WO2003006985A1 - Procede d'examen pour le diagnostic du polyarthrite rhumatoide, et reactif pour cet examen de diagnostic - Google Patents

Procede d'examen pour le diagnostic du polyarthrite rhumatoide, et reactif pour cet examen de diagnostic Download PDF

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Publication number
WO2003006985A1
WO2003006985A1 PCT/JP2002/006931 JP0206931W WO03006985A1 WO 2003006985 A1 WO2003006985 A1 WO 2003006985A1 JP 0206931 W JP0206931 W JP 0206931W WO 03006985 A1 WO03006985 A1 WO 03006985A1
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WIPO (PCT)
Prior art keywords
rheumatoid arthritis
gtp
diagnosis
examination
reagent
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PCT/JP2002/006931
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English (en)
Japanese (ja)
Inventor
Yasuyuki Ishizuka
Shunpei Niida
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Applied Cell Biotechnologies, Inc.
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Publication of WO2003006985A1 publication Critical patent/WO2003006985A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/91045—Acyltransferases (2.3)
    • G01N2333/91074—Aminoacyltransferases (general) (2.3.2)
    • G01N2333/9108—Aminoacyltransferases (general) (2.3.2) with definite EC number (2.3.2.-)

Definitions

  • 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 ⁇ -gnoretamino retranspeptidase ( ⁇ -G ⁇ ⁇ ) as a marker.
  • ⁇ -G ⁇ ⁇ ⁇ -gnoretamino retranspeptidase
  • rheumatoid rheumatism rheumatoida darthrtis
  • This rheumatoid arthritis is an inflammation of the synovium lining the joint.
  • 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. ⁇ I was unable to deny gussets.
  • 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.
  • the present invention provides a novel and definitive method based on the detection of this enzyme, based on the technical idea of using ⁇ -daltamyl transpeptidase as a novel marker (indicator) expressed from patients with 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
  • the present invention employs the following solutions.
  • 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 ⁇ -GTP as a marker is provided.
  • y-GTP The y_daltamyl transpeptidase enzyme is hereinafter abbreviated as “y-GTP”.
  • This ⁇ -GTP is known as an enzyme that hydrolyzes ⁇ -glutamyl peptides such as glutathione and transfers a ⁇ -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 ⁇ -GTP protein and the method of confirming expression of mRNA encoding ⁇ -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.
  • the present invention provides a diagnostic reagent for rheumatoid arthritis, which contains at least an anti- ⁇ -GT ⁇ antibody (an antibody that specifically binds to ⁇ -GT ⁇ ).
  • the first technology that can propose a completely novel use of ⁇ -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.
  • 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 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 ⁇ There can be mentioned a method of confirming by an activity measuring method.
  • pannus means that the synovium grew pathologically.
  • ⁇ -GT GT activity measurement method is that ⁇ -(DL- ⁇ -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 ⁇ -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.
  • 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.
  • 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)
  • topology linear, type of sequence: protein, method for determining the characteristics of the sequence: S
  • the anti- ⁇ -GT ⁇ antibody is not particularly limited as long as it specifically binds to GT GT protein. By directly detecting specific ⁇ -GT ⁇ protein specifically expressed from patients with rheumatoid arthritis, Diagnosis of rheumatoid arthritis can be reliably performed.
  • a reagent having a configuration containing at least an anti-V-GT ⁇ antibody that specifically binds to v-GT ⁇ can be considered.
  • 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.
  • ⁇ -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 mR ⁇ of ⁇ -GT ⁇ 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).
  • RNA extraction method such as the AGPC method.
  • mice 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 ⁇ g) as templates.
  • ⁇ -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.
  • Example 1 “Expression of ⁇ -GTP protein in inflamed sites in model animals of rheumatoid arthritis” (Relationship between progression of inflammation and bone breakout and osteoclasts and ⁇ -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.
  • 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- ⁇ -GTP antibody was performed according to a standard method using a goat anti-rat ⁇ -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 ⁇ -GT T protein was observed.
  • ⁇ -GT ⁇ was also clearly expressed in capillaries (Capillary) and plasma cells (Plasmaceles) in the synovium and pannus.
  • 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.
  • Fig. 6 a photograph substituted for a drawing
  • y_GTP was remarkably expressed in the synovial cells and plasma cells.
  • Example of measuring P activity to confirm the relationship between RA symptoms and ⁇ -GTP activity Example of detecting V-GTP protein expression in human synovial fluid by immunoprecipitation using antibodies, Clarifying the relationship between RA symptoms and ⁇ -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 ⁇ _GTP protein is clarified by detection by the ⁇ ⁇ method.
  • ⁇ -GT ⁇ as a marker for rheumatoid arthritis
  • the method for measuring or detecting ⁇ -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.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Rehabilitation Therapy (AREA)
  • Rheumatology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

On a récemment trouvé que la η-glutamyl transpeptidase est exprimée ou que son activité est élevée dans les tissus synoviaux humains, etc. du polyarthrite rhumatoïde. Sur la base de cette constatation, on a développé un procédé d'examen du diagnostic du polyarthrite rhumatoïde en utilisant la η-glutamyl transpeptidase (η-GTP) comme marqueur, ainsi qu'un réactif pour l'examen du diagnostic du polyarthrite rhumatoïde, ledit réactif contenant au moins une substance réagissant avec la η-GTP.
PCT/JP2002/006931 2001-07-13 2002-07-09 Procede d'examen pour le diagnostic du polyarthrite rhumatoide, et reactif pour cet examen de diagnostic WO2003006985A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2001213281A JP2003024099A (ja) 2001-07-13 2001-07-13 慢性関節リウマチの検査診断方法及び検査診断薬
JP2001-213281 2001-07-13

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102971631A (zh) * 2010-12-15 2013-03-13 株式会社凯蒂生物 类风湿性关节炎的检查方法及类风湿性关节炎检查用试剂盒

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
RAMBABU K. ET AL.: "gamma-Glutamyl transpeptidase in synovial fluid, serum and urine of patients with rheumatoid arthritis", BIOCHEM. MED. METAB. BIOL., vol. 43, no. 3, 1990, pages 183 - 192, XP002956379 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102971631A (zh) * 2010-12-15 2013-03-13 株式会社凯蒂生物 类风湿性关节炎的检查方法及类风湿性关节炎检查用试剂盒
US9733243B2 (en) 2010-12-15 2017-08-15 Kayteebio, Co. & Ltd. Test method for rheumatoid arthritis and kit for rheumatoid arthritis test

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