JP3267893B2 - Diagnostics for autoimmune diseases - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a high mobility group protein-1 (H) to which an antibody of an autoimmune hepatitis patient reacts.
MG-1), high mobility group protein-2 (HMG-2),
Alternatively, a diagnostic agent or a diagnostic kit for autoimmune hepatitis (AIH) using fragments of those polypeptides,
And a method for detecting an antibody of the patient.

[0002]

2. Description of the Related Art Recently, the inventors of the present invention have revealed that a new pANCA-associated antigen is present in the serum of a patient with ulcerative colitis (UC), and have conducted a structural analysis.
high mobility group protein-1 (HMG-1) and high mob
ility group protein-2 (HMG-2) (Sobajima J. et al. Clin. Exp. Immunol. 105: 120-124,1
996, Sobajima J. et al. Clin. Exp. Immunol. 107: 135-14
0, 1997). By constructing an ELISA system using these HMG-1 and HMG-2, rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, Behcet's disease, scleroderma, primary biliary cirrhosis, microscopic polyangiitis / nodule Polymyelitis polyarthritis, ulcerative colitis, and anti-HMG1 / 2 antibody in the serum of patients with Crohn's disease showed a high positive rate in all diseases, furthermore, this ELISA system,
Compared to the method for measuring antinuclear antibodies by the indirect fluorescent antibody method, it shows that it is relatively sensitive, simple, reliable and objective, so that the detection of antibodies against the antigen is useful for diagnosis of the above-mentioned diseases. (Japanese Patent Application No. 8-266431).

[0003] However, it is not known whether there is an anti-HMG1 / 2 antibody-positive disease other than the above-mentioned diseases.

[0004]

Therefore, the present inventors have expanded the investigation diseases and have developed anti-HMG-1 / 2 in other autoimmune diseases.
The disease applicable as a diagnostic method in the primary screening was investigated by measuring the positive rate of the antibody. In the present invention, anti-HMG-1 / 2 antibodies were measured in liver diseases.

[0005]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies to overcome the above-mentioned conventional problems, and as a result, the antigens have been obtained by using an ELISA system using HMG-1 and HMG-2. On the other hand, by showing that the AIH patient has a high positive rate, the inventors have found that the detection of an antibody against the antigen can be a diagnostic marker for AIH, and have completed the present invention.

[0006] The present invention provides 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 AIH patient. The present invention relates to a diagnostic agent for the disease, a kit for diagnosing the disease, and a method for detecting an antibody of the patient. In a preferred embodiment, the polypeptide is selected from human, bovine, porcine, and rat HMG-1 or HMG-2.

Thus, the object of the present invention is achieved.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The polypeptide used in the present invention is selected from polypeptides belonging to the HMG-1 family or HMG-2 family, or fragments thereof. The HMG-1 family refers to human H shown in SEQ ID NO: 1.
Refers to a polypeptide having 90% or more amino acid homology to MG-1 and includes, for example, HMG-1 such as bovine HMG-1 (SEQ ID NO: 3), pig (SEQ ID NO: 4), rat (SEQ ID NO: 5), etc. including. Preferably, human HMG-1 is used, but pigs, cows, and rats can be used because of their high homology. FIG. 3 shows a comparison of the amino acid sequences of these HMG-1s.

On the other hand, the HMG-2 family is represented by SEQ ID NO: 2.
Refers to a polypeptide having 80% or more amino acid homology to human HMG-2, and includes porcine HMG-2 (SEQ ID NO: 6), a partial sequence of bovine HMG-2 (SEQ ID NO: 7), and rat HMG- HMG-2 such as 2 (SEQ ID NO: 8). Preferred is human, porcine, bovine and rat HMG-2. FIG. 4 shows a comparison of the amino acid sequences of these HMG-2s.

[0010] A 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 are capable of reacting with antibodies from AIH patients. These fragments refer to fragments of a polypeptide belonging to the HMG-1 family or the HMG-2 family that can react with an antibody of an AIH 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 AIH 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.

