JP3472048B2 - Diagnostics for autoimmune diseases - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to high mobility group protein-1 (HMG) to which antibodies of patients with autoimmune diseases react.
-1), high mobility group protein-2 (HMG-2), or a kit for diagnosing an autoimmune disease or a kit for diagnosing an autoimmune disease using a fragment of the polypeptide, and a method for detecting an antibody in a patient with an autoimmune disease It is a thing.
In particular, rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, Behcet's disease, scleroderma, primary biliary cirrhosis, microscopic polyangiitis / polyarteritis nodosa,
Antibodies from patients with ulcerative colitis and Crohn's disease
Rheumatoid arthritis, systemic lupus erythematosus using HMG-1, HMG-2, or fragments of these polypeptides,
Sjogren's syndrome, Behcet's disease, scleroderma, primary biliary cirrhosis, microscopic polyangiitis / polyarteritis nodosa, ulcerative colitis, and diagnostic kits for Crohn's disease The present invention relates to a method for detecting an antibody.

[0002]

It has been reported that various anti-neutrophil cytoplasmic antibodies (ANCA) are present in autoimmune diseases and inflammatory diseases. ANCA is an autoantibody detected by the indirect fluorescent antibody method (IIF), and its staining pattern shows cytoplasmic-ANCA (cANCA) and perinuclear-ANCA (pANCA).
To be distinguished. cANCA is detected as frequently as 80% in Wegener's granulomatosis, and its antigen is 90% or more of proteinase.
-3 (proteinase-3: PR-3). On the other hand, pANCA is microscopic polyarteritis, pauci-imm
une type necrotizing crescenti
c gromerulonephritis (NCGN), with a high frequency of 80%, and 80% of the antigen is myeloperoxidase (myerop
eroxidase: MPO-ANCA). As described above, the measurement of antibody having high disease specificity enables early diagnosis and differential diagnosis of vasculitis syndrome.

Recently, chronic inflammatory bowel disease (IBD) such as ulcerative colitis (UC), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE),
PANCA is recognized in patients with 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 relationship with the etiology and pathological condition has been studied.
The specificity for A is low, suggesting the presence of other antigens.

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

Lactoferrin (Lactofin) 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, etc., but various reports have been made specifically for these diseases. At present, the corresponding antigen has not yet been determined. Antigens corresponding to other pANCA-positive diseases have not been identified as well.

[0006] The standard clinical diagnosis of ulcerative colitis and Crohn's disease includes endoscopy and X-ray examination. These tests have the disadvantages of being expensive, painful and lengthy to the patient for the patient. On the other hand, the detection of pANCA by the indirect fluorescent antibody method has recently been reported as a serological diagnosis 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 and used, the result depends on the state of the cells and the immobilization technique. It has the drawback of being unreliable and is not yet in widespread use. No autoantibodies have been found for Crohn's disease. As described above, at present, a specific and convenient method for diagnosing ulcerative colitis or Crohn's disease using patient serum has not been developed.

In rheumatoid arthritis and systemic lupus erythematosus, various autoantibodies such as rheumatoid factor or antinuclear antibody are produced, but it is detected in scleroderma, polymyositis, Behcet's disease, periarteritis nodosa, etc. There are few autoantibodies, and clinical symptoms predominate in the diagnosis. In these diseases, diagnosing the disease at an early stage,
It has been pointed out that the prognosis is important to give sufficient treatment so as not to progress to a mature disease, but a serologically convenient diagnostic method for early diagnosis has not been developed.

[0008] In general, when an autoimmune disease is suspected, a test for antinuclear antibody is used as a diagnostic method in primary screening. Antinuclear antibody is a generic term for autoantibodies that use nucleic acids of cell nuclei and various nuclear protein components as antigens, and there are various types. The indirect fluorescent antibody method is mainly used as a method for detecting antinuclear antibodies. As an antinuclear antibody,
Anti-DNA antibody, anti-histone antibody, anti-E
NA antibody, anti-centromere antibody, anti-nucleolar antibody, etc. are presumed. However, it has been pointed out that the measurement of antinuclear antibodies 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 drawback is that this test method is judged by macroscopic observation. Therefore, the judgment standard and the judgment technique are non-objective.

However, the measurement of antinuclear antibody by the indirect fluorescent antibody method is indispensable for diagnosing various clinical diseases such as systemic lupus erythematosus and grasping the clinical picture even though they have such drawbacks. . On the other hand, autoantibodies (autoantigens) that are changed to antinuclear antibodies for the above reasons
It is also true that there is a need for a simple and objective primary screening method for autoimmune diseases, which is characterized by the identification of the above-mentioned, and there is no disparity between institutions.

[0010]

Therefore, identifying an autoantigen of a patient with an autoimmune disease and detecting an autoantibody using the autoantigen is a method of confirming the diagnosis of an autoimmune disease and performing an appropriate treatment. It opens the way for policy establishment.
Therefore, it is desired to identify and isolate a common autoantigen that appears in autoimmune diseases, and to develop a simple antibody detection method using this antigen.

[0011]

[Means for Solving the Problems] As a result of intensive studies to overcome the above-mentioned conventional problems, the present inventors have found that antibodies in the serum of autoimmune diseases, particularly pANCA-positive ulcerative colitis, are detected. The high mobility group protein-1 (H), a protein known as a novel antigen to which this antibody reacts, was used.
MG-1) and high mobility group protein-2 (HMG-2)
The first successful isolation and identification of By constructing an ELISA system using this HMG-1 and HMG-2, rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, Behcet's disease, scleroderma, primary biliary cirrhosis, microscopic Antibodies in patients with experimental polyangiitis / polyarteritis nodosa, ulcerative colitis, and Crohn's disease show a high positive rate, and this ELISA system is compared with the indirect fluorescent antibody assay for antinuclear antibodies , Which is relatively sensitive, simple, reliable and objective, the detection of antibodies to the antigen can be detected in rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, Behcet's disease, scleroderma, primary Biliary cirrhosis, microscopic polyangiitis / polyarteritis nodosa, ulcerative colitis,
They have found that they can be used as markers for diagnosis of patients with Crohn's disease, and have completed the present invention.

The HMG antigen has so far been measured as one of antinuclear antibodies in autoimmune diseases, not as an ANCA antigen. Dennis JS et al. Reported that anti-HMG-1 antibody was 10.3% and anti-HMG-2 antibody was 6.9% in patients with systemic lupus erythematosus, and both antibodies were 0% in mixed connective tissue disease and rheumatoid arthritis ( Science 215, 1245-1247, 1982). In addition, Briolay J. et al. Immunoblotted in systemic lupus erythematosus, rheumatoid arthritis, and scleroderma.
As a result of detecting HMG-1 antibody and anti-HMG-2 antibody, it is reported that both antibodies have no diagnostic value (Autoimmunity
2, 165-176, 1989). There seems to be a problem with the purity of HMG antigen, the ELISA method, the immunoblot method, etc., the condition of the patient measured and the number of samples, but at this point HMG-1 and HMG
The invention of diagnostic drug using -2 has not been completed. But anti
As for HMG-1 antibody and anti-HMG-2 antibody-positive cases, 39% of juvenile rheumatism patients who are antinuclear antibody-positive are reported to be positive for anti-HMG-1 antibody and / or anti-HMG-2 antibody (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 the positive rate was measured in various diseases. As a result, a statistically significant difference was observed in the above 9 diseases as compared with healthy people. As a result, the present invention has been completed.

