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Composition for detecting immune antibodies of rheumatoid arthritis by dot immunogold filtration assay

A technology of immune diafiltration and immune antibody, applied in the direction of peptides, etc., can solve the problems of large-scale clinical application of unfavorable products, the impact of detection sensitivity, and no data to support BiP rheumatoid arthritis

Active Publication Date: 2013-06-19
SHANGHAI RONGSHENG BIOLOGICAL PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

64% of patients with rheumatoid arthritis can produce antibodies directly against BiP, and there are reports that it is highly specific to the disease, but there is no data to support the role of BiP in predicting rheumatoid arthritis, and these reports The BiP antibody still needs to be confirmed by independent clinical research (Clinica Chimica Acta 2004, 350, 17-34)
Since the combination of the antibody molecule to be tested and the labeled antibody / antigen is always in the process of adsorption and desorption throughout the detection process, in practical applications, the detection sensitivity is affected by the detection membrane used, which is not conducive to the large-scale clinical application of the product. application

Method used

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  • Composition for detecting immune antibodies of rheumatoid arthritis by dot immunogold filtration assay
  • Composition for detecting immune antibodies of rheumatoid arthritis by dot immunogold filtration assay
  • Composition for detecting immune antibodies of rheumatoid arthritis by dot immunogold filtration assay

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Experimental program
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Effect test

Embodiment 1

[0096] The synthesis of embodiment 1CCP peptide I and CCP peptide II

[0097] CCP peptides can be synthesized by solid-phase synthesis techniques using Fmoc chemistry. For the specific steps of this method, see Eur.J.Immunol.1994, 24, 3188-3193; J.Org.Chem.1972, 37, 3404-3409; Huang Weide, Chen Changqing Polypeptide Synthesis, Beijing: Science Press, 1985.

[0098] The formation method and steps of disulfide bonds can be found in the literature: Huang Weide, Chen Changqing Peptide Synthesis, Beijing: Science Press, 1985, p85; Michael W.Pennington Peptide Synthesis Protocols (Methods in Molecular Biology), Humana Press, 1994, p91- 169.

[0099] In this example, various CCP peptides were synthesized by Jill Biochemical (Shanghai) Co., Ltd., and the specific sequences are as follows:

[0100] CCP peptide I: His-Gln-Cys-His-Gln-Phe-Arg-Phe-Cit-Gly-Cit-Ser-Arg-Ala-Ala-Cys-Gly, on which the third and sixteenth Cys pass The sulfhydryl group forms a disulfide bond, and makes the po...

Embodiment 2

[0102] Example 2 The Combination of CCP Peptide I and CCP Peptide II with High Molecular Polymers

[0103] Dissolve 2 mg of carrier protein BSA in 200 μl of deionized water, 2 mg of CCP peptide I and CCP peptide II (molar ratio 1:1) in 0.5 ml of coupling buffer (0.1 mol / L MES, 0.9 mol / L NaCl, 0.2 g / L L NaN 3 , pH4.7). Add 0.5 ml of CCP solution to 200 μl of carrier protein solution. Add 1ml of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) solution with a concentration of 10mg / ml into the mixture of CCP and BSA, mix gently, and react at room temperature for 2h , dialyzed with 0.01mol / L pH7.4 phosphate buffer at 4°C for 3 days, and stored at 4°C after aliquoting.

Embodiment 3I

[0104] Example 3 IgG labeled colloidal gold

[0105] Heat 99ml ultrapure water to 80-90°C, add 1% (w / v) HAuCl 4 1ml, continue heating and stirring until boiling. Fill and boil for about 10 seconds, quickly add the existing trisodium citrate solution (0.0236g trisodium citrate, made into a concentration of about 1% (w / v)), stir and boil for 10 minutes. After cooling at room temperature, add ultrapure water to make up to 100ml. Scan the obtained colloidal gold at a wavelength of 400-700nm, and require the absorption value at a wavelength of 525-535nm to reach 0.92-0.98.

[0106] In 100ml of prepared gold sol that has been cooled to room temperature, use 1% (w / v) K 2 CO 3 Adjust the pH to 8.2.

[0107] Add 1.5 mg mouse anti-human IgG (Hangzhou Keelong Biotechnology Co., Ltd.) (0.1 M phosphate buffer diluted to 1 mg / ml), and stir at room temperature for 30 min.

[0108] Add BSA solution to block for 30min (5% (w / v) solution, add 25ml). Centrifuge at 10,000rpm for 25min and ...

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Abstract

The invention relates to a composition for detecting immune antibodies of rheumatoid arthritis by a dot immunogold filtration assay, which comprises a cyclic citrullinated peptide (CCP) composition, a high molecular polymer, a microporous filtering film, a water absorption medium and a carrier medium, wherein the high molecular polymer is combined on the microporous filtering film in a covalent form or a non-covalent form; the CCP composition comprises CCP I and CCP II and is combined with the high molecular polymer in a covalent form or a non-covalent form; and the molar ratio of the CCP I to the CCP composition is 0-1. The composition not only can realize the purpose of quickly detecting the immune antibodies of rheumatoid arthritis in vitro but also is convenient for patients to carry out self-diagnosis, and provides a reference for knowing the progression of the disease in time.

Description

technical field [0001] The invention relates to a composition for detecting rheumatoid arthritis antibodies, in particular to a composition for detecting rheumatoid arthritis immune antibodies by spot gold immunofiltration method, which can realize rapid and accurate detection of rheumatoid arthritis immune antibodies. Background technique [0002] Clinically, autoimmune diseases mainly include systemic lupus erythematosus, rheumatoid arthritis, Sjogren's syndrome, dermatomyositis, polymyositis, systemic (multiple) sclerosis, scleroderma, etc. These diseases were once named " Connective tissue disease", later both abroad and domestically classified them as rheumatic diseases. [0003] Rheumatoid arthritis (RA) is a relatively common systemic autoimmune disease mainly manifested by chronic polyarticular inflammation. Very painful. The incidence of the disease is 0.5-1% worldwide, and the incidence in China is about 0.36%. The age of onset of patients is mostly between 20 a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08
Inventor 朱绍荣
Owner SHANGHAI RONGSHENG BIOLOGICAL PHARM CO LTD
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