[0011] Since HMG-1 and HMG-2 are universal proteins possessed by all cells, they can be used in any organ, tissue,
It can be prepared by extracting even from cells. For example, human thymus, porcine thymus, bovine thymus, human placenta, neutrophils, HL
-60 cell line. Extraction and purification methods are known,
For example, Yoshida et al. (J. Biochem. Tokyo, 95: 117-124,19
80) and Adachi et al. (J. Chromatogr, 530: 39-46, 1992)
Can be prepared. Bovine HMG-1 and HMG
-2 mixture is sold by Wako Pure Chemical Industries, Ltd.

The above-mentioned polypeptide belonging to the HMG-1 family or the 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 cells 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.
As ion exchange chromatography, for example,
A monoQ column (manufactured by Pharmacia) or the like can be used.

In the present invention, autoimmune hepatitis (AI)
H). Autoimmune hepatitis (aut)
oimmune hepatitis (hereinafter AIH) is a typical autoimmune hepatitis together with primary biliary cirrhosis (PBC). PBC
AIH is thought to be an autoimmune response to hepatocyte components in response to an autoimmune response to bile duct epithelial cells, and is more frequent in women, and the emergence of various autoantibodies, including hyperglobulinemia and antinuclear antibodies It shows characteristic clinical findings such as fever, arthralgia, and autoimmune diseases including collagen diseases. Among autoantibodies, antinuclear antibodies (ANA) (70-90%) that react with components of the cell nucleus and antismooth muscle antibodies (66%) that react with smooth muscle are the most common. Antinuclear antibodies are not always specific for AIH, and other autoimmune diseases,
In particular, it is also observed in systemic lupus erythematosus (SLE). Anti-smooth muscle antibodies, unlike antinuclear antibodies, have a low positive rate in autoimmune diseases such as SLE and are relatively specific to AIH.

[0014] The diagnostic agent of the present invention comprises a polypeptide contained in the above HMG-1 family or HMG-2 family, or a fragment 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 AIH patient to form an antigen-antibody complex. Thus, it may contain additional components that can detect the formed antigen-antibody complex.
These components include, for example, precipitation reaction, ELISA, RIA
It is a component that is compatible with methods such as the method and the Western blotting method. A polypeptide contained in the HMG-1 family or HMG-2 family, or a fragment thereof, can be used in a diagnostic kit. The diagnostic kit is, for example, a polypeptide contained in the HMG-1 family or HMG-2 family, or an ELISA plate on which a fragment thereof is immobilized, and an antigen-antibody complex bound to an antibody of an AIH patient. Reagents. 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), human IgG, IgM,
Reacts with gA. These secondary antibodies may be labeled with a labeling agent generally used in an immunoassay.
Such labeling agents include radioisotopes (eg, 32
P, 3H, 125I, etc.), enzymes (eg, β-galactosidase, peroxidase, alkaline phosphatase, glucose oxidase, lactate oxidase, alcohol oxidase, monoamine oxidase, etc.), coenzyme / prosthetic groups (eg, 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 is described in a compendium (for example, “Seizai Chemistry Laboratory Course 5 Immunobiochemical Research Method”, Tokyo Kagaku Dojin, 1986, p102-112).
And can be appropriately selected from known methods as described in the 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 is available from Immunotec
h Available from 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 a sample at a time in an ELISA system.
Type plate, C type plate (Sumitomo Bakelite), maxi soap plate (Nunc) and EIA
/ RIA plate (manufactured by Coaster) and the like.

The binding between the carrier and the antigen can be performed by a known method such as a physical adsorption method, an ionic 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 AIH antibody of the present invention
This is a method including a step of reacting a polypeptide selected from the MG-1 family, a polypeptide selected from the HMG-2 family, or a fragment (antigen) thereof with a body fluid component of AIH. Conditions well known to those skilled in the art are applied as conditions for reacting the antigen with the antibody. Detection of antigen-antibody reactants
Methods 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, washed, washed with an alkaline phosphatase-labeled anti-human IgG antibody as a secondary antibody, and then reacted with an alkaline phosphatase substrate p-nitrophenyl phosphorus. Add acid to develop color, 405n
The anti-HMG-1 and anti-HMG-2 are measured by measuring the absorbance of
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. Hereinafter, anti-HMG-1 by an ELISA system using HMG-1 and HMG-2 antigens
A method for measuring an antibody and an anti-HMG-2 antibody will be described.

