JPH07157500A - Human synovial fluid-derived protein affinitive to hyaluronan and antibody - Google Patents

Abstract

PURPOSE:To obtain the subject new protein existing in knee synovial fluid of a patient with deformed arthritis of knee, rheumatoid arthritis of knee, etc., in the form of a hyaluronan-bonded protein, separable from such knee synovial fluid and enabling diagnosis of arthritis by analyzing the concentration in knee synovial fluid. CONSTITUTION:Knee synovial fluid of a patient with deformed arthritis of knee or rheumatoid arthritis of knee is collected and a protein-denaturing agent is added thereto. The resultant mixture is subjected to ultracentrifugation under a protein non-dissociation condition to obtain a fraction of high-concentration hyaluronan. After addition of a protein-denaturing agent thereto, the obtained fraction is subjected to ultracentrifugation under a protein dissociation condition and the obtained hyaluronan-protein composite material is treated with a hyaluronan lyase, thus affording the objective new protein having a molecular weight shown by two main bands of about 85 kD (X band) and about 83 kD (Y band) in an unreduced state when measured by the SDS-PAGE method and about 6.0 to 7.0 isoelectric point. Protease V8-treated X and Y bands are respectively represented by formula I and formula II (Xaa is unidentified).

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a human synovial fluid-derived hyaluronan-binding protein, a complex of the protein and hyaluronan, an antibody against the protein, a method for measuring the protein, a method for diagnosing arthritis, and a kit for diagnosing arthritis. It is about.

[0002]

2. Description of the Related Art Synovial fluid fills the space between joint cartilage between joint cartilage in the joint capsules of joints in various parts of the body and maintains the smooth motility of the joint and alleviates the impact applied during exercise. have. Most of these functions are actually owed to the action of hyaluronan (hyaluronic acid), which is abundant in the synovial fluid. Hyaluronan in the synovial fluid of normal humans exists almost alone, but it is known that several kinds of proteins are bound to hyaluronan in the synovial fluid of inflammatory diseases such as osteoarthritis and rheumatoid arthritis. Has been. The composition of the synovial fluid is basically the same as the plasma component, except for the abundance of hyaluronan, which is released by synovial cells into the synovial fluid. When inflammation occurs in a joint, the permeability of affected blood vessels changes, and so-called acute-phase inflammatory proteins such as haptoglobin and α1-protease inhibitor penetrate into the synovial fluid. In the report that these proteins bind to hyaluronan, three types of haptoglobin, α2-protease inhibitor, and inter-α-trypsin inhibitor were detected in reactivity with the antibody. It is not clear whether the binding of these proteins to hyaluronan is specific. Hyaluronan in the synovial fluid binds by forming a Schiff base between the reducing aldehyde group and the amino group of the protein,
A stable bond is formed by Amadori transition,
It has also been reported that it forms a complex with albumin and transferrin in a non-specific binding manner. On the other hand, Yoneda et al. Isolated a hyaluronan-binding protein from bovine serum and found that it had a previously unknown property. Serum-derived hyaluronan-binding protein (Serum
Drived Hyaluronan Associated Protein; SHAP) (Yoneda, M. et al., (1990), J. Biol. Chem., 2
65, (9), 5247-5257). However, it has not been reported that a protein having a binding property to hyaluronan has been purified and isolated from the joint fluid of a human arthritis patient.

[0003]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a human synovial fluid-derived hyaluronan-binding protein present in the synovial fluid of an arthritis patient and an antibody thereto. Another object of the present invention is to provide a method for diagnosing arthritis by measuring the human synovial fluid-derived hyaluronan-binding protein present in human synovial fluid using this antibody. Further, an object of the present invention is to provide a diagnostic kit used for diagnosing this arthritis.

[0004]

The present inventors have found that the hyaluronan-binding protein is abundant in the synovial fluid of human arthritis patients, and purified and isolated this substance.
The physical properties have been clarified. In addition, the hyaluronan-binding protein of the present invention has a possibility of becoming a new probe in exploring the importance of hyaluronan-protein interaction in the affected site of arthritis, and it is possible to measure this substance in synovial fluid. Have found that it can be a useful means in differentiating the above arthritis, ie, osteoarthritis of the knee and rheumatoid knee arthritis.

That is, the present invention is present in the knee joint fluid of human patients with osteoarthritis or rheumatoid knee arthritis in association with hyaluronan, is separated from these knee joint fluids, and has the following physicochemical properties: Human synovial fluid-derived hyaluronan-binding protein having the following properties: (A) Molecular weight: sodium dodecyl sulfate-polyacrylamide gel Approximately 85 kilodaltons (X band) and 83 kilodaltons (Y band) in a non-reducing state on electrophoresis.
(B) Partial protein primary structure forming two major bands of: The primary structure of the peptide fragment obtained by treatment of the X band with protease V8 is as follows: Val Asp Gln Val Thr Leu Tyr Ser Tyr Lys Val Gln Ser Thr Ile Thr Ser Arg Met Ala Thr Thr Met Ile Gln Ser Lys Val Val Asn Asn Ser Pro Gln Pro The peptide fragment obtained by treatment of Y band protease V8 is as follows: Ile Val Ile Lys Val Lys Pro Lys Gln Leu Val His His Phe Glu Ile Asp Val Asp Ile Phe Glu Pro Gln Gly Ile Ser Lys Leu Asp Ala Xaa Ala Ser Pro (in the formula, Xaa represents an unidentified amino acid). (C) Isoelectric point: about 6.0 to 7.0 (estimated from pH dependence of binding to DEAE-Cellulofine A800). The present invention is also a complex of the human synovial fluid-derived hyaluronan-binding protein and hyaluronan.

