and the solution is replaced with PBS. From this aqueous solution, the 28 kDa and 29 kDa antigens can be purified. Suitably reverse phase HPLC may be used. Fractionation by reverse phase HPLC using a concentration gradient of acetonitrile can purify 28 kDa antigen and 29 kDa antigen having a purity of 90% or more. The conditions of reverse phase HPLC using a concentration gradient of acetonitrile can be performed under conditions well known to those skilled in the art.

As a result of analyzing the amino acid sequence of the purified protein, the 29 kDa antigen was identified as high-mobility group protein-1 (H
MG-1) and 28 kDa antigen are high-mobility group pro
tein-2 (HMG-2).

After the purified IgG of the antibody-positive patient was absorbed by bovine HMG-1 and HMG-2, the reaction with the purified 28 kDa antigen and the 29 kDa antigen was examined by Western blotting, but no reaction was observed. From this, the antigens were HMG-1 and HMG-2
Was confirmed.

In addition, HMG-
Fractions 1 and HMG-2 were prepared and subjected to western blotting, which revealed that they reacted with patient antibodies.

The amino acid sequence of human HMG-1 differs from porcine, bovine and rat (FIG. 13) in that these amino acids differ from human, because they differ only by two, one and two amino acids, respectively. HMG-1 from animals can be used as an antigen for ELISA. As for HMG-2, human and pig differ only by two amino acids (FIG. 14),
It is considered that it can be used instead of human.
As shown in FIG. 14, only a partial sequence was determined for bovine, and the reported sequence is considered to be uncertain (bovine sequence is reprinted from Protein Data Bank PIR B61611). HMG-1 and HMG-2 are considered proteins whose amino acid sequences are conserved fairly well between species, and considering the high similarity between humans and pigs, it is thought that cattle also differ by more than two. Absent. This is the absorption experiment described above,
Patient antibody is absorbed by bovine HMG-1 and HMG-2 and human HM
It also suggests that it no longer reacts with G-1 and HMG-2.

HMG-1 and HMG-2 can be conveniently prepared from human, porcine, or bovine thymus tissue according to the method described in the literature. The preparation method from human thymus tissue will be briefly described as an example. Slice the thymus tissue of the child into 0.075 M NaCl / 0.02
Suspend the cells in 5M EDTA (pH 7.5) and destroy the cells with a polytron homogenizer. The precipitate containing chromatin is recovered by centrifugation, suspended in 0.35 M NaCl (pH 7), and HMG bound to chromatin is released by homogenizer treatment. The insoluble matter is removed by centrifugation, and the obtained supernatant is made into a 2% trichloroacetic acid solution and left at 4 ° C. for 2 hours to precipitate insoluble proteins other than HMG-1 and HMG-2. The supernatant is collected by centrifugation, subjected to alkaline acetone precipitation with ammonia / acetone, and the precipitated HMG-1 and HMG-2 are collected by centrifugation. The precipitate was washed with 90% acetone and dried. This fraction was dissolved in 6M guanidine hydrochloride and dialyzed.
Substitution with PBS and concentration were performed to obtain HMG-1 and HMG-2 fractions. The HMG-1 and HMG-2 fractions reacted with antibodies from ulcerative colitis patients by Western blotting.

Pig HMG-1 and Pig HMG used in the present invention
-2 is the method of Y. Yoshida et al.
And K. Shimura, J. Biochem. Tokyo 95, 117-124, 19
80, and Y. Adachi et al., J. Chromatogr, 530, 39-46, 19
A sample prepared and purified according to 92) having a purity of 95% or more was used. These reacted with patient serum like human HMG-1 and HMG-2.

In addition, commercially available bovine HMG-1 and HMG-2 (manufactured by Wako Pure Chemical Industries, Ltd.) also reacted with antibodies from patients with ulcerative colitis.
Therefore, HMG-1 and HMG-2 are considered to be antigens of patients with ulcerative colitis.

An ELISA system was constructed using purified porcine HMG-1 and HMG-2 according to the method described above. That is, 96-well ELI
5 μg / ml pig in each well of SA plate (Nunc)
Add HMG-1 or HMG-2 in 50 μl portions, and add
It was left still for 6 hours. After removing excess antigen, blocking with 5% BSA is performed. Patient serum appropriately diluted with 5% BSA is added and left at room temperature for 2 hours. After washing with a washing solution, alkaline phosphatase-labeled goat anti-human IgG F (ab ') ₂ is added, and reacted at room temperature for 2 hours. After washing 5 times with the washing solution,
A paranitrophenylphosphoric acid (Sigma) solution (10% diethanolamine solution) is added, and the mixture is reacted at room temperature for 20 to 25 minutes, and the absorbance at 405 nm is measured.

Using this system, anti-HMG-1 antibodies and anti-HMG
When the standard curve was tested using sera from patients with ulcerative colitis that were positive for both -2 antibodies, concentration-dependent linear lines were obtained for both antigens, enabling measurement of anti-HMG-1 and anti-HMG-2 antibodies. It was found that using this ELISA system
It was suggested that -1 antibody and anti-HMG-2 antibody could be measured.

Therefore, various autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, Behcet's disease, scleroderma, polymyositis / dermatomyositis, primary biliary cirrhosis, microscopic polyangiitis / Anti-HMG-1 antibody and anti-HMG-2 antibody were separately measured in patients with polyarteritis nodosa, ulcerative colitis, Crohn's disease and healthy subjects. As a result, primary biliary cirrhosis, ulcerative colitis,
HMG-1 is HMG in 7 diseases except Behcet's disease
The antigenicity was stronger than -2, and the positive rate was higher. In addition, HMG-1 showed a statistically significant difference compared to healthy subjects in nine diseases other than polymyositis / dermatomyositis. Furthermore, when compared with the antinuclear antibody positive rate by the indirect fluorescent antibody method, the anti-HMG antibody positive rate was equal to or higher than the antinuclear antibody positive rate in 7 out of 10 diseases. This indicated that the measurement of anti-HMG-1 antibody and anti-HMG-2 antibody may be a diagnostic method for autoimmune diseases that can replace the detection of antinuclear antibodies by the indirect fluorescent antibody method. In addition, it is considered that an autoimmune disease can be diagnosed more widely and surely by using it together with an antinuclear antibody test.

HMG-1 and HMG as the corresponding antigens of pANCA
MG-2 was identified, but these were originally identified as nuclear proteins and may be antigens of antibodies previously called antinuclear antibodies. Therefore, not only pANCA-positive disease but also anti-nuclear antibody-positive disease
It is possible that the MG-2 antibody is detected. Furthermore, since the ELISA method is more sensitive than the indirect fluorescent antibody method, anti-HMG-1 and anti-HMG-2 antibodies can be detected in diseases that were previously considered to be ANCA negative or low in the positive rate. there is a possibility. For example, autoimmune hepatitis, hepatitis B, hepatitis C, Wegener's granulomatosis, leukocyte destructive vasculitis, Chur
Examples include g-Strauss syndrome, primary sclerosing cholangitis, mixed connective tissue disease, malignancy, amoeba abscess, Sweet's disease, multiple sclerosis, Alzheimer's disease, Hashimoto's disease, hyperthyroidism, erythroleukemia and the like.