The present invention contains at least one of a polypeptide selected from the HMG-1 family, a polypeptide selected from the HMG-2 family, or a fragment thereof capable of reacting with an antibody of an autoimmune disease patient. The present invention relates to a diagnostic agent for autoimmune diseases, a kit for diagnosing these diseases, and a method for detecting antibodies in patients with autoimmune diseases.

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

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

As a result, the object of the present invention is achieved.

[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 protein contained in the chromatin structure, and is a protein universally contained in all higher animals and plants. It is also known that it is abundant not only in the nucleus but also in the cytoplasm. Although its physiological function is not clearly understood, HMG binds to DNA, but when it binds to DNA, it is not specific to the nucleotide sequence but relaxes the double helix structure.
It is thought that it functions as an extremely wide range of transcription promoting factors and nucleosome relaxing factors, that is, it changes the higher-order structure of the to the optimal structure to enhance transcription activity. There are several types of HMG.

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

HMG-1 (high mobility group protein-
1) Family means human HMG-1 shown in SEQ ID NO: 1
A polypeptide having 90% or more amino acid homology with, for example, bovine HMG-1 (SEQ ID NO: 3),
HMG- such as pig (SEQ ID NO: 4), rat (SEQ ID NO: 5)
Including 1. Human HMG-1 is preferable, but pig, cow, and rat can be used because of its high homology. these
A comparison of the amino acid sequences of HMG-1 is shown in FIG.

On the other hand, HMG-2 (high mobility group prote
in-2) family means human HMG shown in SEQ ID NO: 2.
-2 refers to a polypeptide having 80% or more amino acid homology with porcine HMG-2 (SEQ ID NO: 6), bovine HMG
-2 partial sequence (SEQ ID NO: 7), HMG-2 such as chicken HMG-2 (SEQ ID NO: 8), chicken HMG-2a (SEQ ID NO: 9), mouse HMG-2 (SEQ ID NO: 10). Human, porcine and bovine HMG-2 are preferred. 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 has one or more amino acids deleted, substituted or added, or
Also included are polypeptides that are capable of reacting with antibodies of patients with autoimmune disease.

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

Since HMG-1 and HMG-2 are universal proteins possessed by all cells, they can be prepared by extraction from any organ, tissue or cell. For example, human thymus, pig thymus, calf thymus, human placenta, neutrophil, HL-60 cell line and the like. Extraction and purification methods are known and include, for example, GH Goodwin et al. (Biochemica et Bio
phisica Acta, 405, 280-291, 1975), 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). In addition, a bovine HMG-1 and HMG-2 mixture 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 tissues or cultured cells producing the polypeptide. It can be produced by expressing. Polypeptides with one or more amino acids deleted, substituted, or added can be prepared by well-known methods, for example, site-directed mutagenesis, 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 a deletion mutation using a phage and expressing it.
As the host cell, either a prokaryote or a eukaryote can be used. Examples thereof include bacteria such as Escherichia coli and Bacillus, yeasts, molds, insect cells, mammalian cells and the like.
For purification of the polypeptide, known methods, for example, chromatography such as gel filtration chromatography, ion exchange chromatography, affinity chromatography, reverse phase liquid chromatography and the like can be used alone or in combination. Reverse phase HPLC and ion exchange chromatography can be preferably used. Reverse phase HP
As the LC column, various commercially available columns can be used, but preferably a protein separation column, for example, a YMC-Protein RP column (manufactured by WMC) can be used.
As ion exchange chromatography, for example, po
lybuffer-exchanger PBE94 column chromatography,
Alternatively, a monoQ column (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, and microscopic polyangitis / Polyarteritis nodosa, ulcerative colitis, and Crohn's disease.

The ulcerative colitis referred to in the present invention is one having the following symptoms. First, ulcerative colitis refers to an idiopathic or nonspecific inflammatory disease of the large intestine, particularly the rectum, which mainly affects the mucous membrane and submucosa. The disease is common in adults under the age of 30, but is also found in children and those over the age of 50. The cause is unknown, and the involvement of immunological mechanisms, genetic factors, and psychological factors is considered. It usually presents with bloody diarrhea and various systemic symptoms. There is a tendency for malignant changes over a long period of time when the entire large intestine is removed. What is refractory ulcerative colitis?
Cases in which the above-mentioned ulcerative colitis satisfies the condition of being chronically persistent, still active for more than 6 months after relapse, or having frequent relapses, while undergoing strict medical treatment. Say.

Crohn's disease is an inflammatory bowel disease of unknown origin that may occur in the entire digestive tract including the small intestine and the large intestine.
It is characterized by noncontiguous or segmental lesions, longitudinal ulcers, full-thickness inflammatory lesions (mass or stenosis), noncaseating granulomas, etc., and is often young (15-24 years). No autoantibodies have been found and there is no convenient serological diagnostic method.

Rheumatoid arthritis (RA) is a chronic progressive disease of unknown cause. The essential form of RA is chronic synovitis that does not show a tendency to spontaneously cure, and infiltration of lymphocytes, angiogenesis, and stratification of synovial cells are accompanied by proliferation of synovial cells. Small joints of limbs and large joints such as knees, elbows, shoulders and hips are placed in symmetry. The continuous synovial inflammation and proliferation of inflamed tissues in such joints eventually destroy cartilage and bone, resulting in joint deformation and disability.

Systemic lupus erythematosus is a multi-organ disease, which causes various diseases. Non-infectious inflammatory lesions occur in almost all tissues and organs throughout the body, including skin symptoms such as butterfly erythema, oral ulcers, arthritis, lupus nephritis, and central nervous system lesions. Also, although various autoantibodies are produced, disease-specific autoantibodies have not been identified. Although the appearance rate of antinuclear antibody by the indirect fluorescent antibody method is almost 100% diagnostically, its specificity is not high. It is an overwhelmingly prevalent disease among young women. The 5-year survival rate has exceeded 95% in recent years, but the course is long-term and remissions and exacerbations are often repeated.

Sjogren's syndrome is a chronic inflammatory disease of exocrine glands mainly composed of lacrimal glands and salivary glands, or water-secreting tissues, and is a dry mouth and eye syndrome due to a decrease in tear and salivary secretion. Most of them are only dry syndrome, but some may be associated with disorders such as thyroid, lung, gastrointestinal, liver, and kidney, and about 30 to 40% are rheumatoid arthritis, systemic lupus erythematosus, and scleroderma. These may be duplicated and clinically exhibit various medical conditions. Erythrocyte acceleration, hypergammaglobulinemia, and various autoantibodies are observed. In immunological tests, autoantibodies were diverse, and rheumatoid factor (80
%), Antinuclear antibodies are found in the majority.

