WO2003006985A1

WO2003006985A1 - Examination method for diagnosing rheumatoid arthritis and reagent for examination diagnosis

Info

Publication number: WO2003006985A1
Application number: PCT/JP2002/006931
Authority: WO
Inventors: Yasuyuki Ishizuka; Shunpei Niida
Original assignee: Applied Cell Biotechnologies, Inc.
Priority date: 2001-07-13
Filing date: 2002-07-09
Publication date: 2003-01-23
Also published as: JP2003024099A

Abstract

It is newly found out that Ϝ-glutamyl transpeptidase is expressed or its activity is elevated in human synovial tissues, etc. of rheumatoid arothritis. Based on this finding, it is intended to provide an examination method of diagnosing rheumatoid arthritis with the use of Ϝ-glutamyl transpeptidase (Ϝ-GTP) as a marker and a reagent for examination diagnosis of rheumatoid arthritis at least containing a substance reacting with Ϝ-GTP.

Description

Description: Laboratory diagnostic method and diagnostic agent for rheumatoid arthritis

The present invention relates to a novel test and diagnosis technique for rheumatoid arthritis. More specifically, the present invention relates to a biochemical test and diagnostic method for rheumatoid arthritis and a diagnostic reagent for test and diagnosis of rheumatoid arthritis using γ-gnoretamino retranspeptidase (γ-GΤ Ρ) as a marker. Background art

A typical example of rheumatic diseases is "rheumatoid rheumatism (rheumatoida darthrtis)". It is said that there are about 700,000 patients in Japan at present.

This rheumatoid arthritis is an inflammation of the synovium lining the joint. Specifically, the joint consists of the epiphyseal part of two consecutive bones, the articular cartilage that covers the epiphyses, and the joint capsule of the sac-like tissue that connects both epiphyses, and the lining from the inside. It consists of a thin synovium less than 1 mm thick. Rheumatoid arthritis is an inflammation of the synovium (synovitis), a disease in which the inflammation becomes chronic and gradually spreads to joints throughout the body.

The most prominent methods for testing and diagnosing rheumatoid arthritis at present are based on a combination of specific symptoms and laboratory findings, and are generally based on the American Society of Rheumatoid Arthritis (1980). (Revised version for 7 years). More specifically, tests and diagnosis of rheumatoid arthritis are usually performed on a regular basis, ranging from one to several months, with urine, blood, and x-rays. Blood tests include blood sedimentation, CRP (C-reactive protein), and rheumatism (rheumatoid factor in blood), as well as histological findings and clinical course. In particular, when the value of the above measurement is high, the patient is suspected of rheumatoid arthritis. However, in the early stage of the disease, even if symptoms of joint pain or swelling appear, these measured values may show normal values.ゥ I was unable to deny gussets.

Moreover, not all rheumatoid arthritis patients are positive in the rheumatoid arthritis reaction, and the symptoms may continue to be negative. In rheumatic reactions, hepatitis and other viral infections, and protozoal infections such as malaria may also be positive, and about 5% of healthy people will have a positive reaction. Therefore, it was not a definitive diagnostic method for rheumatoid arthritis and was only one of the reference findings.

X-ray findings and histological findings are based on observing radiographs and joint tissue sections of joints to determine the disease state. The examination takes time and requires skill. In addition, there is a risk that the examiner's arbitrariness may intervene in the judgment, and there is a lack of objectivity.

Thus, at present, there is no definitive test and diagnosis method for rheumatoid arthritis, and in particular, a biochemical test method for rheumatoid arthritis and a diagnosis using the biochemical test results. The method has not been established yet. Therefore, there is a technical problem that it is difficult to detect and treat rheumatoid arthritis early.

Here, in the earlier application from the S home country, Japanese Patent Application Laid-Open No. 11-94883, an immunoreagent for diagnosing rheumatoid arthritis using an anti-aldolase A antibody as a marker for rheumatoid arthritis is described. Is disclosed. However, the technical idea of using y-glutamyl transpeptidase as a marker has not been disclosed or even suggested.

Further, the inventor of the present application disclosed in Japanese Patent Application Laid-Open No. 11-87507 that the differentiation of osteoblasts of γ_glutamyl transpeptidase (γ-GΔΤ) or a derivative thereof was induced. Based on the discovery that it has activity. A new use of the enzyme as an osteoclast differentiation promoter or the like has been proposed. However, this publication does not give any idea about the relationship between γ-glutamyl transpeptidase and rheumatoid arthritis. I couldn't even do it.

