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Rheumatoid arthritis marker

A rheumatoid and arthritis technology, applied in the field of rheumatoid arthritis markers, can solve the problem of low positive rate and achieve the effect of simplicity and precision

Inactive Publication Date: 2017-08-18
NAT INST OF ADVANCED IND SCI & TECH +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, as a specific blood test method, there is a method using anti-collagen antibody, but the positive rate is not so high, and there is a problem that it becomes positive even in rheumatoid arthritis (Non-Patent Document 13)

Method used

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Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0285] The preparation method of the labeled test sample is the same as that of (2-2) for the lectin chip analysis method.

[0286] Next, for example, in each well of an ELISA plate coated with streptavidin, biotinylated jacalin is bound, and the test blood sample or its MMP-3 fraction is added to make it react with the MMP in the sample. -3 to interact. Next, with or without washing with a buffer, blocking was performed using unreacted Jacalin as a blocking agent, and the labeled MMP-3 antibody was reacted.

[0287] Also, in sandwich ELISA, it is preferable to use it in combination with other lectins other than Jacalin, such as ABA, ACA, or ACG lectins, as in the case of lectin chip analysis.

[0288] Furthermore, instead of the lectin-coated ELISA plate, an antibody-coated ELISA plate in which an anti-MMP-3 antibody is immobilized on a support can be used. In this case, after the antigen-antibody reaction is carried out by adding a test blood sample, the labeled jacalin ca...

Embodiment 1

[0324] Example 1: Determination of the MMP-3 Existing Amount in the Test Serum Sample

[0325] As far as this example is concerned, the serum of 32 patients with rheumatoid arthritis and 4 cases of other arthritis was collected, and 30 cases were used as objects.

[0326] The simple fractionation and concentration (enrichment) of MMP-3 in serum is based on the method previously developed by the present inventor (Non-Patent Document 14), and compared with the protein analyzed by the previous inventor, the concentration of MMP-3 in blood is very low Proteins were optimized for conditions as follows.

[0327] (1-1) Pre-cleaning process:

[0328] Among the proteins contained in the serum, a pre-removal operation (pre-washing) of substances non-specifically bound to the magnetic beads using the antigen-antibody reaction field is performed. That is, to 40 μL of serum separated in a 1.5 mL tube, streptavidin-fixed serum washed three times with a reaction buffer (Tris-buffered salin...

Embodiment 2

[0342] Example 2: Sugar chain profiling by antibody-coated lectin chip

[0343] In this example, MMP-3 in the test serum sample MMP-3-IP subjected to simple fractionation and concentration (enrichment) of MMP-3 in Example 1 was compared using a lectin chip. Sugar chain profile analysis, to investigate the changes of sugar chains on MMP-3 associated with rheumatoid disease.

[0344] Take 2 μL of each test MMP-3-IP sample obtained in Example (1-4), and adjust to 60 μL. Add this solution to the lectin chip (LecChip TM (manufactured by Nippon Graiko Technica Co., Ltd.)) each reaction chamber (7 reaction chambers are formed for each piece of glass) was subjected to an interaction reaction at 20° C. for 10 hours or more. As a result, the binding reaction between the sugar chains on the MMP-3 in the sample and the 45 kinds of lectins immobilized on the chip substrate reached an equilibrium state. Thereafter, in order to prevent the sugar chains on the detection antibody from bind...

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Abstract

The present invention addresses the problem of providing a method whereby disease activity of rheumatoid arthritis is evaluated reliably and with good sensitivity by a simple method, and a kit for use in the method. The present invention furthermore provides a method for accurate diagnosis of polymyalgia rheumatica and relapsing polychondritis, which are similar rheumatic diseases, a method for differentiating rheumatoid arthritis, and a kit for use in the same. The present invention provides a Jacalin-binding O-linked oligosaccharide epitope as a negative marker for diagnosing rheumatoid arthritis, and enables more accurate assessment of disease condition by more objectively determining disease activity of rheumatoid arthritis using the amount of variation of a value obtained by multiplying the amount of MMP-3 in the blood or the amount of bound MMP-3 and ABA, ACA, or ACG in the blood by the reciprocal of the amount of bound MMP-3 and Jacalin in the blood. Through the discovery that an LEL or STL-reactive sugar chain on MMP-3 in the blood is a sugar chain marker for diagnosing polymyalgia rheumatica and relapsing polychondritis, the present invention also provides a method for accurate diagnosis of polymyalgia rheumatica and relapsing polychondritis. The present invention furthermore provides a method for accurate diagnosis of rheumatoid arthritis capable of differentiating between similar rheumatic diseases, by measuring the ratio of the amount of LEL or STL bound on MMP-3 and the amount of Jacalin, ACG, or the like bound on MMP-3 in the blood.

Description

technical field [0001] The present invention relates to a simple and accurate disease activity evaluation method and a kit for evaluation of rheumatoid arthritis. Background technique [0002] Rheumatoid arthritis (RA) is a highly frequent disease affecting nearly 1% of the population, and is a progressive and intractable inflammatory disease in which joint destruction occurs with synovial proliferation of joints. Inflammatory cytokines such as IL-1β and TNF-α and various proteases are secreted from the synovial tissue that has proliferated in the joints of the whole body, and the articular cartilage and bone are gradually eroded. If it continues to progress, the joint is destroyed and out of shape. Extracellular matrix metalloproteinases (matrix metalloproteinases: MMPs) secreted by chondrocytes and synoviocytes activated by inflammatory cytokines are strongly involved in cartilage destruction, among which matrix metalloproteinase-3 (MMP-3) is known It is abundantly produ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/564C07K14/42C12N9/64G01N33/53G01N33/573
CPCC07K14/42C12N9/64G01N33/53G01N33/564G01N33/573C12Y304/24017C07K16/40G01N2333/96494G01N2800/102
Inventor 久野敦松田厚志成松久竹内勤铃木胜也竹下胜
Owner NAT INST OF ADVANCED IND SCI & TECH
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