Scleroderma (systemic sclerosis) is a connective tissue disease associated with skin hardening and organ damage, but there are various types of disease, and it is not always systemic or progressive. Extensive skin hardening (general scleroderma) throughout the body and esophagus, intestine, lungs,
There are various types of organs such as kidney and thyroid gland, which have a poor prognosis with rapid progression, to those with a favorable prognosis called CREST syndrome in which hardening of the skin is limited to the face and fingers and internal lesions gradually appear. . Among antinuclear antibodies, anti-Sc1-70 antibody, which is an autoantibody against topoisomerase 1, has high specificity for pervasive this disease (positive rate 30%). Anti-centromere antibody has high diagnostic value for CREST syndrome, and for the overlapping syndrome of this disease and polymyositis, anti-Ku antibody has high diagnostic value.

Primary bile cirrhosis is a chronic disease (symptomatic) characterized by skin pruritus and jaundice, which often occurs in middle-aged and older women, but is asymptomatic without any of these symptoms. There is also a nature. Beginning with the destruction of the interlobular bile duct, the fibrous tissue gradually grows, and progresses to the histological image of cirrhosis. These bile duct-centered changes are reflected in the clinical picture characterized by persistent jaundice. The etiology is unknown. About 30% of asymptomatic and symptomatic autoimmune diseases (Sjogren's syndrome, rheumatoid arthritis, etc.)
Are known to merge.

Polymyositis (PM) is a disease in which skeletal muscle systematically causes non-suppurative inflammation, resulting in myalgia and muscle weakness. Reddish purple erythema of the eyelid (heliotrope) even with the same PM
Those with keratomatous erythema (Gottron sign) on the back of the finger joint are called dermatomyositis (DM). Even in the striated muscles, the proximal muscles are damaged first, making it impossible to stand up or raise the hands. In severe cases, the neck and head cannot be supported. Since a part of this disease is accompanied by cancer and malignant tumor, Behcet's disease, in which treatment of cancer is indispensable and precedent, has recurrent aphthous ulcer of the oral mucosa (incidence rate 99) as a clinical main symptom. %), Skin symptoms (84%)
Erythema nodosum, folliculitis, acne-like eruption, subcutaneous thrombophlebitis, and razor loss and needle reaction as hyperirritability of the skin, and iris cyclitis and retinochoroiditis in eye symptoms (90%). It is a refractory disease of unknown cause that indicates Other side effects include arthritis and digestive 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 accurate treatment early.
Arteritis causes various lesions throughout the body, but the most important ones are skin lesions, renal lesions, digestive tract lesions, and central nervous system lesions. Most of them are male, and no antinuclear antibody is found. The antibody refers to a component that is present in a body fluid of an autoimmune disease and is caused by a specific antigenic substance and contained in the body fluid. For example, the term “antibody for ulcerative colitis patient” or “antibody for rheumatoid arthritis patient” refers to a body fluid component contained in body fluid such as serum of a patient diagnosed with ulcerative colitis or rheumatoid arthritis, respectively. Examples of antibodies for autoimmune diseases include IgM, IgG, IgE, IgD, IgA and the like.

The diagnostic agent of the present invention includes a polypeptide contained in the above HMG-1 family or HMG-2 family, or a fragment thereof. HMG-1 for diagnostic use
It suffices if at least one kind of the polypeptide of the family, the HMG-2 family of polypeptides or a fragment thereof is contained. 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. Therefore, it may contain an additional component capable of detecting 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 polypeptides included in the HMG-1 family or HMG-2 family, or fragments thereof can be used as a diagnostic kit. Diagnostic kits include, for example, a polypeptide for HMG-1 family or HMG-2 family, or an ELISA plate on which these fragments are immobilized, and an antigen-antibody complex bound to an antibody of an autoimmune disease patient. And a reagent for This reagent contains components compatible with methods such as a precipitation reaction method, an ELISA method, a RIA method, and a Western blotting method. As a reagent for detection, in the ELISA method, for example, a secondary antibody reagent can be mentioned. The secondary antibody reagent is goat or mouse anti-human IgG or anti-human (IgA + IgG + IgM)
It reacts with IgG, IgM, and IgA. These secondary antibodies may be labeled with a labeling agent generally used in 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 (eg, FAD, FMN, ATP,
Biotin, heme, etc.), fluorescein derivatives (eg, fluorescein isothiocyanate, fluorescein ophthalvamil, etc.), rhodamine derivatives (eg,
Tetramethylrhodamine B isothiocyanate, etc.),
Umbelliferone and 1-anilino-8-naphthalene sulfonic acid, luminol derivatives (eg, luminol, isoluminol, etc.) and the like can be used. Preferred are alkaline phosphatase and peroxidase, the substrate is para-nitrophenyl phosphate in the former case, and tetramethylbenzidine (TMBZ) in the latter case. The binding between the antibody and the labeling agent is publicly known as described in a textbook (for example, "Seikagaku Chemistry Laboratory 5 Immunobiochemistry Research Method", Tokyo Kagaku Dojin, 1986, p102-112). Can be appropriately selected from the above methods. 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 is such that the antigen is contained in a suitable container, a carrier such as a resin, a membrane or a film,
Alternatively, a form in which the antigen is fixed to a carrier such as a container, a resin, a film or a film may be mentioned. 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, polymethylene methacrylate, dextran derivatives (Sephadex, etc.), agarose gel (Sepharose, biogel, etc.), polysaccharides such as cellulose (paper desk, filter paper, etc.), glass, silica gel, An inorganic polymer compound such as silicone can be exemplified. These may have introduced functional groups such as amino groups, carboxyl groups, carbonyl groups, hydroxyl groups and sulfhydryl groups. As a suitable example, polystyrene,
Examples include polyvinyl chloride.

The shape of the carrier is flat (microtiter plate, disk, etc.), particulate (beads, etc.), tubular (test tube, etc.), fibrous, film-like, fine particle (latex particle, etc.), capsule-like, It may be in any form such as an endoplasmic reticulum, and a carrier having a suitable shape can be appropriately selected according to the measuring method. Suitably, it is a 96-well microtiter plate capable of treating a large amount of sample at a time in an ELISA system, for example, EB plate (manufactured by Lab Systems), H
Type plate, C type plate (Sumitomo Bakelite), Maxisorp plate (Nunc) and EI
An example is A./RIA plate (manufactured by Coaster).

The carrier is bound to the antigen by a known method such as a physical adsorption method, an ionic binding method, a covalent binding method, or an encapsulation method (for example, "immobilized enzyme" edited by Ichiro Chibata, March 20, 1975).
JP, Kodansha Co., Ltd.) may be employed, and the physical adsorption method is particularly preferable because it is simple. The antigen and the carrier may be bound directly, or may be bound between the antigen and the carrier via another substance (spacer) or the like. The immobilized antigen can be treated with a blocking agent such as gelatin or BSA to prevent nonspecific binding.