The amino acid sequence of human HMG-1 differs from pig, bovine and rat (FIG. 3) in that it differs from human, HMG-1 from animals can be used as an antigen for ELISA. Further, regarding HMG-2, since human and pig differ only by two amino acids (FIG. 4), it is considered that HMG-2 can also be used instead of human. As shown in FIG. 4, only partial sequences of bovines have been determined and the reported sequence is not considered to be reliable. (The bovine sequence was reprinted from the protein data bank PIR B61611. Arranged to match the species). HM
G-1 and HMG-2 are considered to be proteins whose amino acid sequences are conserved fairly well between species, and taking into account the high similarity between humans and pigs, bovines are expected to differ by more than two. Absent. In addition, it is considered that mouse HMG-1 and HMG-2 can also be used.

Pig HMG-1 and Pig HMG-2 used in the present invention
Is the method of Yoshida Y., one of the inventors of the present application (Yoshida Y.
J. Biochem. Tokyo 95: 117-124,1980, Adachi Y.
J. Chromatogr. 530: 39-46,1992) and Japanese Patent Application No. 8-2
It can be prepared by the method described in 66431. The preparation method from porcine thymus tissue will be briefly described as an example. Slice the thymus tissue into 0.
Suspend the cells in 14M NaCl (pH 7.5) and destroy the cells with a homogenizer. A precipitate containing nuclei is obtained by centrifugation. This nuclear fraction
The suspension is suspended in 50 mM Tris-HCl (pH 7.5), crushed with a homogenizer, and the precipitate containing chromatin is collected by centrifugation. The chromatin fraction is suspended in 0.35 M NaCl (pH 7), and HMG bound to chromatin is released by a homogenizer treatment.
The insoluble material was removed by centrifugation, and the obtained supernatant was made into a 2% trichloroacetic acid solution and left at 4 ° C. for 2 hours.
Other insoluble proteins are precipitated. The supernatant is collected by centrifugation, and a final 10% trichloroacetic acid solution is left at 4 ° C. for 2 hours to precipitate HMG-1 and HMG-2, which are collected by centrifugation. The precipitate is washed with acetone and dried. This HMG-
MonoQ column (Pharmacia) from fractions 1 and HMG-2
Can be used for purification.

An ELISA system is constructed using purified porcine HMG-1 and HMG-2. That is, a 96-well ELISA plate (Nunc
5 μg / ml porcine HMG-1 or HMG-
2 was added in an amount of 50 μl, and the mixture was allowed to stand at 4 ° C. for 24-36 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 IgGF (ab ') 2 is added and reacted at room temperature for 2 hours. After washing 5 times with a washing solution, a paranitrophenylphosphoric acid (Sigma) solution (10% diethanolamine solution) is added, 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 antibody and anti-HMG-2
When a standard curve was tested using sera from patients with ulcerative colitis that were positive for both antibodies, concentration-dependent linear lines were obtained for both antigens, and anti-HMG-1 and anti-HMG-2 antibodies could be measured. It was shown that anti-HMG-1 antibody and anti-HMG-2 antibody in each disease can be measured using this ELISA system. Therefore, anti-H was used for AIH, hepatitis C, hepatitis B, and healthy subjects.
The present invention is accomplished by measuring the MG-1 and anti-HMG-2 antibodies to show that AIH is significantly more positive than the other two diseases and healthy individuals.

By the way, during the active period of inflammation, the production of HMG antigen may be enhanced not only in neutrophils but also in activated cells in local inflammation.
It is considered that the release of G enhances the production of anti-HMG antibodies. Therefore, measurement of anti-HMG-1 antibody and anti-HMG-2 antibody is considered to be an indicator of disease activity. Further, the present invention includes a kit for measuring anti-HMG-1 antibody and anti-HMG-2 antibody by ELISA, but as another method
Peripheral blood lymphocytes and HMG-1 in patients with AIH or inflammatory disease
By examining the responsiveness to HMG-2 and HMG-2, the disease specificity can be assayed. That is, HMG-1 and HMG-2
Alternatively, disease can be detected by measuring whether T lymphocytes proliferate in response to those immunoreactive synthetic peptides, or whether there is production of γ-interferon by macrophages in the same assay system.