Neutrophils have a high level of motility and exhibit a phagocytic activity. When inflammation occurs in a living body, it is one of the cells that first migrate to the inflamed area, destroys and melts the inflamed tissue, and eventually removes and absorbs the injured tissue. In many cases, neutrophils are locally infiltrated in tissue injury in autoimmune diseases, and it is thought that they play a role in inflammation. Due to frequent neutrophil invasion due to continuous inflammation or repetition of remission and exacerbation and formation of dense spheres due to phagocytosis of injured tissue, proteins inside neutrophils are exposed to T cells and B cells and eventually produce antibodies. It is thought to be done. If the antibodies produced in this way are anti-HMG-1 and anti-HMG-2 antibodies (mainly anti-HMG-1 antibodies), these antibodies are produced from the early onset of autoimmune disease. Therefore, the measurement of anti-HMG-1 antibody and anti-HMG-2 antibody may be able to diagnose early autoimmune diseases.

Furthermore, during the active period of inflammation, the production of HMG antigen may be enhanced not only in neutrophils but also in activated cells in the local area of inflammation. It is possible that the production of anti-HMG antibodies is enhanced. Therefore, measurement of anti-HMG-1 antibody and anti-HMG-2 antibody is considered to be an indicator of disease activity.

The present invention further includes a kit for measuring anti-HMG-1 antibody and anti-HMG-2 antibody by ELISA. Alternatively, the present invention relates to peripheral blood lymphocytes of patients with autoimmune disease or inflammatory disease and HMG. By examining the responsiveness to -1 and HMG-2, disease specificity can be assayed. That is, to determine whether T lymphocytes proliferate in response to HMG-1 and HMG-2 or their synthetic peptides that are immunoreactive, or whether macrophages produce γ-interferon in the same assay system. By doing so, a disease can be detected.

Hereinafter, the present invention will be described with reference to examples.

[0065]

【Example】

Example 1 Detection of Neutrophil Cytoplasmic Antibody (ANCA) by Indirect Fluorescence Antibody Method Peripheral blood was collected from 35 ulcerative colitis patients (16 males and 19 females) and centrifuged (4 ° C., 13 ° C.). For 2 minutes at 2000 rpm) to obtain serum components. About this serum, anti-neutrophil cytoplasmic antibody (AN
CA) was determined by the indirect fluorescent antibody method. Serum components obtained by similarly treating blood collected from 10 Crohn's disease patients (9 males and 1 female) as a control were also measured.

The measurement conditions of the indirect fluorescent antibody method using ethanol-fixed human neutrophils are described below.

[0067] First, peripheral blood was separated neutrophils toward gravity far precipitation method using Ficoll pack, by cytospin, stuck 10 ⁵ cells per slide. This is air-dried with the cool air of a dryer, and PBS (0.8% NaCl / 0.02% KCl / 10mM Na
Wash with ₂ HPO ₄ /1.5 mM KH ₂ PO ₄ pH 7.4). On the other hand, the serum of the sample is diluted 1:10 with PBS, 20 μl of which is placed on the plate and reacted for 1 hour at room temperature in a humid chamber. After the completion of the reaction, the plate is washed with PBS. FITC-labeled rabbit anti-human IgG F
(ab ') ₂ antibody (manufactured by Serotech) was diluted 1:20 with PBS,
Place 20 μl on the plate and place in a humid chamber at room temperature for 30
Let react for minutes. After the completion of the reaction, the plate is washed with PBS. After washing, the cells are embedded in glycerol diluted 1: 9 with PBS and observed with a fluorescence microscope. Table 1 shows the results detected by this method. Of 35 patients with ulcerative colitis, 24 had ANCA (positive rate 69%; see Table 1).

The anti-neutrophil cytoplasmic antibody (ANCA) in the serum components of the 24 positive patients showed almost the same pANCA (22 out of 24 pANCA) and 2 ANCA) (see Table 1). Table 3 below
Two of the patients No. 24 and 25 had Nuclear-ANCA.

[0069]

[Table 1]

(Example 2) Examination of known antigens against ANCA The antigens against ANCA were examined in the 35 patients with ulcerative colitis described above.

Myeloperoxidase (MPO) (Elastin P
roducts) 5μg / ml, cathepsin G (CaG) (INC Bio
5 μg / ml, and lactoferrin (LF)
Prepare 10 μg / ml (manufactured by Sigma), and add 50 μl / well, 50 μl / well and 10 μl respectively to a 96-well microtiter plate.
0 μl / well was injected and coated overnight at 4 ° C. After coating, the solution was removed, PBS containing 5% BSA (fetal calf serum) (5% BSA / PBS) was added, and reacted for 30 minutes. Next, 5% BSA / PBS was removed, and the serum obtained from the patient was diluted 10-fold with 5% BSA / PBS, added to a microtiter plate, and reacted at room temperature for 24 hours. The reaction solution was removed and washed 5 times with 1% BSA / PBS / 0.5% Tween20.
After washing, an alkaline phosphatase (ALP) -labeled sheep anti-human IgG antibody (manufactured by Immunotech SA) was added with 5% BSA / PBS for 1 hour.
It was diluted 000-fold and added, and reacted at room temperature for 24 hours. After completion of the reaction, the wells were washed 5 times with 1% BSA / 0.5% Tween20 / PBS. After washing, p-nitrophenyl phosphate (final concentration 5 mg / ml)
10% diethanolamine solution (diethanolamine 50
ml + distilled water 450 ml) was added, and the color was developed at room temperature for 30 minutes. After color development, the absorbance at 405 nm was measured.

In this method, no anti-MPO antibody was detected in all cases, and of the 24 patients positive for the indirect immunofluorescence assay, 9 anti-CaG antibodies were positive and 3 anti-LF antibodies were positive. It was positive. The other 12 could not identify the corresponding antigen (see Tables 2 and 3 below).

Example 3 Anti-neutrophil cytoplasmic antibody (ANC
Screening of antigen against A) Western blotting using neutrophil lysate was performed on 24 ANCA-positive patients.

[0074] Peripheral blood was collected from a healthy subject, and a neutrophil fraction was prepared by centrifugation using a Ficoll pack. 1
10 ⁶ neutrophils were suspended in 8 μl of PBS per well, and sample buffer (0.2 M Tris-HCl pH 6.8 / 10% SDS / 25%
2-mercaptoethanol / 25% glycerol / 0.01% BP
2 μl of B) was added and immediately boiled for 10 minutes to obtain an antigen solution. This antigen solution was subjected to electrophoresis (SDS-PAGE) using an SDS-polyacrylamide gel. After the electrophoresis, the sample was transferred to an immobilon membrane (manufactured by Millipore) according to a conventional method, and a 5% skim milk solution was added to block nonspecific binding, followed by a reaction for 2 hours. On the other hand, patient serum 320
Apply μl to Prosep A (manufactured by Bio Processing) (10 ml bed volume) and use 0.1 M-glycine (pH 3.0).
The IgG fraction was eluted and purified to obtain a solution of IgG 20 mg / ml.
The immobilon membrane prepared above and 1 ml of an IgG solution diluted 8-fold with 5% skim milk were reacted at 4 ° C. overnight. After washing
Myeloperoxidase-conjugated anti-human IgG antibody (Kirkegaar
d & Perry Laboratories, INC) (13 μg / ml) was further reacted, and the band was detected by chemiluminescence using an ECL kit (Amersham). The results are shown in Tables 2 and 3.