The method of detecting an antibody against autoimmune disease according to the present invention comprises a polypeptide selected from the HMG-1 family, H
A method comprising 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. As the conditions for reacting the antigen with the antibody, conditions well known to those skilled in the art are applied. A method known to those skilled in the art can also be applied to the detection of the antigen-antibody reaction product.
Examples of the detection method include a precipitation reaction method, an ELISA method, a RIA method, and a western blotting method. For example, after reacting appropriately diluted patient serum with an antigen and washing,
Anti-human labeled with secondary phosphatase, alkaline phosphatase
IgG antibody was added and reacted, and then alkaline phosphatase substrate p-nitrophenyl phosphate was added for color development, and the absorbance at 405 nm was measured to measure anti-HMG-
1 and anti-HMG-2 antibody can be measured.

In the kit, in addition to the HMG antigen, if necessary, an appropriate reagent corresponding to the assay method can be appropriately selected from 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 that reacts with the antibody is screened, and the antigen is HM.
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 1 and HMG-2 antigen will be described.

First, blood is collected from a patient with ulcerative colitis to obtain a serum component. Next, the presence of anti-neutrophil cytoplasmic antibody (ANCA) in the serum component is measured by the fluorescent antibody method. On the other hand, the neutrophil fraction is separated from the peripheral blood of healthy individuals by the gravity centrifugation method. Then process this neutrophil fraction,
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, rapidly cooled with ice cold, and then subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). To do. After the electrophoresis, the protein band is transferred onto a nylon membrane by a conventional method, and nonspecific binding is blocked using skim milk or the like.

On the other hand, the serum of ANCA-positive patients was treated with protein A.
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,
The band is detected by chemiluminescence with a detection agent such as ECL kit (manufactured by Amersham), and the presence of the antigen is determined.

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

Alternatively, the above antibody can be used to identify a cell line producing an antigenic peptide. An antigenic peptide can be produced using such a cell line, and can be 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 shown later in the example, 10 out of 24 ANCA positive patients
At least one 28 / 39.5 / 44/47 / 58kDa antigen was detected in humans, of which 7 had 28kDa 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 sera of patients with ulcerative colitis negative for ANCA by the indirect fluorescent antibody method. The molecular weight of the antigen in the present invention is 10% SDS-PAGE.
It means what was decided in.

When the cells producing the 28 kDa antigen were screened by the above-mentioned screening method, the HL-60 cell line (ATCC CCL-
240) has a 28kDa antigen, and the results of Western blotting revealed that 29kDa in addition to the 28kDa antigen.
It was suggested that the antigen was present. From this HL-60 cell line, 28 and 29 kDa antigens can be purified. 28kDa
A known protein purification method can be applied to the purification of the antigen and the 29 kDa antigen. For example, use HL-60 cell line with 5% FC
The cells are cultured in RPMI1640 medium supplemented with S, the cells are dissolved in 6M guanidine hydrochloride, and ultrasonic treatment is performed to inactivate the protease. The protein is completely dissolved and concentrated by dialysis, for example ultrafiltration, and the solution is replaced with PBS. From this aqueous solution, 28 kDa antigen and 29 kDa antigen can be purified. Suitably reverse phase HPLC may be used. The 28 kDa antigen and the 29 kDa antigen having a purity of 90% or more can be purified by fractionating by reverse phase HPLC using a gradient of acetonitrile. Reversed-phase HPLC conditions using a concentration gradient of acetonitrile can be performed under conditions well known to those skilled in the art.

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

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

Furthermore, HMG- from human and porcine thymus
When 1 and HMG-2 fractions were prepared and subjected to Western blotting, it was found to react with the patient antibody.

The amino acid sequence of human HMG-1 differs from that of human because it differs from pig, bovine, and rat (FIG. 13) by only two, one, and two amino acids, respectively. Animal HMG-1 can be used as an antigen for ELISA. Regarding HMG-2, human and pig differ only in two amino acids (Fig. 14),
After all, it is thought that it can be used instead of human.
As for bovine, as shown in FIG. 14, only the partial sequence has been determined, and the sequence reported above is also considered uncertain (the bovine sequence is reproduced from Protein Data Bank PIR B61611). HMG-1 and HMG-2 are considered to be proteins whose amino acid sequences are fairly well conserved among species, and in the light of the high degree of similarity between humans and pigs, it is considered that cows 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 is also suggested by the fact that it did not react 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 methods described in the above references. The preparation method from human thymus tissue will be briefly described as an example. Cut the thymus tissue of the child into small pieces and 0.075M NaCl / 0.02
Suspend the cells in 5M EDTA (pH 7.5) and disrupt the cells with a Polytron homogenizer. The chromatin-containing precipitate is collected by centrifugation, suspended in 0.35M NaCl (pH 7), and HMG bound to chromatin is released by a homogenizer treatment. The insoluble matter is removed by centrifugation, and the resulting 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 carried out to obtain HMG-1 and HMG-2 fractions. The HMG-1 and HMG-2 fractions reacted with the antibody of patients with ulcerative colitis 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 standard with a purity of 95% or more prepared and purified according to (92) was used. These reacted with patient serum as well as human HMG-1 and HMG-2.

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

An ELISA system was constructed according to the method described above using purified pig HMG-1 and HMG-2. That is, 96-well ELI
5 μg / ml pig in each well of SA plate (Nunc)
Add 50 μl of HMG-1 or HMG-2 and add 24-3 at 4 ℃.
Let stand for 6 hours. After removing the excess antigen, blocking with 5% BSA is performed. Add patient serum appropriately diluted with 5% BSA and let stand for 2 hours at room temperature. 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 para-nitrophenyl phosphoric acid (Sigma) solution (10% diethanolamine solution) is added, and the mixture is reacted at room temperature for 20-25 minutes, and the absorbance at 405 nm is measured.

Using this system, anti-HMG-1 antibody and anti-HMG
When a standard curve was assayed using sera from patients with ulcerative colitis, which was positive for both -2 antibodies, a concentration-dependent straight line was obtained for both antigens, and anti-HMG-1 and anti-HMG-2 antibodies can be measured. It was found that there is an anti-HMG in each disease using this ELISA system.
It was suggested that -1 antibody and anti-HMG-2 antibody can 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, and microscopic polyangiitis / Anti-HMG-1 antibody and anti-HMG-2 antibody were separately measured for polyarteritis nodosa, ulcerative colitis, Crohn's disease patients and healthy subjects. As a result, primary biliary cirrhosis, ulcerative colitis,
And 7 diseases except Behcet's disease, HMG-1 is more HMG
The antigenicity was stronger than -2 and the positive rate was higher. Regarding HMG-1, a statistically significant difference was shown in 9 diseases other than polymyositis / dermatomyositis as compared with healthy subjects. Furthermore, when compared with the antinuclear antibody positive rate by the indirect fluorescent antibody method, the anti-HMG antibody positive rate showed a value equal to or higher than the antinuclear antibody positive rate in 7 out of 10 diseases. This indicates that the measurement of anti-HMG-1 antibody and anti-HMG-2 antibody may be an alternative method for diagnosing autoimmune diseases that can be used instead of the detection of antinuclear antibody by the indirect fluorescent antibody method. Moreover, it is considered that autoimmune diseases can be diagnosed more broadly and reliably by using the anti-nuclear antibody test together.