Furthermore, the present invention provides the aforementioned human synovial fluid-derived hyaluronan-binding protein, its X band component, Y band component, peptide fragments thereof, or a complex of the hyaluronan binding protein and hyaluronan, in a mammal other than human. Can be obtained from the body fluid of the animal or produced by the antibody-producing cells of the animal, and cross-react with one or more of the hyaluronan-binding protein, its X band component and Y band component. It is an antibody. The present invention also provides a method for measuring a human synovial fluid-derived hyaluronan-binding protein, which comprises using the above antibody. Furthermore, the present invention is a method for diagnosing arthritis by measuring the content of hyaluronan-binding protein in synovial fluid collected from human joints using the above antibody. Furthermore, the present invention provides a hyaluronan-binding protein derived from a proteoglycan bound to a solid phase (hyaluronan-binding site of proteoglycan; HAB
A kit for diagnosing arthritis, which essentially comprises R), the above-mentioned antibody, and an anti-immunoglobulin antibody to which a labeling substance is bound or can be bound. The present invention will be described in detail below.

In the present specification, amino acids and peptides are represented by the abbreviations adopted by the IUPAC-IUB Biochemical Nomenclature Commission (CBN). For example, the following abbreviations are used. When amino isomers may have optical isomers, the L form is shown unless otherwise specified. Ala: Alanine Arg: Arginine Asn: Asparagine Asp: Aspartic acid Gln: Glutamine Glu: Glutamic acid Gly: Glycine His: Histidine Ile: Isoleucine Leu: Leucine Lys: Lys: Lysine Lys: Lys: Lys: Lys: Lys: Lys: Methionine Phe: Phenylalanine : Tyrosine Val: Valine

The hyaluronan-binding protein of the present invention was found in large amounts in the knee joint fluid of human patients with knee osteoarthritis or rheumatoid knee arthritis.
This substance can be purified by the following method. That is, the synovial fluid containing the complex of hyaluronan-binding protein of the arthritis patient and hyaluronan is collected,
A low concentration protein denaturant (for example, about 0.4 M guanidine hydrochloride) is added thereto, and ultracentrifugation (associative ultracentrifugation) is performed in a state where the protein and hyaluronan are associated (under non-dissociation conditions). Next, a higher concentration of protein denaturant (for example, guanidine hydrochloride of about 4M) is added to the fraction containing a large amount of hyaluronan, and the mixture is subjected to ultracentrifugation (dissociative ultracentrifugation) under protein dissociation conditions to increase the amount of hyaluronan. Collect the containing fractions. As this protein denaturant, guanidine hydrochloride,
Urea or the like is used. The hyaluronan-rich fraction is desalted and saturated with 75% ethanol to obtain a precipitate. This precipitate is a complex of hyaluronan and a hyaluronan-binding protein. This complex does not dissociate under protein dissociation conditions.

Next, this complex is decomposed with a mucopolysaccharide degrading enzyme capable of decomposing hyaluronan as a substrate, and if necessary, dialysis treatment, molecular sieving treatment (gel filtration, ultrafiltration, etc.), and electrophoresis. The hyaluronan-binding protein is purified and isolated by treatment. As the mucopolysaccharide degrading enzyme capable of degrading hyaluronan as a substrate, hyaluronidase, chondroitinase and the like are used.

To prepare the antibody of the present invention, a human synovial fluid-derived hyaluronan-binding protein, an X band component or a Y band component which is considered to be a subunit thereof, and a peptide obtained by enzymatically or chemically decomposing these A fragment, or a complex of hyaluronan and the above protein is used as an antigen (immunogen), and this is administered to an animal other than human (preferably a mammal) to immunize the animal, and then produced in the body of the animal. Antibodies can be obtained from the body fluids (such as blood) (eg, as antisera). Alternatively, monoclonal antibodies can be obtained by collecting antibody-producing cells (spleen cells, lymph node cells, peripheral blood cells, etc.) of immunized animals.

Specifically, the above-mentioned antigen is dissolved in an appropriate solvent such as phosphate buffered saline (PBS) and the like, and usually an adjuvant (Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum adjuvant, Bordetella pertussis adjuvant, etc.) is added thereto. ) Are mixed, and animals other than humans (preferably mammals), for example, rabbit, mouse, rat, guinea pig, hamster, goat, sheep, cow, horse, chicken, duck etc. (preferably rabbit, rat, mouse, Goat) lymph node, subcutaneous, intradermal, intravenous or intraperitoneal. After the first administration, if booster immunization is performed 1 to 5 times once every 1 to 5 weeks in the same manner, an antibody against the above-mentioned antigen (immunogen) is produced in the immunized animal. The antibody titer of blood or the like is examined by immunoassay (ELISA, etc.) using a microplate coated with an antigen, and blood or the like is collected after the antibody titer rises. In order to separate the antiserum from the blood, the collected blood can be left standing and then centrifuged. When it was necessary to further purify such antiserum, selective fractionation method utilizing difference in solubility (salting out with neutral salts such as sodium sulfate and ammonium sulfate, low temperature alcohol precipitation, etc.) and difference in charge were used. Fractionation method (isoelectric focusing, electrophoresis, ion exchange chromatography, etc.), fractionation method that utilizes the difference in specific affinity with antibodies and immunoglobulins (Protein A
Etc.), a method of utilizing the difference in adsorption coefficient to the carrier (adsorption chromatography using a hydroxyapatite adsorbent, etc.),
Separation and purification can be performed by appropriately combining methods usually used as a method for purifying antiserum, such as a fractionation method utilizing a difference in molecular weight (gel filtration method, ultracentrifugation method, etc.).