[0075]

[Table 2]

[0076]

[Table 3]

As a result, anti-neutrophil cytoplasmic antibodies (ANC
In 3/24 sera of positive patients with A), 28 / 39.5 / 44/4
It was found that there was an antigen binding to any of the 7/50/58 kDa bands. In particular, it was confirmed that a substance binding to the 28 kDa band was present in the sera of 7 out of 11 persons. Five of the seven patients with this 28 kDa band were patients diagnosed with intractable ulcerative colitis (see FIG. 1).

On the other hand, from the serum of a patient with ulcerative colitis in which ANCA was not detected by the indirect fluorescent antibody method (ANCA negative), 28
No antibody binding to kDa was detected. No antibody binding to 28 kDa was detected in the serum of a patient with Crohn's disease used as a control.

To summarize the results, patients with ulcerative colitis
Twenty-four of the 35 were ANCA-positive by indirect immunofluorescence, and Western blotting confirmed that 11 of 35 had an antigen that binds to ANCA. Was positive). In addition, there are 7 patients with intractable ulcerative colitis in 35 patients, of which 6
Antigen binding to was detected by Western blotting, and a band of 28 kDa was observed in 5 out of 6 persons. No positive band was observed in the serum of a Crohn's disease patient as a control (see FIG. 1).

At this point, the results of these experiments
This suggests that the appearance of an antibody against the 28kDa antigen may be a marker for predicting the severity (refractoryness) of ulcerative colitis, isolation analysis of the antigen, and a simple antibody detection system using the isolated antigen This demonstrates the importance of development.

Example 4 HL-60 cells were treated with 28 kDa and 29
Confirmation of Presence of kDa Antigen The HL-60 cell line, which is a neutrophil cell derived from promyelocytic leukemia, was cultured in RPMI1640 medium supplemented with 5% FCS, and the method described in Example 3 was used. Neutrophil lysate was prepared. Using this lysate, Western blotting was performed in the same manner as in Example 3. The results are shown in FIG. 2 together with the positive control using neutrophil lysate. This binds to the antibody fraction obtained from ulcerative colitis patients
It was confirmed that the 28 kDa antigen was present. Also, this 28k
The Da band was always detected as a thick band, suggesting the presence of a 29 kDa antigen in close proximity to the 28 kDa antigen.

Example 5 Purification of 28 kDa and 29 kDa Antigens from HL-60 Cells The HL-60 cell line in which the presence of the antigen was confirmed in Example 4 was 5% F
The cells were cultured in an RPMI1640 medium supplemented with CS. When 1 × 10 ⁵ cells became 2 × 10 ⁶ cells per 75 cm ² flask, the cells were collected by centrifugation so that the total number of cells became 2 × 10 ⁸ cells. 10 ml of 6M guanidine hydrochloride was added and dissolved, and sonication (USP600 manufactured by Shimadzu Corporation) was performed for 1 minute.
With this operation, the cells were completely lysed. After diluting twice by adding the same amount of distilled water as this solution, the supernatant was collected by centrifugation at 80,000 × g for 30 minutes. The membrane YM3 (manufactured by Amicon) was used to remove the supernatant to remove a molecular weight of 3,000 or less. ) Was transferred to an Amicon ultrafiltration device (manufactured by Amicon), filtered while adding PBS, and then concentrated to a final volume of 4 ml.
Was used as a PBS solution. This PBS solution was centrifuged at 80,000 × g for 30 minutes to remove the precipitate, and the supernatant was recovered. The collected supernatant was used as an antigen-containing sample, and the 28 kDa and 29 kDa fractions were fractionated from this sample using HPLC. Using a YMC-Pack Protein RP column (YMC), the protein was eluted with a gradient of acetonitrile concentration of 16% to 48%. The results are shown in FIG. The peak of No. 9 in FIG. 3 was collected and freeze-dried. The freeze-dried sample was redissolved in PBS, and the protein was eluted again using a YMC-Pack Protein RP column with a gradient of acetonitrile concentration of 24% to 36%. The HPLC system was performed using a Shimadzu LC-7A system. The results are shown in FIG. No.5 and N
The peak of o.6 was collected and concentrated and dried by centrifugation. The sample that had been centrifuged, concentrated and dried was electrophoresed by SDS-PAGE, transferred to a PVDF membrane (manufactured by Amersham) by western blotting, stained with Ponceau S, and then the 28 kDa and 29 kDa bands were cut out and collected. FIG. 5 shows the results of Western blotting of the purified antigen. SDS-PA of purified antigen
In GE, the two proteins were separated and identifiable. Because both proteins reacted with patient serum, SDS-PAGE from cell lysates could not be separated due to their close molecular weights, or could not be detected due to the low neutrophil 29kDa antigen content. Can be

(Example 6) Determination of partial amino acid sequence and homology analysis The membrane containing the bands of 28 kDa and 29 kDa recovered in Example 5 was dried and then used for determination of the amino acid sequence. The amino acid sequence was determined using a fully automatic protein primary structure analyzer manufactured by Shimadzu.
This was performed using a PPSQ-10 system. As a result, the N-terminal 32 partial amino acid sequence of the 28 kDa band was determined. The sequence was as follows:

Gly Lys Gly Asp Pro Asn Lys Pro Arg Gl
y Lys Met Ser Ser Tyr Ala Phe PheVal Gln Thr Xaa A
rg Glu Glu His Lys Lys Lys His Pro Asp (SEQ ID NO: 1
1) Similarly, when the amino acid sequence of the 29 kDa band was analyzed, 32 amino acid sequences from the N-terminus were determined.
The sequence was as follows:

Gly Lys Gly Asp Pro Lys Lys Pro Arg Gl
y Lys Met Ser Ser Tyr Ala Phe PheVal Gln Thr Xaa A
rg Glu Glu His Lys Lys Lys His Pro Asp (SEQ ID NO: 1
2)

(Example 7) Homology analysis of partial amino acid sequence Homology analysis was performed on the amino acid sequence obtained in Example 6. Altschul, SF and other BLAST programs (J.
Mol. Biol. 205, 403-410, 1982), a homology search was performed for all amino acid sequences contained in the known database PIR. As a result, a 29 kDa antigen was identified as a non-histone nuclear protein HMG-1 (Reeck, GR Nucleic Acids Res. 17,
1197-1214, 1989) and 31 out of 32 amino acids. The 28 kDa antigen is HMG-2 (Majumdar, A. et al., Nucleic Aci
ds Res. 19, 6643, 1991) and 31 out of 32 amino acids were identical. No cysteine at position 22 could be identified for either antigen. Since cysteine cannot be detected without modifying the thiol group, conversely, the 22nd is considered to be cysteine and SDS-
Taking into account the molecular weight by PAGE, the 28 kDa and 29 kDa antigens were identified as HMG-2 and HMG-1, respectively.