HMG-1 and H as the corresponding antigens of pANCA
Although MG-2 was identified, these were originally identified as nuclear proteins and may be antigens of an antibody that has been called an antinuclear antibody until now. Therefore, not only for pANCA-positive diseases but also for antinuclear antibody-positive diseases, anti-HMG-1 and H
It is considered that MG-2 antibody is detected. Furthermore, since the ELISA method has higher sensitivity than the indirect fluorescent antibody method, anti-HMG-1 antibody and anti-HMG-2 antibody can be detected even in diseases that have been considered to have a low ANCA negative or positive rate until now. 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, malignant tumor, amoeba abscess, Sweet disease, multiple sclerosis, Alzheimer's disease, Hashimoto's disease, hyperthyroidism, and erythroleukemia.

Neutrophils have a high degree of motility and exhibit a strong phagocytosis. It is one of the cells that first migrates to the local inflammation when inflammation occurs in the body, destroys and melts the inflamed tissue, and finally removes and absorbs the injured tissue. Even in tissue injury in autoimmune diseases, there are many cases where neutrophils locally infiltrate, and it is considered to play a role in inflammation. Proteins inside neutrophils are exposed to T cells and B cells due to frequent neutrophil infiltration due to continuation of inflammation or repeated remission and exacerbation and formation of dense spheres due to phagocytosis of injured tissue, and antibodies are eventually produced. It is thought to be done. If the antibodies thus produced are anti-HMG-1 antibody and anti-HMG-2 antibody (mainly anti-HMG-1 antibody), these antibodies are produced from the early onset of autoimmune disease. There is a possibility that early autoimmune diseases can be diagnosed by measuring anti-HMG-1 antibody and anti-HMG-2 antibody.

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

Further, the present invention also includes a kit for measuring anti-HMG-1 antibody and anti-HMG-2 antibody by ELISA, but as an alternative method, peripheral blood lymphocytes and HMG of patients with autoimmune disease or inflammatory disease are also included. The disease specificity can be assayed by examining the responsiveness to -1 and HMG-2. That is, whether T lymphocytes proliferate in response to HMG-1 and HMG-2 or their immunoreactive synthetic peptides, or whether γ-interferon is produced by macrophages in the same assay system By doing so, the disease can be detected.

The present invention will be described below with reference to examples.

[0065]

【Example】

(Example 1) Detection of neutrophil cytoplasmic antibody (ANCA) by indirect fluorescent antibody method Peripheral blood was collected from 35 patients (16 men, 19 women) with ulcerative colitis and centrifuged (4 ° C, 13 ° C). (2000 rpm for 1 minute) to obtain a serum component. About this serum, anti-neutrophil cytoplasmic antibody (AN
The positive rate of CA) was measured by the indirect fluorescent antibody method. As a control, serum components obtained by similarly treating blood collected from 10 patients with Crohn's disease (9 males and 1 female) were also measured.

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

First, peripheral blood is subjected to specific gravity centrifugation using Ficoll pack to separate neutrophils, and 10 ⁵ cells are attached per slide by cytospin. This was air-dried with cold air from a dryer, and PBS (0.8% NaCl / 0.02% KCl / 10mM Na
₂ Wash with 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 allowed to react for 1 hour at room temperature in a humid chamber. After the reaction is complete, wash with PBS. FITC-labeled rabbit anti-human IgG F
(ab ') ₂ antibody (manufactured by Serotech) was diluted 1:20 with PBS,
Place 20 μl of the solution on the plate and incubate at room temperature in a moist chamber.
React for minutes. After the reaction is complete, wash with PBS. After washing, it is embedded in glycerol diluted 1: 9 with PBS and observed with a fluorescence microscope. The results of detection by this method are shown in Table 1. Of the 35 patients with ulcerative colitis, 24 had ANCA (69% positive rate; see Table 1).

The antineutrophil cytoplasmic antibody (ANCA) in the serum components of 24 positive patients was mostly pANCA (22 out of 24 pANCA, 2 out of 20 nuclea) as a staining pattern of the indirect fluorescent antibody method. ANCA) (see Table 1). Table 3 below
Patients Nos. 24 and 25 had Nuclea-ANCA.

[0069]

[Table 1]

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

Myeloperoxidase (MPO) (Elastin P
roducts) 5 μg / ml, cathepsin G (CaG) (INC Bio
chemical) 5 μg / ml, and lactoferrin (LF)
(Sigma) 10 μg / ml was prepared, and 50 μl / well, 50 μl / well and 10 μl / well were added to a 96-well microtiter plate, respectively.
0 μl / well was injected and coating was performed at 4 ° C. overnight. After coating, the solution was removed, and PBS containing 5% BSA (fetal bovine serum) (5% BSA / PBS) was added and reacted for 30 minutes. Then, 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, use alkaline phosphatase (ALP) -labeled sheep anti-human IgG antibody (Immunotech SA) with 5% BSA / PBS.
It was diluted 000 times and added, and the mixture was reacted at room temperature for 24 hours. After completion of the reaction, the plate was washed 5 times with 1% BSA / 0.5% Tween 20 / PBS. After washing, p-nitrophenyl phosphate (final concentration 5 mg / ml)
10% diethanolamine solution (diethanolamine 50
(100 ml of distilled water + 450 ml of distilled water) was added and color was developed for 30 minutes at room temperature. After color development, the absorbance at 405 nm was measured.

By this method, anti-MPO antibody could not be detected in all cases, and out of 24 indirect fluorescent antibody method-positive patients, 9 were positive for anti-CaG antibody and 3 were anti-LF antibody. It was positive. The other 12 were unable to identify the corresponding antigens (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.

Peripheral blood was collected from a healthy person and the neutrophil fraction was prepared by centrifugation using Ficoll pack. 1
10 ⁶ neutrophils were suspended in 8 μl of PBS per well and sample buffer (0.2M Tris-HCl pH6.8 / 10% SDS / 25%
2-mercaptoethanol / 25% glycerol / 0.01% BP
2 μl of B) was added and immediately boiled for 10 minutes to give an antigen solution. This antigen solution was subjected to electrophoresis (SDS-PAGE) using SDS-polyacrylamide gel. After the electrophoresis, the protein 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 reaction for 2 hours. On the other hand, patient serum 320
Apply μl to ProSep A (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 20 mg / ml IgG solution.
The immobilon membrane prepared above was reacted with 1 ml of an IgG solution diluted 8-fold with 5% skim milk at 4 ° C. overnight. After washing
Myeloperoxidase-conjugated anti-human IgG antibody (Kirkegaar
d & Perry Laboratories, INC) 13 μg / ml was further reacted, and chemiluminescence was performed with an ECL kit (Amersham) to detect bands. The results are shown in Tables 2 and 3.

[0075]

[Table 2]

[0076]

[Table 3]

As a result of these results, anti-neutrophil cytoplasmic antibody (ANC
28 / 39.5 / 44/4 in the serum of 11 out of 24 patients positive for A)
It was found that there was antigen bound to either of the 7/50/58 kDa bands. In particular, it was confirmed that a substance that binds to the 28 kDa band was present in the serum of 7 out of 11 subjects. Five out of seven patients having this 28 kDa band were patients diagnosed with refractory ulcerative colitis (see FIG. 1).