The antibody of the present invention may be a monoclonal antibody. For the monoclonal antibody, the antibody-producing cells can be prepared by a known method (hybridoma method; G. Kohler et al, Eu.
r. J. Immunol., 6, 511-519 (1976); Nature, 256, 495-4.
97 (1975); M. Shulman et al, Nature, 276, 269-270 (197
8); “Monoclonal antibody-hybridoma and ELISA
-", Kodansha Co., Ltd., published on February 20, 1983), and allowed to grow for a long period of time, and by culturing the cells, they can be produced and accumulated in the medium. Also, the hybridomas were transferred to pristane (2, 6, 1) in animals with major histocompatibility compatibility, such as BALB / c mice.
(0,14-tetramethylpentadecane) may be pre-administered and then intraperitoneally administered to form an ascites tumor to accumulate the antibody in the ascites. The monoclonal antibody thus produced can be purified by the same method as the above antiserum.

The antibody of the present invention prepared as described above is
Human synovial fluid-derived hyaluronan-binding protein, X
It shows cross-reactivity with one or more of band component and Y band component and should be used for differential diagnosis of human arthritis as osteoarthritis or rheumatoid arthritis, or prognosis of arthritis treatment. You can The antibody of the present invention is not limited to an immunized animal, a production method, etc., as long as it has the above-mentioned antigen specificity, and includes a polyclonal antibody (such as antiserum) and a monoclonal antibody. Further, a fragment (for example, F (ab ′) ₂ , Fab ′, Fab, etc.) obtained by subjecting such an antibody to limited digestion with a protease such as papain, pepsin, or trypsin, which has the characteristics of the antibody of the present invention, Those retained are included in the antibody of the present invention.

In this diagnostic method, synovial fluid is collected from the joints of patients with arthritis and the amount of hyaluronan-binding protein complex contained therein is quantified. When the amount is large, rheumatoid arthritis (RA) In some cases, it is a method that can diagnose osteoarthritis (OA). As the quantification method, a known immunoassay method, dot blot method (Western blot method) or the like is used.

Known immunoassays (immunoassays) used in the present invention are limited as long as they can quantitatively or qualitatively measure human synovial fluid-derived hyaluronan-binding properties using the above-mentioned antibodies of the present invention. Not done. That is,
Immunoturbidimetric method (Tur
bidimetricImmunoassay), an immunodiffusion method in which an antigen and an antibody are diffused by using a gel as a support to form a sedimentation line or a sedimentation ring, an antigen is adsorbed on a carrier, and this is reacted with the antibody Western blot method (dot blot method) for secondary detection of anti-immunoglobulin antibody, protein A, etc.
Alternatively, any measurement system of a labeled immunoassay using an antigen or antibody labeled with a substance that can be easily detected by fractionation can be applied to the measurement method of the present invention. More specifically, the immunoturbidimetric method includes, for example, an agglutination reaction method using polystyrene microparticles (latex) to which the antibody of the present invention is bound, erythrocytes, etc. as carrier particles; The agarose gel, agarose gel, cellulose acetate membrane, dextran gel, etc. are used as the carrier for the antibody of O., such as the Ouchterlony diffusion method and the Uudan diffusion method; the Western blotting method (dot blotting method) is a nitrocellulose membrane or polyvinylidene method. The antibody of the present invention is reacted with the antigen by adsorbing the antigen on the fluoride membrane, and then the antibody of the present invention is detected with an enzyme-labeled anti-immunoglobulin antibody (eg, peroxidase-labeled anti-IgG antibody) or a radioisotope-labeled protein A. As a labeled immunoassay, an enzyme is used as a labeled substance. Use as an enzyme immunoassay (enzyme immunoassay, EIA or ELISA (Enzyme Linke
d Immunosorbent Assay)); a radioimmunoassay using a radioisotope as a labeling substance (radioimmunoassay, RIA), a fluorescent immunoassay using a fluorescent substance as a labeling substance (fluorescence immunoassay), and the like.

These measuring methods are known methods [edited by Minoru Irie, "Continued Radioimmunoassay", Kodansha, 1979.
Issued May 1, 2010; Eiji Ishikawa et al., "Enzyme Immunoassay",
Second Edition, Medical Institute, December 15, 1982; Method
s in Enzymology, Vol.92, (1983), Immunochemical Te
chniques, Part E: Monoclonal Antibodies and Genera
l Immunoassay Methods, published by Academic Press, Inc.].