(Example 8) Absorption test of patient antibody with HMG antigen The HMG-1 and HMG-2 antigens purified in Example 5 and neutrophil lysate were subjected to SDS-PAGE in the same manner as in Example 3, followed by And transferred to Immobilon membrane. IgG purified from HMG antigen-positive ulcerative colitis patient (0.024 mg / ml) as primary antibody
And a bovine HMG-1 / 2 mixture (0.5 mg / ml) at a ratio of 1: 2, and an antibody solution reacted at 4 ° C. overnight was used. After washing as in Example 3, a secondary antibody was reacted and detected by ECL. As a result, in the antibody absorbed with the bovine HMG mixed solution, 28 kDa and 29 kDa
Band and the neutrophil 28 kDa band disappeared (FIG. 6).
From this, the 29 kDa and 28 kDa antigens are HMG-
Probably 1 and HMG-2.

Example 9 Preparation of HMG-1 and HMG-2 Fractions from Human Thymic Tissue HMG-1 and HMG-2 were prepared from human, porcine and bovine thymic tissues.
It can be conveniently prepared according to the method of the above-mentioned literature. Slice the thymus tissue (13 g) of the child into small sections, and use 30 ml of 0.075 M NaCl / 0.025 M
Suspended in EDTA (pH 7.5) and suspended in a polytron homogenizer (Pol
cells were destroyed for 2 minutes in ice water (ytron) (speed 1
0). The precipitate containing chromatin was recovered by centrifugation at 2,000 xg for 30 minutes at 4 ° C. Repeat this operation four more times (however, the homogenization time is 1 minute), and precipitate 30 ml.
Suspended in 0.35 M NaCl (pH 7) and treated with a homogenizer (s
Peed 5, 1 minute) to release HMG bound to chromatin. This operation was repeated twice more to obtain a 90 ml solution. The insoluble material was removed by centrifugation at 2,000 xg for 30 minutes at 4 ° C, the resulting supernatant was converted to a 2% trichloroacetic acid solution, and left at 4 ° C for 1 hour to remove insoluble proteins other than HMG-1 and HMG-2. Settled. The supernatant was collected by centrifugation (4 ° C., 2000 × g, 30 minutes), and 2-mercaptoethanol was added to a final concentration of 0.
01M was added. 1.5 ml of 30% ammonia was added, and immediately 300 ml of acetone was added, stirred, and left at 4 ° C. overnight. Precipitated HMG-1 and HMG-2 were recovered by centrifugation, washed with 90% acetone, and dried. This fraction
It was dissolved in 6M guanidine hydrochloride, replaced with PBS by dialysis and concentrated to obtain HMG-1 and HMG-2 fractions. The HMG-1 and HMG-2 fractions reacted with antibodies from ulcerative colitis patients by Western blotting (FIG. 7). In addition, commercially available bovine HMG-1 and HMG-2 (manufactured by Wako Pure Chemical Industries, Ltd.) also reacted with antibodies from ulcerative colitis patients (FIG. 7). Therefore, HMG-1 and HMG-2 are considered to be antigens of patients with ulcerative colitis.

(Example 10) HMG-1 and HMG derived from porcine thymus
Purification of MG-2 HMG-1 and HMG-2 derived from porcine thymus were purified by M. Yoshida and KS
himura (J. Biochem. Tokyo, 95, 117-124, 1980), and Y. Adachi et al. (J. Chromatogr, 530, 39-46, 1992).
Purified according to the method described above. The purification method is briefly described. Chromatin fraction obtained in the same manner as in Example 9, 0.35M NaCl
The suspension was suspended in (pH 7) / 1 mM PMSF, homogenized with a Potter-Elvehjem PTFE homogenizer, centrifuged at 5,000 × g for 20 minutes, and the supernatant was collected. This operation was repeated twice, and the obtained supernatants were mixed. This fraction was made into a 2% trichloroacetic acid solution, and the precipitate was removed by centrifugation.
HMG-1 and HMG precipitated as trichloroacetic acid solution
The precipitate containing -2 was collected by centrifugation. 3ml of precipitate 10m
It was dissolved in M Tris-HCl (pH 7.8), applied to a Mono Q column (5 x 50 mm, manufactured by Pharmacia) previously washed and equilibrated with the same buffer, and separated (Pharmacia).
FPLC system was used). Elution was performed with a linear gradient of NaCl from 0 to 1 M. FIG. 8 shows the elution pattern. By this fractionation method, HMG-1 and HMG-2 were obtained with a purity of about 95% or more.

The obtained HMG was confirmed to react with the patient antibody by Western blotting.
HMG-1 fraction is HMG-2, and HMG-2 fraction is HMG-1
Was not mixed (FIG. 7).

(Example 11) Detection of anti-HMG-1 antibody and anti-HMG-2 antibody in serum of patients with intractable ulcerative colitis by western blotting Western blotting using swine HMG-1 and swine HMG-2 mixture as an antigen Was done. A mixture of porcine HMG-1 (0.5 μg) and porcine HMG-2 (0.5 μg) was dissolved in a sample buffer (Example 3), heat-treated according to a conventional method, and then subjected to SDS-PAGE.
Was done. After SDS-PAGE, transfer to PVDF membrane, serum of 5 patients with intractable ulcerative colitis, 28kDa positive, HRP-labeled anti-human
Reaction was performed in the order of IgG antibody, and detection was performed by ECL. As a result, four of the five refractory ulcerative colitis patients were positive, and one was 28 kDa positive, but the antigen was HMG-1 and HMG-.
It was not 2 (FIG. 9). From these results, anti-HMG-1 antibody and / or anti-HMG-1 antibody was found in the serum of patients with refractory ulcerative colitis.
It was shown that the HMG-2 antibody was present.