On the other hand, from the sera of patients with ulcerative colitis in which ANCA was not detected by the indirect fluorescent antibody method (ANCA negative), 28
No antibody that bound to kDa was detected. In the control sera of patients with Crohn's disease, no antibody that binds to 28 kDa was detected.

Summarizing these results, patients with ulcerative colitis
Twenty-four of 35 were positive for ANCA by indirect immunofluorescence, and Western blotting confirmed that 11 of 35 had antigens that bind to ANCA (all 11 of these were ANCA by indirect immunofluorescence). Was positive). There are 7 patients with refractory ulcerative colitis out of 35, and 6 out of these 7 have ANCA.
An antigen that binds to was detected by Western blotting, and a band of 28 kDa was observed in 5 out of 6 people. No positive band was observed in the control sera of Crohn's disease patients (see FIG. 1).

At this point, these experimental results show that neutrophils
We suggest that the appearance of antibodies against 28kDa antigen may serve as a marker for predicting the severity (refractory degree) of ulcerative colitis. Isolation analysis of the antigen and a simple antibody detection system using the isolated antigen It shows the importance of the development of.

Example 4 In HL-60 cells, 28 kDa and 29 were obtained.
Confirmation of the presence of the kDa antigen The HL-60 cell line, which is a neutrophil-derived cell derived from promyelocytic leukemia, was cultured in RPMI1640 medium supplemented with 5% FCS, and the method described in Example 3 was used. Neutrophil lysates were made. Using this lysate, Western blotting was carried out in the same manner as in Example 3. The results are shown in Figure 2 along with the positive control using neutrophil lysate. This binds to the antibody fraction obtained from patients with ulcerative colitis
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 the 29 kDa antigen in close proximity to the 28 kDa antigen.

Example 5 Purification of 28kDa and 29kDa Antigens from HL-60 Cells HL-60 cell line confirmed to have antigen in Example 4 was treated with 5% F.
The cells were cultured in 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 was 2 × 10 ⁸ . 10 ml of 6 M guanidine hydrochloride was added and dissolved, followed by ultrasonic treatment (USP600 manufactured by Shimadzu Corporation) for 1 minute.
By this operation, the cells were completely lysed. The same amount of distilled water as this solution was added to dilute the solution twice, and the supernatant was recovered by centrifugation at 80,000 xg for 30 minutes. The supernatant YM3 (Amicon) ) Is attached to an Amicon ultrafilter (manufactured by Amicon), filtered while adding PBS, and then concentrated to a final volume of 4 ml.
Of PBS solution. This PBS solution was centrifuged at 80,000 xg 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. Proteins were eluted using a YMC-pack protein RP column (manufactured by YMC) with a concentration gradient of acetonitrile concentration of 16% to 48%. The results are shown in Fig. 3. The No. 9 peak 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 concentration gradient of acetonitrile concentration of 24% to 36%. The HPLC system used was an LC-7A system manufactured by Shimadzu. The results are shown in Figure 4. No.5 and N
The peak of o.6 was collected and centrifugally concentrated and dried. The sample concentrated and dried by centrifugation was electrophoresed on 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. The result of Western blotting of the purified antigen is shown in FIG. Purified antigen SDS-PA
In GE, the two proteins were separated and became distinguishable. Since both proteins reacted with patient sera, it was considered that they could not be separated by SDS-PAGE from cell lysates due to their close molecular weight, or could not be detected due to the low content of 29 kDa antigen in neutrophils. To be

Example 6 Determination of Partial Amino Acid Sequence and Homology Analysis The membrane containing the 28 kDa and 29 kDa bands recovered in Example 5 was dried and then used for amino acid sequence determination. Amino acid sequence is determined by Shimadzu's fully automated primary protein structure analyzer
This was done using a PPSQ-10 system. As a result, in the 28 kDa band, the N-terminal 32 partial amino acid sequences were determined. The sequences were 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-terminal were determined.
The sequences were 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 The amino acid sequence obtained in Example 6 was subjected to homology analysis. BLAST program (J.
Mol. Biol. 205, 403-410, 1982) was used to perform a homology search for all amino acid sequences contained in the known database PIR.As a result, the 29 kDa antigen was the non-histone nuclear protein HMG-1 (Reeck, GR NucleicAcids Res .17,
1197-1214, 1989) and 31 of 32 amino acids were in agreement. The 28-kDa antigen was HMG-2 (Majumdar, A. et al. Nucleic Aci
ds Res. 19, 6643, 1991) and 31 of the 32 amino acids were in agreement. The 22nd cysteine could not be identified for both antigens. Since cysteine cannot be detected without modifying the thiol group, the 22nd position is considered to be cysteine, and SDS-
The 28kDa and 29kDa antigens were identified as HMG-2 and HMG-1, respectively, in consideration of the molecular weight by PAGE.

(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, and then, And transferred to the immobilon film. IgG (0.024mg / ml) purified from HMG antigen-positive ulcerative colitis patient as the primary antibody
And a bovine HMG-1 / 2 mixture (0.5 mg / ml) were mixed at 1: 2 and reacted overnight at 4 ° C. to use an antibody solution. After washing as in Example 3, a secondary antibody was reacted and detected by ECL. As a result, the antibody absorbed in the bovine HMG mixed solution was 28 kDa and 29 kDa.
And the 28 kDa band of neutrophils disappeared (Fig. 6).
This also indicates that the 29 kDa and 28 kDa antigens are HMG-
1 and HMG-2.

(Example 9) Preparation of HMG-1 and HMG-2 fractions from human thymus tissue HMG-1 and HMG-2 were prepared from human, porcine and bovine thymus tissue, respectively.
It can be conveniently prepared according to the method described in the above literature. Child thymus tissue (13 g) is sliced into 30 ml of 0.075M NaCl / 0.025M
Suspend in EDTA (pH 7.5) Polytron homogenizer (Pol
Cells were disrupted for 2 minutes in ice water using 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 4 more times (however, homogenizing time is 1 minute), and precipitate 30 ml.
Suspended in 0.35M NaCl (pH7) and treated with a homogenizer (s
HMG bound to chromatin was released by peed 5, 1 min). This operation was repeated twice more to make a 90 ml solution. Insoluble matter was removed by centrifugation at 4 ° C, 2,000 xg for 30 minutes, and the resulting supernatant was made into a 2% trichloroacetic acid solution and left at 4 ° C for 1 hour. Then, insoluble proteins other than HMG-1 and HMG-2 were removed. Allowed to settle. The supernatant was collected by centrifugation (4 ° C, 2000 xg, 30 minutes), and 2-mercaptoethanol was added to a final concentration of 0.
Added to be 01M. 1.5 ml of 30% ammonia was added, 300 ml of acetone was immediately added, and the mixture was stirred and left overnight at 4 ° C. The 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 the antibodies of patients with ulcerative colitis by Western blotting (Fig. 7). Further, commercially available bovine HMG-1 and HMG-2 (manufactured by Wako Pure Chemical Industries, Ltd.) also reacted with the antibody of patients with ulcerative colitis (FIG. 7). Therefore, HMG-1 and HMG-2 are considered to be antigens for patients with ulcerative colitis.