In the case of the labeled immunoassay, the antibody or anti-immunoglobulin antibody of the present invention is treated with an enzyme (peroxidase, alkaline phosphatase, β-galactosidase, etc.), a radioactive isotope ( ¹²⁵ I, ¹³¹ I, ^3).
H, etc.), fluorescent substances (fluorescein isothiocyanate, umbelliferone, etc.), chemiluminescent substances (luminol, etc.), or other substances (biotin-avidin (or streptavidin) system, It is labeled with these labeling substances indirectly via an immunoglobulin antibody or the like) and then used for the measurement. After the antigen-antibody reaction, separation of the antibody bound to the antigen in the sample (including the sandwich bound product) and the unbound free antibody (B
When F separation is required, a liquid phase method (using a secondary antibody, polyethylene glycol, dextran charcoal powder, etc.) or a solid phase method (using a protein or antibody having a binding property to the immobilized measurement control substance) is used. ), But the solid phase method is preferred.

As the immunoassay method of the present invention, the following method is particularly preferable because it is simple and preferable. That is, a hyaluronan-binding protein (HABR) derived from proteoglycan is immobilized on a solid phase, and this HABR is reacted with a complex of hyaluronan and a hyaluronan-binding protein present in a measurement sample to allow the complex to react with the hyaluronan-binding protein. An antibody that is specifically bound to the complex that is captured on the solid phase is known by binding the complex of the present invention and the antibody of the present invention that have been captured on the solid phase via a moiety. It is a method of detecting and measuring by the method. As a method for detecting and measuring an antibody, a method for measuring a labeling substance using the antibody of the present invention to which the above-described labeling substance is bound in advance as an antibody; A method in which an antibody bound to the body is reacted with an anti-immunoglobulin antibody bound to a labeling substance to measure the labeling substance; or an unlabeled antibody is used in the above immunoreaction, and the antibody bound to the complex is converted to biotin or the like. The method of reacting with the anti-immunoglobulin antibody bound to the substance of, and further reacting the binding substance of avidin (or streptavidin) that specifically reacts with the substance such as biotin and the labeling substance to measure the labeling substance Can be mentioned. As the solid phase, a test tube, beads, microplate, filter paper, latex or the like is used, and as a method for immobilizing HABR, antibody or the like on the solid phase, a physical adsorption method, a covalent bond method, an encapsulation method, etc. A general method for preparing an immobilized enzyme [“immobilized enzyme”, 1975
March 20, 2000, Kodansha Co., Ltd., pp. 9-75]. The physical adsorption method is particularly preferable because it is simple. It should be noted that it is preferable to block the part to which HABR, antibody, etc. are not bound with serum albumin, gelatin or the like.

Further, the kit for diagnosing arthritis of the present invention can be used for quantifying the complex of the hyaluronan-binding protein and hyaluronan. Such a kit for diagnosing arthritis includes, for example, an anti-immunoglobulin antibody (preferably an anti-IgG antibody) to which a proteoglycan-derived hyaluronan-binding protein bound to a solid phase, the above-mentioned antibody, and a labeling substance can be bound or bound. antibody)
A kit for diagnosing arthritis consisting essentially of:

[0020]

EXAMPLES Hereinafter, specific examples of the present invention will be described.
The present invention is not limited to this. Example 1 Preparation of hyaluronan-binding protein 1) Purification of hyaluronan-hyaluronan-binding protein complex from synovial fluid The synovial fluid of a patient with rheumatoid knee arthritis was collected, lightly centrifuged to remove insolubles, and then treated with Hank's solution. Diluted 5-6 times. To this was added 1/10 volume of 4M guanidine hydrochloride solution, a small amount of sodium azide was added, and then cesium chloride was added to make 50% (w / v). After being placed in an ultracentrifuge tube and allowed to stand at 4 ° C., ultracentrifugation at 4 ° C. and 123,000 × g was carried out for 48 hours (ultracentrifugation under protein non-dissociation conditions; associative ultracentrifugation). After ultracentrifugation, fractions divided into 10 equal parts were taken from the bottom of the tube, and the specific gravity of each of them, hyaluronan and protein concentration were measured, and the results are shown in FIG. 1.

In FIG. 1, the solid line represents the protein concentration (unit: mg / ml) of each fraction quantified by the Lowry method, and the broken line represents the hyaluronan concentration (unit: μg / ml) quantified by the method using actinomycete hyaluronidase.
ml). Hyaluronan collected in a fraction with a low specific gravity (specific gravity around 1.30) is considered to be a low molecular weight hyaluronan fragment due to joint inflammation. specific gravity
30% or more of hyaluronan was present in the 1.43 or more fraction, and 93% or more of the protein component was distributed in the fraction having a specific gravity of less than that.

Fractions having a specific gravity of 1.43 or more containing a large amount of hyaluronan were collected, and guanidine hydrochloride was further added to give a final guanidine hydrochloride concentration of 4M. This was subjected to ultracentrifugation again (ultracentrifugation under protein dissociation conditions; dissociative ultracentrifugation), divided into 10 equal fractions, and the results of measuring specific gravity and hyaluronan and protein concentrations are shown in FIG. . In FIG. 2, the solid line and the broken line represent the protein concentration (unit: mg / ml) and hyaluronan concentration (unit: μg / ml), respectively.