Accordingly, the present invention provides a novel and definitive method based on the detection of this enzyme, based on the technical idea of using γ-daltamyl transpeptidase as a novel marker (indicator) expressed from patients with rheumatoid arthritis. By providing biochemical diagnostic methods and diagnostic reagents for rheumatoid arthritis, the aim is to realize early detection and early treatment of rheumatoid arthritis and contribute to the development of the medical and pharmaceutical industries. I do. Disclosure of the invention

In order to achieve the above object and solve the technical problem, the present invention employs the following solutions.

First, in the present invention, the present inventor has completely novel the fact that y-dartamyl transpeptidase enzyme is expressed or its activity is increased in synovial tissue and the like of human rheumatoid arthritis disease. Based on the findings of the present invention, a method for testing and diagnosing rheumatoid arthritis characterized by using this γ-GTP as a marker is provided.

The y_daltamyl transpeptidase enzyme is hereinafter abbreviated as “y-GTP”. This γ-GTP is known as an enzyme that hydrolyzes γ-glutamyl peptides such as glutathione and transfers a γ-glutamyl group to another amino acid peptide, and is often abbreviated as:

The y-GTP used as a marker in the present invention is a method for measuring y_GTP activity, which is currently generally used in laboratory diagnosis of liver diseases, etc. Antibody) and the direct detection method utilizing the reaction between γ-GTP protein and the method of confirming expression of mRNA encoding γ-GTP by PCR (polymerase chain reaction). Based on one method selected from, it is possible to detect easily and reliably. That is, there is an advantage that the detection or measurement of T / -GTP can be easily and reliably performed by an established known means. In addition, the present invention provides a diagnostic reagent for rheumatoid arthritis, which contains at least an anti-γ-GTΡ antibody (an antibody that specifically binds to γ-GTΡ). This makes it possible to easily and promptly confirm the expression of γ-G Τ Τ in the synovial tissue of human rheumatoid arthritis disease. In addition, it is possible to provide a test kit dedicated to test diagnosis of rheumatoid arthritis, which is provided with the test diagnostic agent.

As described above, according to the present invention, in which γ-GTP is used as a marker, the first technology that can propose a completely novel use of γ-GTP as a marker for rheumatoid arthritis is provided. Significance, and V-GT III measurement or detection methods have already been established in the medical field, etc., so that biochemical diagnostic tests for rheumatoid arthritis can be easily and quickly applied to the medical field. It has the second technical significance that it can be spread, and the third technical significance that it can provide a definitive biochemical diagnostic method for rheumatoid arthritis. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram (a photograph as a substitute for a drawing) showing the result of detecting D-band by 1% agarose gel electrophoresis in Example 1.

FIG. 2 is a diagram showing a tissue section of phase 2 in Example 2 (a photograph as a substitute for a drawing).

FIG. 3 is a diagram (a photograph as a substitute for a drawing) showing a tissue section of phase 4 in Example 2.

FIG. 4 is a view showing a tissue section of Phase 5 in Example 2 (a photograph as a substitute for a drawing).

FIG. 5 is a diagram showing a tissue section of Phase 6 in Example 2 (a photograph as a substitute for a drawing).

FIG. 6 is a drawing (photograph substituted for a drawing) showing the expression of y-GTP in human RA synovial tissue in Example 3. BEST MODE FOR CARRYING OUT THE INVENTION First, as a preferred embodiment of the method for testing and diagnosing rheumatoid arthritis according to the present invention, expression in the inflammatory site (synovium, pannus, articular cartilage, plasma cells) and urine of human rheumatoid arthritis disease is preferred. Y_GTP, particularly preferably GT-expressed in plasma cells, synovium, pannus and articular cartilage, is a known _protein commonly used in the detection and diagnosis of liver diseases and the like. GT Ρ There can be mentioned a method of confirming by an activity measuring method. In addition, pannus means that the synovium grew pathologically.

When this measured value is high, it is possible to perform examination and diagnosis of rheumatoid arthritis. The principle of γ-GT GT activity measurement method is that 、-(DL-γ-glutamyl) aniline (I) is used as a substrate, amino acid (II) is added as a receptor, and the azo dye of the reaction product is added. This can be carried out using a commercially available kit as appropriate. Also, in the present invention, "Since there may exist proteins having the same V-GTP activity but different proteins, a direct antigen-antibody reaction between an anti-y_GTP antibody and a specific γ-GTP protein may be used. An embodiment in which the expression of the y-GTP protein is directly confirmed by a detection method (immunological technique) and the test and diagnosis of a rheumatoid arthritis disease are surely performed can be adopted.

Here, the y-GTPP protein expressed in relation to osteoclast-forming activity and bone fracture is considered to be suitable as a marker for rheumatoid arthritis.