Example 12 Measurement of Anti-HMG-1 Antibody and Anti-HMG-2 Antibody by ELISA Method ELISA using a mixture of porcine HMG-1, porcine HMG-2, human HMG-1 and human HMG-2 as an antigen Was done. 50 μl of porcine HMG-1 or porcine HMG-2 serially diluted from 6.25 μg / ml was added to each well of a 96-well maxi soap plate (manufactured by Nunc).
In the case of humans, 50 μl of HMG-1 and HMG-2 (equivalent mixture) serially diluted from 6.25 μg / ml were added, and the mixture was allowed to stand at 4 ° C. for 24-36 hours. After removing excess antigen, add 200 μl of 5% BSA
Blocking was carried out by leaving still for 30 minutes or more. The serum of a patient with intractable ulcerative colitis positive for anti-HMG-1 antibody and / or anti-HMG-2 antibody was used as a standard serum, and 50 μl of the serum diluted 40-fold with 5% BSA was added. 0.05% Tween / 0.02
After washing 5 times with% azide / PBS (washing solution), 1000-fold diluted alkaline phosphatase-labeled goat anti-human IgG F (ab ')
100 µl of ₂ (Immunotech SA) was added and reacted at room temperature for 2 hours. After washing 5 times with a washing solution, 100 μl of a 0.1% paranitrophenylphosphoric acid (Sigma) solution (10% diethanolamine solution) was added, and reacted at room temperature for 20 to 25 minutes.
The absorbance at 5 nm was measured. FIG. 10 shows the calibration curve of the positive control. Dose-dependent linear lines were obtained for all three ELISAs. From these calibration curves, porcine HMG-1 (Fig. 10-1) and HMG-2
It was found that the anti-HMG-1 antibody and / or the anti-HMG-2 antibody could be measured by ELISA using a mixture of human HMG-1 and human HMG-2 (FIG. 10-3) (FIG. 10-2). Further, the calibration curve suggested that a measurement having a concentration of 5 μg / ml at which the absorbance becomes almost 1.0 may be used for the measurement.

Example 13 Measurement of Anti-HMG-1 Antibody and Anti-HMG-2 Antibody in Each Disease Antibodies in patients with autoimmune diseases and inflammatory diseases were measured using this ELISA system. The diseases used are as follows.
50 rheumatoid arthritis, 47 systemic lupus erythematosus,
12 Sjogren's syndrome, 32 Behcet's disease, 15 polymyositis / dermatomyositis, 20 scleroderma, primary biliary cirrhosis
41, microscopic polyangitis / polyarteritis nodosa 19,
There are 62 ulcerative colitis and 15 Crohn's disease. On the other hand, serum of 33 healthy persons was used as a control. Pig HMG-1 and HMG-2 as antigens, respectively, 5 μg / ml
Used at a concentration of As serum, a standard serum diluted 40-fold was used for measurement. Separately from the test serum, put the same standard curve antigen as in Example 12 on each plate, and use the standard serum (40-fold dilution) at the same time as the test serum.
The measurement was performed according to 12. Only when a linear calibration curve as shown in FIG. 10 was obtained from each plate, the measured value of the sample in the plate was regarded as valid. The value obtained by subtracting the absorbance of the blank well to which no antigen was added from the absorbance of the standard serum was taken as 100%. Similarly, the ratio was calculated from the absorbance value of the test serum from which the blank value was subtracted, and the ratio was used as the value of the test serum. If the value was too high at a 40-fold dilution of the serum, the measurement was performed at a dilution of at least 80-fold and the value was calculated. As a result, a value exceeding mean + 2 sd of a healthy person was defined as positive.

In order to compare the anti-HMG antibody positive rate and the anti-nuclear antibody positive rate, an anti-nuclear antibody was measured for the same sample by the indirect fluorescent antibody method. The measurement was performed using a fluoro HEPANA test manufactured by MBL, and a dilution of 40 times or more was regarded as positive.

Table 4 shows the positive rates by antigen and disease, and FIG.
FIG. 12 shows a scatter diagram for each disease using HMG-1 as an antigen, and FIG. 12 shows a scatter diagram for each disease using HMG-2 as an antigen.

[0096]

[Table 4]

In primary biliary cirrhosis, ulcerative colitis and Behcet's disease, the anti-HMG-1 antibody positive rate and the anti-HMG-2 antibody positive rate showed almost the same value. However, among the other seven diseases, the anti-HMG-1 antibody positive rate was higher (Table 4), indicating that HMG-1 had stronger antigenicity. In particular, in Sjogren's syndrome, HMG-1 is 66.7% and HMG-2 is 16.7%, which is overwhelmingly significant for HMG-1. This indicates that it can be used for narrowing down Sjögren's syndrome. HMG-
In 1, all nine diseases except polymyositis / dermatomyositis showed a statistically significant difference compared to healthy subjects. on the other hand,
With HMG-2, rheumatoid arthritis, systemic lupus erythematosus, Behcet's disease, primary biliary cirrhosis and ulcerative colitis showed statistically significant differences compared to healthy subjects. Most anti-HMG-2 antibody-positive patients were anti-HMG-1 antibody-positive, but a small number of anti-HMG-2 antibody-positive and anti-HMG-1 antibody-negative patients existed. Therefore, in order to perform a diagnosis with higher sensitivity, it is considered better to measure both antibodies.

The results of adding anti-HMG-1 antibody and anti-HMG-2 antibody positive showed a very high positive rate in primary biliary cirrhosis, rheumatoid arthritis, Sjogren's syndrome, and systemic lupus erythematosus. In primary biliary cirrhosis, the positive rate is considered to be equal to or higher than that of anti-mitochondrial antibody, and no antigen showing such a high positive rate has been found so far in the remaining three diseases. In addition, the scleroderma, ulcerative colitis, Crohn's disease, microscopic polyangitis / polyarteritis nodosa, and Behcet's disease have a high positive rate. Antigen is the first time. For microscopic polyangiitis / polyarteritis nodosa, myeloperoxidase is the antigen with the highest positive rate, while HMG is the next antigen. In the present invention, anti-HMG-1 antibody and anti-HMG-2 antibody are used for polymyositis /
The antibodies were found to be detectable simultaneously in 9 diseases except dermatomyositis.

Therefore, this time, antinuclear antibodies were simultaneously measured for each disease by the indirect fluorescent antibody method and compared with the HMG antigen positive rate (Table 4). Seven out of the ten diseases showed values equal to or higher than the antinuclear antibody positive rate. From this, HMG-1 and HMG
The measurement of -2 was shown to be a diagnostic method for autoimmune diseases that can replace the detection of antinuclear antibodies by the indirect fluorescent antibody method.
In addition, it is considered that an autoimmune disease can be diagnosed more widely and surely by using it together with an antinuclear antibody test.

[0100]

EFFECTS OF THE INVENTION Rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, Behcet's disease, scleroderma,
The ability to identify common counterpart antigens HMG-1 and HMG-2 in primary biliary cirrhosis, microscopic polyangiitis / polyarteritis nodosa, ulcerative colitis, and Crohn's disease was used. Simple antibody detection has become possible. Furthermore, a method and a measurement kit for measuring antibodies to both antigens using HMG-1 and HMG-2 have been shown to be diagnostic agents for these diseases.