Hyaluronan was distributed with a specific gravity of 1.45 to 1.46 as the center, and more than 60% of the total hyaluronan was concentrated in the range of specific gravity of 1.4 to 1.5, so fractions in this range were collected. This fraction was desalted and a precipitate was obtained by precipitation with 75% ethanol. This precipitate is a complex of hyaluronan and a hyaluronan-binding protein (HA
-Hyaluronan-binding protein complex), and the amount of protein contained therein was about 0.03% of the amount contained in the original synovial fluid. When this fraction was treated with hyaluronidase derived from actinomycetes, sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) shown in (3) below was obtained.
11%) Electrophoresis shows a molecular weight of about 85,000 daltons
Two bands of 83,000 daltons were shown (X band and Y band respectively) (see Figure 3).

The markers in the figure are electropherograms of a standard molecular weight kit manufactured by Pharmacia. The molecular weight of 94,000 dalton is phosphorylase b, 67,000 dalton is bovine serum albumin, 43,000 dalton is ovalbumin, 30,000 dalton is carbonic anhydrase, 20,100 dalton is soybean trypsin inhibitor, 14,400 dalton is α-
Lactalbumin is shown respectively. Lane 3 is an electrophoretic sample obtained by a preparation method that includes heating hyaluronidase to the purified HA-hyaluronan-binding protein complex, followed by heating. Lane 2 is an electrophoretic sample prepared by performing the same treatment as the sample in lane 3 except that the enzyme was not added to the complex. Lane 1 is an electrophoretic sample prepared without adding any enzyme to the complex and without heating or heating at all. No stained band appeared in either lane 1 or lane 2, and two bands appeared only in lane 3. Therefore, the band appearing in lane 3 is not a substance generated by heating during enzyme treatment or heating during electrophoresis sample preparation, but HA-
It was found that the hyaluronan of the hyaluronan-binding protein complex represents the protein released by digestion with hyaluronidase.

2) Method for separating hyaluronan-binding protein from hyaluronan-hyaluronan-binding protein complex HA-hyaluronan-binding protein complex is dissolved in an appropriate buffer, and chondroitinase ABC is added to formyl-cellulofine ( After applying hyaluronan to a column packed with a carrier immobilized under a trade name, manufactured by Chisso Co., Ltd., sold by Seikagaku Corporation, the digested product is dialyzed to remove the hyaluronan decomposition product, and hyaluronan-binding property is obtained. A protein alone solution was obtained.

3) Hyaluronan-Specific Assay Method To a sample solution (100 μl to 300 μl), 3 volumes of 95% ethanol containing 1.3% potassium acetate was added and stirred, and the mixture was stirred at ice temperature.
After leaving still for 30 minutes, it was centrifuged at 15,000 rpm for 30 minutes, and the supernatant was removed. 50 mM acetic acid / sodium acetate buffer (pH 5.0) was added to the resulting precipitate to dissolve it, and actinomycete-derived hyaluronidase 2.5 to 5.0 TRU (turbidity reduction unit) was added to the precipitate.
Digested at 30 ° C for 30 minutes. After digestion, the enzyme reaction was stopped by heating at 100 ° C. for 3 minutes, and the mixture was centrifugally filtered with a φ0.22 μm filter. The filtrate was applied to a post-column reaction type sugar detection HPLC system (Tosoh Corp.) to quantify the hyaluronan degradation product. For the quantification, sodium hyaluronate-derived sodium hyaluronate was dissolved in the same buffer and treated with the enzyme at the same time as the sample, which was used as a standard.

4) Molecular weight measurement method Segrest et al. [Method in Enzymolgy, Volume 28-B, 54
-63 (1972)], Tris / Glycine / SDS
10 μl on SDS-polyacrylamide gel at pH 8.3
The sample was applied and electrophoresed. HA-
The hyaluronan-binding protein complex treated with hyaluronidase to remove hyaluronan was used. Stain with Coomassie Brilliant Blue R-250,
The molecular weight was observed based on the electrophoretic mobility of the standard molecular weight kit (Pharmacia) in the same electrophoresis. The result is shown in FIG. As described above, the hyaluronan-binding protein of the present invention contains about 85,000 daltons (X band) and about 85,000 daltons (X band).
Showing two bands with 3,000 daltons (Y band),
None of them were found in the sample that had not been treated with hyaluronidase, which was a band at a position different from that of hyaluronidase. In addition, this result shows that even if the sample is reduced,
It was the same without doing it.

5) Measurement of isoelectric point When hyaluronan of the HA-hyaluronan-binding protein complex of the present invention is digested with an enzyme (hyaluronidase) to give a hyaluronan-binding protein alone, an anion exchanger column (DEAE-cellulofine) is used. A800: manufactured by Chisso Co., Ltd., sold by Seikagaku Corporation), pH7.
At 0, all bound, and at pH 6.0, those bound and not bound were at about the same ratio. From this phenomenon,
The isoelectric point of the protein was determined to be between about pH 6.0 and 7.0, especially about pH 6.0-6.5.

6) Determination of partial amino acid primary structure The hyaluronan-binding protein of the present invention was separated into X band and Y band by SDS-polyacrylamide gel electrophoresis, and each was treated with protease V8 according to a known method. From the obtained peptide fragments, one peptide from the X band and one peptide from the Y band are selected, and the amino acid sequencer manufactured by Applied Biosystems is used to carry out the amino acid from the amino terminus by the solid phase method. The sequence was determined. The amino acid sequence from the amino terminus of each peptide was as follows.