Specifically, the DNA (cDNA) base sequence shown in SEQ ID NO: 1 of the attached Sequence Listing (number of strands: double-stranded, hypothetical sequence: No, antisense: No, Specified by the method for determining the characteristics of the sequence: S) and the amino acid sequence shown in SEQ ID NO: 2 (topology: linear, type of sequence: protein, method for determining the characteristics of the sequence: S) Select y-GTP protein as a marker and confirm its reactivity with anti-γ-GTP antibody.

The anti-γ-GTΡ antibody is not particularly limited as long as it specifically binds to GT GT protein. By directly detecting specific γ-GTΡ protein specifically expressed from patients with rheumatoid arthritis, Diagnosis of rheumatoid arthritis can be reliably performed.

Furthermore, based on a method for confirming expression of mRNA encoding γ-GTP protein by RT-PCR (polymerase chain reaction), expression of y_GTP was confirmed, and rheumatoid arthritis disease was confirmed. An embodiment in which the test and the diagnosis of the above are performed can also be adopted. It should be noted that any method other than the above can be adopted as one embodiment of the present invention as long as the method can surely detect the γ-GT GT protein. For example, immunological assays using antibodies include radioimmunoassay (RIΑ), enzyme-linked immunosorbent assay (ELISΑ), and competitive enzyme-antibody assay without radioisotope (Ε Ε Ε). . It is also possible to detect γ-GTΡ protein using a biosensor device such as Biacore.

Next, as a preferred embodiment of the reagent for testing and diagnosing rheumatoid arthritis according to the present invention, for example, a reagent having a configuration containing at least an anti-V-GTΡ antibody that specifically binds to v-GTΡ Can be considered.

The specific form of this reagent is widely interpreted, and furthermore, a diagnostic test kit equipped with this reagent, a biosensor chip having a detection surface equipped with a ligand such as an anti-y_GTP antibody, etc. are also provided. can do.

Example 1>

An experiment was conducted to confirm the expression of the T / -GTP gene in the inflamed site of a model animal of rheumatoid arthritis (hereinafter abbreviated as “RA”).

Γ-GTP mRNA expression was confirmed at the RA-like inflammation site (mainly the ankle joint) of a CIA (Collagen induced arthritis) mouse, which is an RA model animal, by the RT-PCR method. Before the onset of normal mice and RA-like inflammation, the expression of mRΝΝ of γ-GTΡ was not confirmed at all.

CIA mice were prepared according to the method of Suzuki et al. (Yu Suzuki at al., J Rheumatol., 1998; 25: 1154-60).

That is, 5-11 weeks old, DB AZ 1 J mouse (Japanese charles river) of 0.1111 _§ type 1 collagen (collagen technology workshop) and Freund's complete adjuvant, H -37 Ra (Difco) Immunized at the base. Further, after 3 weeks, the same amount of collagen II as before was mixed with Freund's incomplete adjuvant to avoid carotening.

Since the peak of inflammation occurred 10 days after the onset of the disease, it was euthanized with CO ₂ gas, the site of inflammation (mainly joints) was collected, and RNA was prepared. At this time, the collected site was immediately frozen with liquid nitrogen, crushed with a freezer mill, and the total RNA was isolated and purified by an RNA extraction method such as the AGPC method.

For comparison, DBA / 1J mouse kidney (kidney), pre-immune joint (controljoint), and post-immune joint skeletal muscle (Skeletal mu scle) inflamed mice were subjected to the same method as above. And total RNA was prepared.

RT-PCR (kit from Perkin Elraer) was performed using these four types of Tota1 RNA (0.5 μg) as templates. For the amplification of γ-GTP, two types of primers were used, a forward primer (5'-ATCATCGGCCTCTGTATCTG-3 ') and a reverse primer (5'-GCTGTTGTAGATGGTGAAGA-3'). . This ^: the DNA amplified is 228 base pairs.

G3PD D (or DAP DH: Clontech) was used as the control. The DNA amplified by this primer is 983 base pairs. The RT—PCR reaction was performed according to the conditions attached to the kit. After completion of the reaction, the desired DNA band was detected by electrophoresis of 1/10 volume of the reaction product in 1% agarose gel. Figure 1 attached shows this result.

Here, as shown in FIG. 1 (a photograph substituted for a drawing), the expression levels of G3PDHmRNA are almost equal in the four types of tissues, and it is considered that all RNAs are almost equal. Thus, it was revealed that y-GTPP was expressed in RA-like inflammatory sites except in the kidney where it was constantly expressed. Example 2>

“Expression of γ-GTP protein in inflamed sites in model animals of rheumatoid arthritis” (Relationship between progression of inflammation and bone breakout and osteoclasts and γ-GTP) CIA mice (Example 1) were categorized as follows, after immunization with Papillae type II collagen. Phase 1 the day before the first immunization, Phase 2 the day before the booster, Phase 3 when macroscopic swelling is observed, Phase 4 10 days after onset, Phase 5 20 days after onset 60 days after that, it was designated as phase 6.