[0101]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 214 Sequence type: Amino acid Sequence type: Peptide Sequence characteristics: HMG-1 Origin: Human Sequence Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr Ala Phe Phe 5 10 15 Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro Asp Ala Ser Val Asn 20 25 30 35 Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg Trp Lys Thr Met Ser Ala Lys 40 45 50 Glu Lys Gly Lys Phe Glu Asp Met Ala Lys Ala Asp Lys Ala Arg Tyr Glu Arg 55 60 65 70 Glu Met Lys Thr Tyr Ile Pro Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp 75 80 85 90 Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr 95 100 105 Arg Pro Lys Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys 110 115 120 125 Lys Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr Glu 130 135 140 Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala Ala Tyr Arg 145 150 155 160 Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val Val Lys Ala Glu Lys Ser 165 170 175 180 Lys Lys Lys Lys Glu Glu Glu Glu Asp Glu Glu Asp Glu Glu Asp Glu Glu Glu 185 190 195 Glu Glu Asp Glu Glu Asp Glu Asp Glu Glu Glu Asp Asp Asp Asp Glu 200 205 210

SEQ ID NO: 2 Sequence length: 208 Sequence type: amino acid Sequence type: Peptide Sequence features: HMG-2 Origin: Human sequence Gly Lys Gly Asp Pro Asn Lys Pro Arg Gly Lys Met Ser Ser Tyr Ala Phe Phe 5 10 15 Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro Asp Ser Ser Val Asn 20 25 30 35 Phe Ala Glu Phe Ser Lys Lys Cys Ser Glu Arg Trp Lys Thr Met Ser Ala Lys 40 45 50 Glu Lys Ser Lys Phe Glu Asp Met Ala Lys Ser Asp Lys Ala Arg Tyr Asp Arg 55 60 65 70 Glu Met Lys Asn Tyr Val Pro Pro Lys Gly Asp Lys Lys Gly Lys Lys Lys Asp 75 80 85 90 Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu His 95 100 105 Arg Pro Lys Ile Lys Ser Glu His Pro Gly Leu Ser Ile Gly Asp Thr Ala Lys 110 115 120 125 Lys Leu Gly Glu Met Trp Ser Glu Gln Ser Ala Lys Asp Lys Gln Pro Tyr Glu 130 135 140 Gln Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala Ala Tyr Arg 145 150 155 160 Ala Lys Gly Lys Ser Glu Ala Gly Lys Lys Gly Pro Gly Arg Pro Thr Gly Ser 165 170 175 180 Lys Lys Lys Asn Glu Pro Glu Asp Glu Glu Glu Glu Glu Glu Glu Glu Asp Glu 185 190 195 Asp Glu Glu Glu Glu Asp Glu Asp Glu Glu 200 205

SEQ ID NO: 3 Sequence length: 214 Sequence type: amino acid Sequence type: peptide Sequence characteristics: HMG-1 Origin: bovine sequence Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr Ala Phe Phe 5 10 15 Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro Asp Ala Ser Val Asn 20 25 30 35 Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg Trp Lys Thr Met Ser Ala Lys 40 45 50 Glu Lys Gly Lys Phe Glu Asp Met Ala Lys Ala Asp Lys Ala Arg Tyr Glu Arg 55 60 65 70 Glu Met Lys Thr Tyr Ile Pro Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp 75 80 85 90 Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr 95 100 105 Arg Pro Lys Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys 110 115 120 125 Lys Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr Glu 130 135 140 Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala Ala Tyr Arg 145 150 155 160 Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val Val Lys Ala Glu Lys Ser 165 170 17 5 180 Lys Lys Lys Lys Glu Glu Glu Glu Asp Glu Glu Asp Glu Glu Asp Glu Glu Glu 185 190 195 Glu Glu Asp Glu Glu Asp Glu Glu Glu Glu Glu Asp Asp Asp Asp Glu 200 205 210

SEQ ID NO: 4 Sequence length: 214 Sequence type: amino acid Sequence type: peptide Sequence characteristics: HMG-1 Origin: Pig sequence Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr Ala Phe Phe 5 10 15 Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro Asp Ala Ser Val Asn 20 25 30 35 Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg Trp Lys Thr Met Ser Ala Lys 40 45 50 Glu Lys Gly Lys Phe Glu Asp Met Ala Lys Ala Asp Lys Ala Arg Tyr Glu Arg 55 60 65 70 Glu Met Lys Thr Tyr Ile Pro Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp 75 80 85 90 Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr 95 100 105 Arg Pro Lys Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys 110 115 120 125 Lys Leu Gly Glu Met Trp Asn Asn Thral Ala Ala Asp Asp Lys His Pro Tyr Glu 130 135 140 Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala Ala Tyr Arg 145 150 155 160 Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val Val Lys Ala Glu Lys Ser 165 170 175 180 Lys Lys Lys Lys Glu Glu Glu Glu Asp Glu Glu Asp Glu Glu Asp Glu Glu Glu 185 190 195 Glu Glu Asp Glu Glu Asp Glu Glu Glu Glu Glu Asp Asp Asp Asp Glu 200 205 210

SEQ ID NO: 5 Sequence length: 214 Sequence type: amino acid Sequence type: peptide Sequence characteristics: HMG-1 Origin: rat Sequence Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr Ala Phe Phe 5 10 15 Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro Asp Ala Ser Val Asn 20 25 30 35 Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg Trp Lys Thr Met Ser Ala Lys 40 45 50 Glu Lys Gly Lys Phe Glu Asp Met Ala Lys Ala Asp Lys Ala Arg Tyr Glu Arg 55 60 65 70 Glu Met Lys Thr Tyr Ile Pro Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp 75 80 85 90 Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr 95 100 105 Arg Pro Lys Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys 110 115 120 125 Lys Leu Gly Glu Met Trp Asn Asn Thral Ala Ala Asp Asp Lys His Pro Tyr Glu 130 135 140 Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala Ala Tyr Arg 145 150 155 160 Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val Val Lys Ala Glu Lys Ser 165 170 175 180 Lys Lys Lys Lys Glu Glu Glu Asp Asp Glu Glu Asp Glu Glu Asp Glu Glu Glu 185 190 195 Glu Glu Glu Glu Glu Asp Glu Glu Glu Glu Glu Asp Asp Asp Asp Glu 200 205 210

SEQ ID NO: 6 Sequence length: 209 Sequence type: amino acid Sequence type: Peptide Sequence characteristics: HMG-2 Origin: Pig sequence Gly Lys Gly Asp Pro Asn Lys Pro Arg Gly Lys Met Ser Ser Tyr Ala Phe Phe 5 10 15 Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro Asp Ser Ser Val Asn 20 25 30 35 Phe Ala Glu Phe Ser Lys Lys Cys Ser Glu Arg Trp Lys Thr Met Ser Ala Lys 40 45 50 Glu Lys Ser Lys Phe Glu Asp Met Ala Lys Ser Asp Lys Ala Arg Tyr Asp Arg 55 60 65 70 Glu Met Lys Asn Tyr Val Pro Pro Lys Gly Asp Lys Lys Gly Lys Lys Lys Asp 75 80 85 90 Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu His 95 100 105 Arg Pro Lys Ile Lys Ser Glu His Pro Gly Leu Ser Ile Gly Asp Thr Ala Lys 110 115 120 125 Lys Leu Gly Glu Met Trp Ser Glu Gln Ser Ala Lys Asp Lys Gln Pro Tyr Glu 130 135 140 Gln Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala Ala Tyr Arg 145 150 155 160 Ala Lys Gly Lys Gly Glu Ala Gly Lys Lys Gly Pro Gly Arg Pro Thr Gly Ser 165 170 17 5 180 Lys Lys Lys Asn Glu Pro Glu Asp Glu Glu Glu Glu Glu Glu Glu Glu Glu Asp 185 190 195 Glu Asp Glu Glu Glu Glu Asp Glu Asp Glu Glu 200 205