Primary structure of the peptide fragment obtained by treatment of the X band with protease V8. Val Asp Gln Val Thr Leu Tyr Ser Tyr Lys Val Gln Ser Thr Ile Thr Ser Arg Met Ala Thr Thr Met Ile Gln Ser Lys Val Val Asn Asn Ser Pro Gln Pro Primary structure of peptide fragment obtained by treatment of Y band protease V8. Ile Val Ile Lys Val Lys Pro Lys Gln Leu Val His His Phe Glu Ile Asp Val Asp Ile Phe Glu Pro Gln Gly Ile Ser Lys Leu Asp Ala Xaa Ala Ser Pro (Xaa represents unidentified amino acid) Protease V8 (Protease V8) is an enzyme prepared from Staphylococcus aureus V8, which is an endoproteinase (to the end of the carboxyl group terminal side of aspartic acid or glutamic acid) in a phosphate buffer (pH 7.8). Suitable pH 4.0 and 7.8) [Manufacturer
ICN Biomedicals Inc.).

Example 2 Preparation of Antibodies to Human Joint Fluid Hyaluronan-Binding Protein The HA-hyaluronan-binding protein complex obtained according to Example 1 was dissolved in 50 mM acetic acid / sodium acetate buffer (pH 5.0). After digestion with hyaluronidase derived from actinomycete, the digested product was treated with SDS- in the same manner as in Example 14).
Polyacrylamide gel electrophoresis was performed. The X band component and the Y band component of the hyaluronan-binding protein were extracted. 0.5 ml of phosphate buffered saline (PBS) containing each of these as an antigen was mixed with an equal volume of Freund's complete adjuvant and injected into rabbit knee bulk lymph nodes.
Then, every two weeks, 50 μl of PBS was mixed with the same volume of Freund's incomplete adjuvant twice and injected at the same site. Weekly ELISA (EnzymeLinked Immunosorbent Assa
The antibody titer was examined by y) and blood was collected two weeks after the second boost to obtain two kinds of antisera. This antiserum was used as an antibody. The obtained antibody reacted to both the X component and the Y component to the same extent regardless of whether the X component or the Y component was used as an antigen. Further, these antibodies also reacted with bovine serum and mouse serum with components corresponding to the hyaluronan-binding protein of the present invention contained therein. In addition, HA
-When the hyaluronan-binding protein complex is used as an antigen, an antibody having the same antigen specificity can be obtained.

Example 3 Immunoassay of Hyaluronan-binding Protein in Synovial Fluid of Arthritis Patients (1) Comparison of the amount of hyaluronan-binding protein contained in synovial fluid of patients with osteoarthritis and synovial fluid of rheumatoid arthritis by dot blot Patients with osteoarthritis of knee (hereinafter abbreviated as OA) (3 cases) and patients with rheumatoid knee arthritis (hereinafter abbreviated as RA) (3 cases)
Then, the hyaluronan-binding protein of the present invention was measured by the following dot blot method. The synovial fluid of each patient was provided by Professor Iwata of the Department of Orthopedic Surgery, Nagoya University School of Medicine.

Each patient's joint fluid was diluted with 20 mM phosphate buffered saline (PBS) (pH 7.2), and 5 μl of each was spotted on a nitrocellulose membrane or a polyvinylidene fluoride (PVDF) membrane. This membrane was immersed in PBS containing 1% bovine serum albumin and blocked at 37 ° C. for 2 hours. After that, it was reacted in PBS containing the anti-hyaluronan-binding protein antibody prepared in Example 2 at room temperature for 1 hour, washed well, and then reacted with peroxidase-conjugated anti-IgG antibody for 1 hour at room temperature to bind to the membrane. Peroxidase was developed with hydrogen peroxide and diaminobenzidine. The result is shown in FIG. Figure 4
In the figure, indicates the intensity of color development, where ● indicates that the color is dark, ○ indicates that the color is clearly identifiable, Δ indicates that the color appears light, and ∴ indicates that the color is almost invisible. Shown respectively.

As a result, the concentration of hyaluronan-binding protein in the synovial fluid of RA patients was clearly higher than that in the synovial fluid of OA patients, and there was a difference of several times to several tens of times as judged from the dilution ratio of the sample. there were.

(2) ELISA (Enzy) of the amount of hyaluronan-hyaluronan-binding protein complex contained in synovial fluid of patients with osteoarthritis and synovial fluid of patients with rheumatoid arthritis
Comparison by me Linked Immunosorbent Assay) In each of 2 cases of OA synovial fluid and RA synovial fluid in which the concentration of hyaluronan-binding protein was different by the dot blotting of Example 3 (1), it was bound to hyaluronan by ELISA. The amount of hyaluronan binding protein (HA-hyaluronan binding protein complex) was compared.

(A) Purification of hyaluronan-binding protein derived from proteoglycan A known method (Tenglad, A., Biochim. Biophys. Acta, 578, P from commercially available bovine nasal cartilage proteoglycan monomer).
281, 1979) and the method described in JP-A-64-469, a hyaluronan-binding protein (hyaluronan-binding site of proteoglycan; hereinafter abbreviated as HABR) was purified.
That is, 300 g of bovine nasal septal cartilage was cut into small pieces with scissors, and guanidine hydrochloride 4M which had been previously kept at 4 ° C was used.
The mixture was added to a 0.5 M solution of sodium acetate (pH 5.8) containing, and the mixture was kept at 4 ° C. and extracted with stirring overnight. Extract at 13,000Xg
After centrifugation for 20 minutes, the supernatant was collected. The supernatant was dialyzed against purified water, freeze-dried, and the resulting solid content was crushed to obtain a crude extract powder.