In addition, the destruction of neutrophils in the local area of inflammation causes HMG
In the case where is released, it is considered that the measurement of HMG itself may serve as an indicator of disease activity. HM
Examples of the method for measuring G include sandwich ELISA and immunoprecipitation. In the sandwich ELISA, for example, a monoclonal anti-HMG antibody is immobilized on a plate and, after binding to purified HMG, reacted with patient serum, and the bound anti-HMG antibody may be detected with an anti-Ig antibody labeled with HRP or the like. In the case of immunoprecipitation, HMG and patient serum can be reacted in a solution, reacted with an anti-Ig antibody labeled with I125 or the like, and measured by measuring the radioactivity of the precipitated antigen-antibody reactant.

[0027]

The present invention will be described below with reference to examples. (Example 1) Purification of HMG-1 and HMG-2 derived from porcine thymus 1) Preparation of porcine HMG HMG-1 and HMG-2 derived from porcine thymus were purified from Adachi et al. (J. Chromatog
r, 530: 39-46, 1992). The purification method is briefly described. 600g of frozen porcine thymus was added to 3L of cold SSC
(0.14M NaCl, 0.01M sodium citrate, 5mM NaHSO3) using a Waring blender for 3 times at 15,000rpm for 30 seconds, and centrifuged at 2,500g for 15 minutes to obtain a crude precipitate of nuclei. . The precipitate was further suspended in 600 ml of cold SSC, triturated using a Waring blender, and centrifuged.
The same operation was performed twice more, and the nuclei were sufficiently washed. The nuclei were ground in a hypotonic cold 100 ml of Tris-HCl (pH 7.6) using a Waring blender and centrifuged to obtain a precipitate. The same operation was performed once again to obtain a chromatin precipitate.

Chromatin was suspended in 600 ml of cold 0.35 M NaCl, ground using a Waring blender, centrifuged, and the supernatant was recovered. The same extraction was performed twice for the precipitate, and each supernatant was mixed. The supernatant is the final concentration
After adding 100% (w / v) TCA to 2% (w / v) TCA and stirring for 1 hour, the mixture was centrifuged at 7,000 g for 20 minutes, and the supernatant was collected. The supernatant was further made up to 10% TCA and stirred for 1 hour.
The precipitate was collected by centrifugation for 20 minutes. The obtained precipitate was washed with acetone and air-dried to obtain HMG1 and HMG2 powder. 2) Purification of HMG1 / 2 HMG1 and HMG2 were purified using a MonoQ column (Pharmacia). Elution was performed with a linear concentration gradient of NaCl from 0 to 1M. FIG. 1 shows the elution pattern. By this fractionation method, HMG-1 and HMG-2 were obtained with a purity of about 95% or more. (Example 2) Anti-HMG-1 antibody and anti-HMG- by ELISA method
2. Measurement of Antibody ELISA was performed using porcine HMG-1 and HMG-2 as antigens. 96
Porcine HMG-1 or pig serially diluted from 6.25 μg / ml into each well of a maxi-soap plate (Nunc)
50 μl of HMG-2 was added, and the mixture was allowed to stand at 4 ° C. for 24-36 hours. After removing excess antigen, 200 μl of 5% BSA was added, and the mixture was allowed to stand for 30 minutes or more to perform blocking. Anti-HMG-1 antibody and / or anti-HMG-
The serum of a patient with 2-antibody-refractory ulcerative colitis was used as a standard serum, and 50 µl of a 40-fold dilution of the serum with 5% BSA was added, followed by standing at room temperature for 2 hours. After washing 5 times with 0.05% Tween / 0.02% azide / PBS (washing solution), 1000-fold diluted alkaline phosphatase-labeled goat anti-human IgGF (ab ') 2 (Immunotech SA
100 μl) and reacted at room temperature for 2 hours. After washing 5 times with a washing solution, 0.1% paranitrophenyl phosphate (Sigma)
(A 10% diethanolamine solution) was added, and the mixture was reacted at room temperature for 20 to 25 minutes, and the absorbance at 405 nm was measured. FIG. 2 shows the calibration curve of the positive control. From this calibration curve, pig HMG-1 (FIG. 2-1) and HMG-2 (FIG. 2-2)
-HMG-1 antibody and / or anti-HMG-
It turned out that 2 antibodies can be measured. 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 3) Anti-HMG-1 antibody and anti-HMG- in each disease
2. Measurement of Antibody Antibodies in AIH patients, hepatitis B patients, and hepatitis C patients were measured using this ELISA system. On the other hand, serum of 33 healthy persons was used as a control. Porcine HMG-1 and HMG as antigens
Each of -2 was used at a concentration of 5 μg / ml. As serum, a standard serum diluted 40-fold was used for measurement. 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. When the value was too high at a 40-fold dilution of serum, the measurement was performed at a dilution of 80-fold or more and the value was calculated. The result is a healthy person
Values greater than mean + 2 sd were considered positive. In addition, 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. Measurement is MBL
Using a FluoroHEPANA test manufactured by Co., Ltd., a dilution of 80-fold or more was positive for AIH and a dilution of 20-fold or more for hepatitis B and C. Table 1 shows the positive rates by antigen and disease.