SEQ ID NO: 7 Sequence length: 186 Sequence type: amino acid Sequence type: peptide Sequence characteristics: partial sequence of HMG-2 Origin: bovine sequence Gly Lys Gly Asp Pro Asn Lys Pro Arg Gly Lys Met Ser Ser Tyr Ala Phe Phe 5 10 15 Val Gln Thr Ser Arg Glu Glu His Lys Lys Lys His Pro Asp Ala Ser Val Asn 20 25 30 35 Phe Ser Glu / Arg Trp Lys Thr Met Ser Ala Lys Glu Lys Ser Lys Phe Glu Asp 40 45 50 Met Ala Lys Ser Asp Lys Ala Arg Tyr Asp Arg Glu Met Lys Asn Tyr Val Pro 55 60 65 70 Pro Lys Gly Asp Lys Lys Gly Lys Lys Lys Asp Pro Asn Ala Pro Lys Arg Pro 75 80 85 90 Pro Ser Ala Phe Phe Leu Phe Ser Ala Glu His Arg Pro Lys Ile Lys Ala Glu 95 100 105 His Pro Gly Leu Ser Ile Gly Asp Thr Ala Lys Lys Leu Gly Glu Met Trp Ser 110 115 120 125 Gln Gln Ser Ala Lys Asp Lys Gln Pro Tyr Glu Gln Lys Ala Ser Lys Leu Lys 130 135 140 Glu Lys Tyr Glu Lys Xaa Ala Ala Tyr Arg Ala Lys Gly Lys Ser Glu Ala Gly 145 150 155 160 Lys Lys Gly Pro Gly Arg Pro Thr Gly Ser Lys Lys Lys Asn Glu Pro Glu Asp 165 170 175 180 Glu Glu Glu Glu Glu Glu Glu 185

SEQ ID NO: 8 Sequence length: 206 Sequence type: amino acid Sequence type: Peptide Sequence characteristics: HMG-2 Origin: Chicken sequence Gly Lys Gly Asp Pro Asn Lys Pro Arg Gly Lys Met Ser Ser Tyr Ala Tyr Phe 5 10 15 Val Gln Thr Cys Pro Arg Glu His Lys Lys Lys His Pro Asp Ser Ser Val Asn 20 25 30 35 Phe Ala Glu Phe Ser Arg Lys Cys Ser Glu Arg Trp Lys Thr Met Ser Ser Lys 40 45 50 Glu Lys Gly Lys Phe Glu Glu Met Ala Lys Gly Asp Lys Ala Arg Tyr Asp Arg 55 60 65 70 Glu Met Lys Asn Tyr Val Pro Pro Lys Gly Glu Lys Lys Gly Lys Lys Lys Asp 75 80 85 90 Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu His 95 100 105 Arg Pro Lys Ile Lys Asn Asp His Pro Gly Leu Ser Ile Gly Asp Thr Ala Lys 110 115 120 125 Lys Leu Gly Glu Met Trp Ser Glu Gln Ser Ala Lys Asp Lys Gln Pro Tyr Glu 130 135 140 Gln Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala Ala Tyr Arg 145 150 155 160 Ala Lys Ser Lys Ser Asp Ala Gly Lys Lys Gly Pro Gly Arg Pro Ala Gly Ser 165 170 175 180 Lys Lys Lys Ala Glu Pro Glu Glu Glu Glu Glu Glu Glu Glu Asp Glu Glu Glu 185 190 195 Glu Glu Glu Glu Glu Glu Asp Glu Glu 200 205

SEQ ID NO: 9 Sequence length: 201 Sequence type: amino acid Sequence type: Peptide Sequence characteristics: HMG-2a Origin: Chicken Ala Lys Gly Asp Pro Lys Lys Pro Lys Gly Lys Met Ser Ala Tyr Ala Phe Phe 5 10 15 Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys Asn Pro Glu Val Pro Val Asn 20 25 30 35 Phe Ala Glu Phe Ser Lys Lys Cys Ser Glu Arg Trp Lys Thr Met Ser Ser Lys 40 45 50 Glu Lys Ala Lys Phe Asp Glu Met Ala Lys Ala Asp Lys Val Arg Tyr Asp Arg 55 60 65 70 Glu Met Lys Asp Tyr Gly Pro Ala Lys Gly Gly Lys Lys Lys Lys Asp Pro Asn 75 80 85 90 Ala Pro Lys Arg Pro Pro Ser Gly Phe Phe Leu Phe Cys Ser Glu Phe Arg Pro 95 100 105 Lys Ile Lys Ser Thr Asn Pro Gly Ile Ser Ile Gly Asp Val Ala Lys Lys Leu 110 115 120 125 Gly Glu Met Trp Asn Asn Leu Ser Asp Gly Glu Lys Gln Pro Tyr Asn Asn Lys 130 135 140 Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Val Ala Asp Tyr Lys Ser Lys 145 150 155 160 Gly Lys Phe Asp Gly Ala Lys Gly Ala Ala Thr Lys Ala Ala Arg Lys Lys Val 165 170 175 1 80 Glu Glu Glu Asp Glu Glu Glu Glu Glu Asp Glu Glu Glu Glu Asp Glu Asp Asp 185 190 195 Asp Asp Glu 200

SEQ ID NO: 10 Sequence length: 208 Sequence type: amino acid Sequence type: peptide Origin: mouse Sequence characteristics: HMG-2 Gly Lys Gly Asp Pro Ile Lys Pro Leu Gly Lys Met Ser Ser Tyr Ala Phe Phe 5 10 15 Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro Asn Ser Ser Val Asn 20 25 30 35 Phe Ala Glu Ile Ser Lys Lys Cys Ser Lys Arg Trp Lys Thr Met Ser Ala Lys 40 45 50 Glu Asn Ser Lys Phe Glu Asp Leu Ala Lys Ser Asp Lys Ala Cys Tyr Tyr Arg 55 60 65 70 Glu Met Lys Asn Tyr Val Ser Pro Lys Gly Asp Lys Lys Gly Lys Lys Lys Asp 75 80 85 90 Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala Phe Cys Leu Phe Cys Ser Glu Asn 95 100 105 Arg Pro Lys Ile Lys Ile Glu Tyr Pro Gly Leu Ser Ile Gly Asp Thr Ala Lys 110 115 120 125 Lys Leu Gly Glu Met Trp Ser Glu Gln Ser Ala Lys Glu Lys Gln Pro Tyr Glu 130 135 140 Gln Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Phe Ala Ala Tyr Arg 145 150 155 160 Val Lys Gly Lys Ser Glu Ala Gly Lys Lys Gly Pro Gly Arg Pro Ala Gly Ser 165 170 175 180 Lys Lys Lys Asn Asp Ser Glu Asp Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu 185 190 195 Asp Glu Glu Gly Glu Glu Glu Asp Glu Glu 200 205