Mice of each phase euthanized by co ₂ gas was examined immunochemically were taken joint site. That is, after the collected joints were embedded in paraffin, sections were continuously formed in the longitudinal direction of the lower limb.

According to Endocrinology, 122, 1373 (1988), trap (TRAP) staining was performed by reacting a substrate (Naphthol AS-MXphosphate) with a dye (Fast redviolet LB salt) at 37 ° C in the presence of tartaric acid after deparaffinization. . Subsequent operations are dehydration, clearing, encapsulation, and tissue sections. The nuclei were stained with methyl green. Immunostaining (immunostatic) using an anti-γ-GTP antibody was performed according to a standard method using a goat anti-rat γ-GTP antibody. As shown in FIG. 2 (a photograph substituted for a drawing), in the tissue section of the phase 2, there were no TRAP-positive osteoclasts, and almost no γ-GT T protein was observed.

As shown in Fig. 3 (drawing substitute photograph), in phase 4, remarkable osteoclasts were observed at the joint surface of the articular cartilage and at the boundary between cartilage and bone marrow. In addition, γ-GTP is observed in S-swelled synovium (Synovialmemmbrane) and pannus (Pannus).

As shown in Fig. 4 (drawing substitute photograph) and Fig. 5 (drawing substitute photograph), in phases 5 and 6, it was found that both osteoclasts and γ-G Τ 検出 increased as the detection sites expanded. . In particular, γ-GTΡ was also clearly expressed in capillaries (Capillary) and plasma cells (Plasmaceles) in the synovium and pannus.

Thus, it was found that the expression of γ-GTP increased in proportion to the progress of the symptom (bone destruction). Example 3>

“Expression of γ-GTP in synovial tissue of patients with rheumatoid arthritis” With the consent of RA patients, the unnecessary unnecessary synovial tissue removed was embedded in paraffin during joint replacement surgery. According to the method described above, immunostaining using an anti-human γ-GTP antibody and HE (hematoxylin and eosin) staining were performed.

For HE staining, after deparaffinization, stain with methoxylin solution, wash with water, lightly pass through 80% ethanol, stain with eosin solution, and wash gently with water. Then, it was dehydrated, transparent, and sealed to obtain tissue sections.

Polo.

As shown in Fig. 6 (a photograph substituted for a drawing), it was revealed that many inflammatory cells infiltrated the synovial tissue, and that y_GTP was remarkably expressed in the synovial cells and plasma cells. .

The above Examples 1 to 3 fully demonstrated the feasibility of the present invention. In this example, the expression of y_GTΡ mRNA in human synovial tissue was confirmed by RT_PCR as in Example 1, and T / -GT of human synovial fluid (lymph fluid collected in the joint space) was confirmed. Example of measuring P activity to confirm the relationship between RA symptoms and γ-GTP activity, Example of detecting V-GTP protein expression in human synovial fluid by immunoprecipitation using antibodies, Clarifying the relationship between RA symptoms and γ-GT-activity by detecting urinary Y-GTP activity Example, ELIS using antibodies against V-GTP protein and partial degradation products in synovial fluid of RA patients An example can be given in which the relationship between the R R symptom and the amount of γ_GTP protein is clarified by detection by the Α · method. Industrial applicability

According to the present invention, a completely novel use of γ-GTΡ as a marker for rheumatoid arthritis can be proposed. The method for measuring or detecting γ-GTP is based on a biochemical test / diagnosis method and a test / diagnosis reagent for rheumatoid arthritis using a technique already established in the medical field. Can be easily and quickly spread to the field. Furthermore, it can provide a definitive biochemical examination / diagnosis technology for rheumatoid arthritis, and can be used for early diagnosis and early treatment of rheumatoid arthritis.

Claims

The scope of the claims

1. A diagnostic method for rheumatoid arthritis using γ-glutamyl transpeptidase as a marker.

2. The method for testing and diagnosing rheumatoid arthritis according to claim 1, wherein the y-glutamyl transpeptidase is detected based on a method for measuring γ-GΤ activity.

3. The method for detecting rheumatoid arthritis according to claim 1, wherein the γ-glutamyl transpeptidase protein is detected based on a direct detection method using an anti-γ-glutamyl transpeptidase antibody. Inspection and diagnosis method.

4. The method for testing and diagnosing rheumatoid arthritis according to claim 1, wherein the mRNA expression of the γ-glutamino retranspeptidase is confirmed based on the PCR method.

5. A diagnostic reagent for rheumatoid arthritis, which contains at least a substance that reacts with glutamyl transpeptidase.