Example 10 Pig Thymus-Derived HMG-1 and H
Purification of MG-2 HMG-1 and HMG-2 derived from porcine thymus were treated with M. Yoshida and K. S.
himura (J. Biochem. Tokyo, 95, 117-124, 1980), and Y. Adachi et al. (J. Chromatogr, 530, 39-46, 1992)
Purified according to the method of. The purification method will be briefly described. The chromatin fraction obtained in the same manner as in Example 9 was treated with 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 resulting supernatants were mixed. This fraction was made into a 2% trichloroacetic acid solution, centrifuged to remove the precipitate, and the supernatant was further added to 10%.
HMG-1 and HMG precipitated as a trichloroacetic acid solution
The precipitate containing -2 was collected by centrifugation. 3 ml 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) that had been washed and equilibrated with the same buffer solution in advance, and separation was performed (manufactured by Pharmacia).
FPLC system was used). Elution was performed with a linear concentration gradient of NaCl from 0 to 1 M. Figure 8 shows the elution pattern. By this fractionation method, HMG-1 and HMG-2 were obtained with a purity of approximately 95% or higher.

The HMG obtained here was confirmed to react with a patient antibody by Western blotting,
In addition, HMG-1 for the HMG-1 fraction and HMG-1 for the HMG-2 fraction
It was confirmed that there was no contamination (Fig. 7).

(Example 11) Detection of anti-HMG-1 antibody and anti-HMG-2 antibody in serum of patients with refractory ulcerative colitis by Western blotting Western blotting using swine HMG-1 and swine HMG-2 mixture as antigen I went. A mixture of porcine HMG-1 (0.5 μg) and porcine HMG-2 (0.5 μg) was dissolved in a sample buffer (Example 3), and after heat treatment according to a conventional method, SDS-PAGE.
I went. After SDS-PAGE, sera from 5 patients with 28kDa positive refractory ulcerative colitis, transferred to PVDF membrane, HRP-labeled anti-human
IgG antibodies were reacted in this order, and detection was performed by ECL. As a result, 4 out of 5 refractory ulcerative colitis patients were positive and 1 was 28 kDa positive, but the antigens were HMG-1 and HMG-.
It was not 2 (Fig. 9). From these results, the serum of patients with refractory ulcerative colitis has anti-HMG-1 antibody and / or anti-HMG-1 antibody.
The presence of HMG-2 antibody was shown.

(Example 12) Measurement of anti-HMG-1 antibody and anti-HMG-2 antibody by ELISA method ELISA using porcine HMG-1, porcine HMG-2, human HMG-1 and human HMG-2 mixture as an antigen. I went. 50 μl of porcine HMG-1 or porcine HMG-2 serially diluted from 6.25 μg / ml to each well of a 96-well maxisorp plate (manufactured by Nunc),
In the case of human, 50 μl of HMG-1 and HMG-2 (equal amount mixture) serially diluted from 6.25 μg / ml was 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. Using anti-HMG-1 antibody and / or anti-HMG-2 antibody-positive sera from refractory ulcerative colitis as standard serum, 50 μl of 40-fold diluted with 5% BSA was added, and the mixture was allowed to stand at room temperature for 2 hours. 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 the washing solution, 100 μl of 0.1% para-nitrophenylphosphoric acid (Sigma) solution (10% diethanolamine solution) was added and reacted at room temperature for 20-25 minutes, 40
Absorbance at 5 nm was measured. The calibration curve of the positive control is shown in FIG. A dose-dependent straight line was obtained with all three ELISAs. From this calibration curve, pig HMG-1 (Fig. 10-1) and HMG-2 were obtained.
(FIG. 10-2) Alternatively, it was found that the anti-HMG-1 antibody and / or the anti-HMG-2 antibody can be measured by an ELISA method using a mixture of human HMG-1 and human HMG-2 (FIG. 10-3). In addition, this calibration curve suggested that the concentration of 5 μg / ml that gives an absorbance of about 1.0 may be used for the measurement.

(Example 13) Measurement of anti-HMG-1 antibody and anti-HMG-2 antibody in each disease Using this ELISA system, the antibodies of patients with autoimmune diseases and inflammatory diseases were measured. The diseases used are as follows.
Rheumatoid arthritis 50 people, systemic lupus erythematosus 47 people,
Sjogren's syndrome 12 people, Behcet's disease 32 people, polymyositis / dermatomyositis 15 people, scleroderma 20 people, primary biliary cirrhosis
41 people, microscopic polyangiitis / nodular polyarteritis 19 people,
There are 62 ulcerative colitis and 15 Crohn's disease. On the other hand, the sera of 33 healthy persons were used as controls. Pig HMG-1 and HMG-2 were used as antigens at 5 μg / ml
Used at a concentration of. As the serum, a standard serum diluted 40 times was used for the measurement. Separately from the test serum, each plate was provided with an antigen for the same calibration curve as in Example 12, and standard serum (40-fold dilution) was used to simultaneously test serum and
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 validated. 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 set as 100%, and the ratio was calculated from the absorbance value of the test serum from which the blank value was similarly subtracted to obtain the value of the test serum. When the value was too high at 40-fold dilution of serum, measurement was performed at 80-fold or more dilution to calculate the value. As a result, a value higher than mean + 2 sd in healthy subjects was regarded as positive.

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

Table 4 shows the positive rates by antigen and disease, and Fig. 11
Fig. 12 shows a scatter diagram by disease using HMG-1 as an antigen, and Fig. 12 shows a scatter diagram by 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, in the other seven diseases, the anti-HMG-1 antibody positive rate was higher (Table 4), and it was found that HMG-1 had stronger antigenicity. In particular, in Sjogren's syndrome, HMG-1 was 66.7% and HMG-2 was 16.7%, which was overwhelmingly significant for HMG-1, which indicates the possibility of being able to be used for narrowing down Sjogren's syndrome. HMG-
In 1, all 9 diseases except polymyositis / dermatomyositis showed a statistically significant difference when compared with a healthy person. 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 with normal subjects. Most of the anti-HMG-2 antibody-positive patients were anti-HMG-1 antibody-positive, but there were a small number of anti-HMG-2 antibody-positive and anti-HMG-1 antibody-negative patients. Therefore, in order to make a diagnosis with higher sensitivity, it is considered better to measure both antibodies.

As a result of adding anti-HMG-1 antibody and anti-HMG-2 antibody positivity, a very high positive rate was shown in primary biliary cirrhosis, rheumatoid arthritis, Sjogren's syndrome, and systemic lupus erythematosus. In primary biliary cirrhosis, it is considered that the positive rate is equal to or higher than that of anti-mitochondrial antibody, and no antigen showing such a high positive rate in the remaining 3 diseases has been discovered so far. In addition, the positive rate is also high in scleroderma, ulcerative colitis, Crohn's disease, microscopic polyangiitis / polyarteritis nodosa, and Behcet's disease, and the antigens showing such a high positive rate for the previous 3 diseases are HMG. It is the first time for an antigen. For microscopic polyangiitis / polyarteritis nodosa, myeloperoxidase is the antigen with the highest positive rate, but HMG is the next. In the present invention, the anti-HMG-1 antibody and the anti-HMG-2 antibody are polymyositis /
It was revealed that the antibody can be simultaneously detected in 9 diseases except dermatomyositis.