SEQ ID NO: 11 Sequence length: 32 Sequence type: amino acid Sequence type: peptide Fragment type: 28 KDa N-terminal fragment Origin Cell type: neutrophil cell derived from promyelocytic leukemia Cell line: neutrophil Cell line (ATCC CCL-240) Sequence characteristics Sequence determination method: E sequence Gly Lys Gly Asp Pro Asn Lys Pro Arg Gly Lys Met Ser Ser Tyr Ala Phe Phe 5 10 15 Val Gln Thr Xaa Arg Glu Glu His Lys Lys Lys His Pro Asp 20 25 30

SEQ ID NO: 12 Sequence length: 32 Sequence type: amino acid Sequence type: peptide Fragment type: 29 KDa N-terminal fragment Origin Cell type: neutrophil cell derived from promyelocytic leukemia Cell line: neutrophil Cell line (ATCC CCL-240) Sequence characteristics Sequence determination method: E Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr Ala Phe Phe 5 10 15 Val Gln Thr Xaa Arg Glu Glu His Lys Lys Lys His Pro Asp 20 25 30

[Brief description of the drawings]

FIG. 1. Purified Ig obtained from sera of five patients with ulcerative colitis and three healthy persons after electrophoresis of neutrophil cytoplasmic lysate (antigen) isolated from peripheral blood of healthy subjects by SDS-PAGE.
It is a figure which shows the result at the time of performing western blotting using G (antibody).

FIG. 2 is a diagram showing the results of Western blotting using neutrophils and HL-60 cell lysate as antigens, and using serum of a 28 kDa antigen-positive patient as a probe.

FIG. 3 is a view showing an HPLC pattern in a step of isolating an antigen from HL-60 cells. No. 9 peak 2
Includes the 8 kDa and 29 kDa antigens.

FIG. 4 is a view showing an HPLC pattern in a step of isolating an antigen from HL-60 cells. No. 5 peak at 2
The peak of No. 6 of the 8 kDa antigen contains the 29 kDa antigen.

FIG. 5. Purified 28 kDa antigen (lane 2) and purified 29 kD
FIG. 9 is a view showing a western blotting pattern of a antigen (lane 3).

FIG. 6 is a diagram showing an absorption test of a patient antibody by HMG-1 and HMG-2. In the positive control, a band was detected (1, 3), but when the antibody was absorbed, the band (2, 3) was detected.
4) disappeared.

FIG. 7: HMG-1 and HMG-2 prepared from human thymus tissue
HMG-1 and HMG- prepared from porcine thymus tissue
FIG. 2 shows the results of Western blotting of a commercially available mixture of bovine HMG-1 and HMG-2. All human, porcine, and bovine HMG antigens reacted with sera from patients with refractory ulcerative colitis. Only in this example, 15% of polyacrylamide in SDS-PAGE was used.

FIG. 8 shows purification of HMG-1 and HMG-2 from porcine thymus.

FIG. 9 is a diagram showing the results of Western blotting of 5 patients with intractable ulcerative colitis and 3 healthy subjects using a mixture of swine HMG-1 and swine HMG-2 as an antigen.

FIG. 10: Anti-HMG-1 antibody and anti-HMG- by ELISA
It is a figure showing the measurement result of two antibodies. Pig HMG-1 (Fig. 10-
1), HMG-2 (Fig. 10-2) and human HMG-1 and human HMG-
In the two mixtures (FIG. 10-3), a dose-dependent straight line is obtained.

FIG. 11 is a diagram showing the distribution of each disease when HMG-1 is used as an antigen.

FIG. 12 is a diagram showing the distribution of each disease when HMG-2 is used as an antigen.

FIG. 13 is a diagram comparing the amino acid sequences of human, pig, bovine and rat HMG-1.

FIG. 14 is a diagram comparing the amino acid sequences of human, pig, bovine and mouse HMG-2.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Above ▲ Too ▼ Yuko 2-C, Urushinodate Kobei, Urushino-kan Kobei, Matsugasaki, Sakyo-ku, Kyoto-shi, Kyoto (72) Inventor Mao Tanaka Nakagyo, Kyoto-shi, Kyoto 9 Grande Acai I3-B, Nakanomachi, Sanjo-dori Yanagaba-ba, Higashiiri-ku, Tokyo (72) Inventor Kazukazu Nakao 4-1-2, Oeda-Kitakutsukakecho, Nishikyo-ku, Kyoto, Kyoto (72) Inventor Mitsuteru Yoshida Nagareyama, Chiba Prefecture Nishihatsuishi 3-chome 472-21 (72) Inventor Hitoshi Shirakawa 1-14-2-505 Nishibenzai, Asaka-shi, Saitama Prefecture

Claims (9)

[Claims] 1. A polypeptide selected from the HMG-1 family, a polypeptide selected from the HMG-2 family, or a fragment thereof, which contains at least one fragment that can react with an antibody of an autoimmune disease patient. Diagnostic agent for autoimmune diseases. 2. The autoimmune disease includes rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, Behcet's disease, scleroderma, primary biliary cirrhosis, microscopic polyangitis / polyarteritis nodosa, ulcerative colon The diagnostic agent according to claim 1, wherein the agent is inflammation or Crohn's disease. 3. The polypeptide according to claim 1, wherein the polypeptide is human, bovine, porcine, chicken, mouse, or rat HMG-1 or HMG-2.
The diagnostic agent according to claim 1, which is selected from the group consisting of: 4. A polypeptide selected from the HMG-1 family, a polypeptide selected from the HMG-2 family, or a fragment thereof, containing at least one fragment that can react with an antibody of an autoimmune disease patient. A kit for diagnosing an autoimmune disease. 5. The autoimmune disease includes rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, Behcet's disease, scleroderma, primary biliary cirrhosis, microscopic polyangitis / polyarteritis nodosa, ulcerative colon The kit according to claim 4, wherein the kit is inflammation or Crohn's disease. 6. The method according to claim 6, wherein the polypeptide is human, bovine, porcine, chicken, mouse, or rat HMG-1 or HMG-2.
The kit according to claim 4, which is selected from: 7. A method for detecting an antibody of a patient with an autoimmune disease, which comprises a polypeptide selected from the HMG-1 family, a polypeptide selected from the HMG-2 family, or a fragment thereof, A method comprising reacting a reagent containing at least one fragment capable of reacting with an antibody of an immune disease patient with a body fluid component of an autoimmune disease patient. 8. The autoimmune disease includes rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, Behcet's disease, scleroderma, primary biliary cirrhosis, microscopic polyangitis / polyarteritis nodosa, ulcerative colon 8. The method according to claim 7, wherein the disease is inflammation or Crohn's disease. 9. The method of claim 8, wherein said polypeptide is selected from human, bovine, porcine, chicken, mouse, rat HMG-1 or HMG-2.