1600 mg of the obtained crude extract powder and 0.8 mg of trypsin (manufactured by Sigma) were added to 25 ml of 0.1 M Tris-hydrochloric acid buffer solution (pH 7.3) containing 0.1 M of sodium acetate, and the mixture was added at 37 ° C. Stored for 2 hours. Then soybean-derived trypsin inhibitor 1 mg (manufactured by Sigma) and guanidine hydrochloride 19.1 g
Was added, and then 0.1 M sodium acetate solution was further added to make the total volume 50 ml. 50 ml of the trypsinized HABR-containing solution thus obtained was mixed with 50 ml of hyaluronic acid-bound Sepharose, immediately put in a dialysis membrane, cooled to 4 ° C., and dialyzed overnight in 9 volumes of purified water. It was
After dialysis, fill the whole gel into a column with an inner diameter of 3.2 cm and
It was washed with a saline solution of M to remove unadsorbed components.

Further, it is washed with a 1M to 3M saline solution successively to remove unnecessary proteins, and then the above-mentioned guanidine hydrochloride 4M is added.
The adsorbed portion was eluted with a 0.5 M sodium acetate solution (pH 5.8) containing and recovered. The recovered solution was concentrated with DIAFLO PM-10 (manufactured by Amicon) to a total volume of 4 ml, and Sepharose 6B was added.
After passing through a column (φ 3.1 × 43 cm), the components showing the second and third peaks were collected, and each was collected by DIAFLO PM-
The product was separated and concentrated at 10 to obtain a purified sample of HABR.

(B) ELISA The HABR prepared in (A) was added to 20 mM PBS at pH 7.2.
The solution was dissolved in a concentration of 5 μg / ml, 100 μl / well of this solution was dispensed into a 96-well microplate, and left standing at 4 ° C. overnight for coating. After washing the plate, dispense 1% bovine serum albumin / PBS solution at 200 μl / well, 37
Blocking was carried out by incubating at 0 ° C. for 2 hours. The sample synovial fluid (2 synovial fluids from OA and RA patients) is P
Each hyaluronan concentration was combined with BS to prepare a dilution series, 100 μl / well was dispensed to the blocked plate, and the plate was incubated at 37 ° C. for 1 hour. After washing the plate, the anti-hyaluronan-binding protein antibody prepared in Example 2 (using the X band component as an antigen)
Dispense (approx. 1 μg / ml) 100 μl / well at 37 ℃
After incubating for an hour, further peroxidase-conjugated anti-rabbit IgG antibody (manufactured by Jackson Co., Ltd., sold by Seikagaku Corporation; diluted to 1/5000 in commercial stock solution) was dispensed at 100 µl / well and incubated at 37 ° C for 1 hour. did. The plate was thoroughly washed, the peroxidase bound to the microplate was developed with ortho-phenylenediamine and hydrogen peroxide, and the absorbance at 490 nm was compared with a plate reader. The results are shown in FIGS. 5 and 6 for the joint fluids of RA and OA patients, respectively. In each figure, the solid and dotted lines are
The synovial fluid of two patients with different diseases is shown. FIG. 5 is for RA synovial fluid, and FIG. 6 is for OA synovial fluid. OA synovial fluid has 10 times higher hyaluronan concentration than RA synovial fluid. As a result, the amount of HA-hyaluronan-binding protein complex in OA synovial fluid was far smaller than that in RA synovial fluid, which was 1/50 or less.

[0040]

【The invention's effect】

1) Currently, the diagnosis of rheumatism is largely dependent on the findings of the doctor and the patient's awareness, and there is a strong demand for objective and quantitative diagnostic methods. The most common diagnostic method used at present is the 11-item diagnostic criteria of the American College of Rheumatology, and whether rheumatism or not is diagnosed depending on how many of the criteria are met. However, the items included in it are, based on the subjectivity of doctors and patients as described above, and rheumatoid factors,
Those that are detected in other diseases such as liver disease and cancer patients, or those that can be detected in healthy people are also included. Although it is very difficult to establish an absolute objective and quantitative diagnostic method, a combination of several objective and quantitative diagnostic methods will make the diagnosis of RA or OA more reliable and more reliable. Doing early in disease is considered to be an important step in resolving such difficulties. An immunoassay (immunoassay) using an antibody against the hyaluronan-binding protein of the present invention can clearly distinguish OA and RA. By applying this measurement system to a diagnostic reagent, objective and quantitative diagnostic criteria from a new viewpoint can be provided to the conventional diagnostic method. 2) By screening a substance that suppresses the synthesis of hyaluronan-binding protein or a substance that scavenges the hyaluronan-binding protein in body fluid,
It is predicted that new therapeutic agents can be developed.