Therefore, this time, antinuclear antibodies were simultaneously measured by the indirect fluorescent antibody method for each disease and compared with the HMG antigen positive rate (Table 4). Seven out of 10 diseases showed a value equal to or higher than the antinuclear antibody positive rate. From this, HMG-1 and HMG
It was shown that the measurement of -2 could be a diagnostic method for autoimmune diseases, which can be an alternative to the detection of antinuclear antibody by the indirect fluorescent antibody method.
In addition, it is considered that autoimmune diseases can be diagnosed more broadly and reliably by using the anti-nuclear antibody test together.

[0100]

EFFECTS OF THE INVENTION Rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, Behcet's disease, scleroderma,
The use of common antigens HMG-1 and HMG-2 for primary biliary cirrhosis, microscopic polyangiitis / polyarteritis nodosa, ulcerative colitis, and Crohn's disease It became possible to detect antibodies easily. Furthermore, the method and assay kit for measuring antibodies against both antigens using HMG-1 and HMG-2 have been shown to be potential diagnostic agents for these diseases.

[0101]

[Sequence list]

Sequence number 1 Array length: 214 Sequence type: Amino acid Sequence type: Peptide Sequence features: HMG-1 Origin: Human Array 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 Array length: 208 Sequence type: Amino acid Sequence type: Peptide Sequence features: HMG-2 Origin: Human Array 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 Array length: 214 Sequence type: Amino acid Sequence type: Peptide Sequence features: HMG-1 Origin: Bovine Array 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 Glu Glu Glu Glu Asp Asp Asp Asp Glu     200 205 210

SEQ ID NO: 4 Array length: 214 Sequence type: Amino acid Sequence type: Peptide Sequence features: HMG-1 Origin: Pig Array 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 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 Array length: 214 Sequence type: Amino acid Sequence type: Peptide Sequence features: HMG-1 Origin: rat Array 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 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 Array length: 209 Sequence type: Amino acid Sequence type: Peptide Sequence features: HMG-2 Origin: Pig Array 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         165 170 175 180 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 Array length: 186 Sequence type: Amino acid Sequence type: Peptide Sequence characteristics: Partial sequence of HMG-2 Origin: Bovine Array 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                 185

SEQ ID NO: 8 Array length: 206 Sequence type: Amino acid Sequence type: Peptide Sequence features: HMG-2 Origin: chicken Array 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 Asp Glu Glu     200 205

SEQ ID NO: 9 Array length: 201 Sequence type: Amino acid Sequence type: Peptide Sequence features: 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         165 170 175 180 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 Array length: 208 Sequence type: Amino acid Sequence type: Peptide Origin: mouse Sequence features: 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 Array length: 32 Sequence type: Amino acid Sequence type: Peptide Fragment type: 28KDa N-terminal fragment origin Cell type: Promyelocytic leukemia-derived neutrophil cells Cell line: Neutrophil cell line (ATCC CCL-240) Sequence features How the sequence was determined: E Array 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 Array length: 32 Sequence type: Amino acid Sequence type: Peptide Fragment type: 29KDa N-terminal fragment origin Cell type: Promyelocytic leukemia-derived neutrophil cells Cell line: Neutrophil cell line (ATCC CCL-240) Sequence features How the sequence was determined: 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 drawings]

[Fig. 1] Purified Ig obtained from sera of 5 patients with ulcerative colitis and 3 sera of healthy subjects after SDS-PAGE of neutrophil cytosolic lysate (antigen) isolated from peripheral blood of healthy subjects.
It is a figure which shows the result at the time of Western blotting using G (antibody).

FIG. 2 is a diagram showing the results of Western blotting using neutrophils and HL-60 cell lysates as antigens and the serum of 28 kDa antigen-positive patients as probes.

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

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

FIG. 5: Purified 28 kDa antigen (lane 2), and purified 29 kDa
It is a figure which shows the pattern of Western blotting of a antigen (lane 3).

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

FIG. 7: HMG-1 and HMG-2 prepared from human thymus tissue
Of HMG-1 and HMG- prepared from porcine thymus tissue
FIG. 2 shows the results of Western blotting of 2 and a mixture of commercially available bovine HMG-1 and HMG-2. All human, porcine, and bovine HMG antigens reacted with serum from patients with refractory ulcerative colitis. In this example only, 15% polyacrylamide of 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 refractory ulcerative colitis and 3 healthy subjects with a mixture of porcine HMG-1 and porcine HMG-2 as an antigen.

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

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

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

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

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

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Mao Tanaka               Sanjo-dori Ryomaba Higashiiri, Nakagyo-ku, Kyoto City, Kyoto Prefecture               Nakanomachi 9 Grande Acai I3-B (72) Inventor Kazukazu Nakao               Kyoto Prefecture Kyoto City Saikyo Ward Oeda Kitagakekakecho 4-1               -2 (72) Inventor Mitsuteru Yoshida               21-72-3, Nishi-Hatsuishi, Nagareyama-shi, Chiba (72) Inventor Hitoshi Shirakawa               1-14-2-505 Nishibensai, Asaka City, Saitama Prefecture (72) Inventor Fumio Kosakada               2-19-2 Hiji-cho, Himeji City, Hyogo Prefecture               Raza Himeji Higashi 305                (56) References BETTINA WITTEMAN               N, et. al, AUTOANTIB               ODIES TO NONISTON               E CHROMOSOMAL PROT               EINS HMG-1 AND HMG               -2 IN SERA OF PATI               ENTS WITH JUVENILE                 , Arthritis and Rh               eumatism, 1990, Vol.               33, No. 9, P1378-1383

Claims (9)

(57) [Claims] 1. 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 capable of reacting with an antibody of an autoimmune disease patient, Diagnostic agent for autoimmune diseases. 2. The autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, Behcet's disease, scleroderma, primary biliary cirrhosis, microscopic polyangiitis / polyarteritis nodosa, ulcerative large intestine. The diagnostic agent according to claim 1, which has inflammation or Crohn's disease. 3. 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: 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 capable of reacting with an antibody of an autoimmune disease patient, A kit for diagnosing autoimmune diseases. 5. The autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, Behcet's disease, scleroderma, primary biliary cirrhosis, microscopic polyangiitis / polyarteritis nodosa, ulcerative large intestine. The kit according to claim 4, wherein the kit is flame or Crohn's disease. 6. The polypeptide is human, bovine, porcine, chicken, mouse or rat HMG-1 or HMG-2.
The kit of claim 4, selected from 7. A method for detecting an antibody of an autoimmune disease patient, 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 diseased patient with a body fluid component of an autoimmune diseased patient. 8. The autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, Behcet's disease, scleroderma, primary biliary cirrhosis, microscopic polyangiitis / polyarteritis nodosa, ulcerative large intestine. The method according to claim 7, wherein the method is flame or Crohn's disease. 9. The method of claim 8, wherein the polypeptide is selected from human, bovine, porcine, chicken, mouse, rat HMG-1 or HMG-2.