[0029]

[Table 1]

Hepatitis C was as high as 86% and hepatitis C was as high as 27%. On the other hand, hepatitis B was 12%, which was not significantly different from a healthy person. From this, hepatitis B can be ruled out in hepatitis, and anti-HMG-1 / 2 antibody positive A
It became clear that IH was likely. In comparison with the antinuclear antibody, the positive rate of the anti-HMG-1 / 2 antibody was higher than that of the antinuclear antibody in AIH and hepatitis C. From this
It became clear that AIH and hepatitis C can be diagnosed with higher sensitivity than antinuclear antibodies.

[0031]

The method and assay kit for measuring antibodies to both antigens using HMG-1 and HMG-2 have been shown to be diagnostic agents for autoimmune hepatitis.

[0032]

[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 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 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 characteristics: 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 Ly s 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 165 170 175 180 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 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 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 Sequence 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 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 Sequence length: 209 Sequence Type: Amino acid Sequence type: Peptide Sequence features: 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 165 170 175 180 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 Sequence length: 186 Sequence type: Amino acid Sequence type: Peptide Sequence characteristics: HMG-2 Partial sequence 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 Tla 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 Sequence length: 209 Sequence type: Amino acid Sequence type: PE Peptide sequence features: HMG-2 Origin: rat 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 Val 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 Asp 185 190 195 Glu Asp Glu Glu Glu Glu Asp Glu Asp Glu Glu 200 205

[Brief description of the drawings]

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

FIG. 2 shows the results of measurement of anti-HMG-1 antibody and anti-HMG-2 antibody by ELISA. Pig HMG-1 (Fig. 2-1), HMG-
In FIG. 2 (FIG. 2-2), a dose-dependent straight line is obtained.

FIG. 3 is a diagram comparing the amino acid sequences of human, pig, bovine and rat HMG-1.

FIG. 4 is a diagram comparing the amino acid sequences of human, pig, bovine and rat HMG-2.

Continued on the front page (72) Inventor Mitsuteru Yoshida 3-472-21 Nishihatsuishi, Nagareyama City, Chiba Prefecture (72) Inventor Hitoshi Shirakawa 1-1-14-505 Nishibenzai, Asaka City, Saitama Prefecture (72) Inventor Fumio Kosakada Hyogo Prefecture 2-19-2 Hijicho, Himeji City Asahi Plaza Himeji Higashi 305 (56) Reference International Publication 96/41183 (WO, A1) Clin. Exp. Immunol. , Vol. 107, (1997) p. 135− 140 (58) Field surveyed (Int. Cl. ⁷ , DB name) G01N 33/48-33/98

Claims (6)

(57) [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 against autoimmune hepatitis. A diagnostic agent for the disease. 2. The polypeptide is selected from human, bovine, porcine, or rat HMG-1 or HMG-2,
The diagnostic agent according to claim 1. 3. 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 hepatitis patient. And a kit for diagnosing the disease. 4. The polypeptide is selected from human, bovine, porcine, or rat HMG-1 or HMG-2.
The kit according to claim 3. 5. A method for detecting an antibody of a patient with autoimmune hepatitis, 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 the patient with a body fluid component of the patient. 6. The method of claim 1, wherein the polypeptide is selected from human, bovine, porcine, rat HMG-1 or HMG-2.
The method according to 5.