[0041]

[Sequence list]

SEQ ID NO: 1 Sequence length: 35 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: X band protease V8 degradation fragment Origin: Human knee joint fluid Sequence Val Asp Gln Val Thr Leu Tyr Ser Tyr Lys Val Gln Ser Thr Ile 1 5 10 15 Thr Ser Arg Met Ala Thr Thr Met Ile Gln Ser Lys Val Val Asn 20 25 30 Asn Ser Pro Gln Pro 35

SEQ ID NO: 2 Sequence length: 35 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Y band protease V8 degradation fragment Origin: Human knee joint fluid Sequence Ile Val Ile Lys Val Lys Pro Lys Gln Leu Val His His Phe Glu 1 5 10 15 Ile Asp Val Asp Ile Phe Glu Pro Gln Gly Ile Ser Lys Leu Asp 20 25 30 Ala Xaa Ala Ser Pro 35

[Brief description of drawings]

1 shows the relationship between the specific gravity of each associative ultracentrifuged fraction according to Example 1 and the concentrations of hyaluronan and protein.

[Explanation of symbols]

 ─── Protein concentration (mg / ml) ……… Hyaluronan concentration (μg / ml)

FIG. 2 shows the relationship between the specific gravity of each fraction of the associative ultracentrifuged fraction of Example 1 having a specific gravity of 1.43 or more and further dissociative ultracentrifugation, and hyaluronan and protein concentration.

[Explanation of symbols]

 ─── Protein concentration (mg / ml) ……… Hyaluronan concentration (μg / ml)

FIG. 3 shows an SDS-PAGE electrophoretogram of the treated product of the hyaluronan-hyaluronan-binding protein complex of Example 1 with actinomycete-derived hyaluronidase.

[Explanation of symbols]

Lane 1 Sample that was not hyaluronidase-treated or heat-treated Lane 2 Sample that was heat-treated without hyaluronidase-treated Lane 3 Sample that was hyaluronidase-treated

[Fig. 4] Hyaluronan-binding properties of joint fluid of 3 patients each of rheumatoid arthritis (RA1 to RA3) and osteoarthritis (OA1 to OA3) of Example 3 by dot plot using anti-hyaluronan-binding protein antibody. The comparison schematic of protein concentration is shown.

[Explanation of symbols]

●, ○, △, ∴ indicate the coloring intensity. ● ＞ ○ ＞ △ ＞ ∴

FIG. 5: Concentration of hyaluronan in synovial fluid of rheumatoid arthritis of Example 3 and absorbance of peroxidase color development (490 mm)
Shows the relationship with.

[Explanation of symbols]

 ─── Patient A ……… Patient B

FIG. 6 shows the relationship between the concentration of hyaluronan in the synovial fluid of osteoarthritis of Example 3 and the absorbance of peroxidase coloring (490 mm).

[Explanation of symbols]

 ─── Patient C ……… Patient D

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location G01N 33/577 B // C12P 21/08 9161-4B

Claims (7)

[Claims] 1. A human, which is present in the knee joint fluid of a human patient with osteoarthritis or rheumatoid knee arthritis in association with hyaluronan, is separated from these knee joint fluids, and has the following physicochemical properties. Synthetic fluid-derived hyaluronan-binding protein: (A) Molecular weight: Sodium dodecyl sulfate-polyacrylamide gel Two major proteins, about 85 kilodaltons (X band) and about 83 kilodaltons (Y band) in non-reducing state on electrophoresis. (B) Partial protein primary structure forming a band: The primary structure of a peptide fragment obtained by treating the X band with protease V8 is as follows: Val Asp Gln Val Thr Leu Tyr Ser Tyr Lys Val Gln Ser Thr Ile Thr Ser Arg Met Ala Thr Thr Met Ile Gln Ser Lys Val Val Asn Asn Ser Pro Gln Pro A peptide band obtained by treating the Y band with protease V8 Primary structure is as follows: Ile Val Ile Lys Val Lys Pro Lys Gln Leu Val His His Phe Glu Ile Asp Val Asp Ile Phe Glu Pro Gln Gly Ile Ser Lys Leu Asp Ala Xaa Ala Ser Pro (where Xaa is Unidentified amino acids are shown). (C) Isoelectric point: about 6.0 to 7.0 (estimated from pH dependence of binding to DEAE-Cellulofine A800) 2. Synthetic fluid of a human arthritis patient is collected, a protein denaturant is added thereto, and ultracentrifugation is performed under non-protein dissociation conditions to collect a fraction having a high hyaluronan concentration, and the protein denaturant is added to the fraction. It can be obtained by performing ultracentrifugation under protein dissociation conditions and decomposing the resulting complex of hyaluronan and a hyaluronan-binding protein with a mucopolysaccharide degrading enzyme capable of degrading hyaluronan as a substrate. A protein having the physicochemical properties of 3. A complex of the human synovial fluid-derived hyaluronan-binding protein according to claim 1 or 2 and hyaluronan. 4. The human synovial fluid-derived hyaluronan-binding protein according to claim 1, its X band component, Y band component,
A non-human mammal can be immunized with a peptide fragment thereof or the complex according to claim 3 and collected from the body fluid of the animal, or the hyaluronan-binding protein produced by antibody-producing cells of the animal, That X
An antibody that cross-reacts with one or more of the band component and the Y band component. 5. A method for measuring a human synovial fluid-derived hyaluronan-binding protein, which comprises using the antibody according to claim 4. 6. A method for diagnosing arthritis, which comprises diagnosing arthritis by measuring the content of hyaluronan-binding protein in synovial fluid collected from a human joint using the antibody of claim 4. 7. A hyaluronan-binding protein derived from proteoglycan, which is bound to a solid phase, and the antibody of claim 4.
A kit for diagnosing arthritis, which essentially consists of a labeling substance or an anti-immunoglobulin antibody capable of binding.