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Composition for detecting rheumatoid arthritis immune antibody by colloidal gold lateral chromatography

A lateral chromatography, immune antibody technology, applied in the direction of peptides, etc., can solve the problems of lack of data to support BiP rheumatoid arthritis, waiting for further independent clinical research, long time for testing, etc.

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)
A single ELISA test usually requires a large number of samples, and the test takes a long time. The test process also requires professional personnel to operate, and it relies heavily on medical institutions, which is not conducive to the self-help of ordinary patients. monitor

Method used

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  • Composition for detecting rheumatoid arthritis immune antibody by colloidal gold lateral chromatography
  • Composition for detecting rheumatoid arthritis immune antibody by colloidal gold lateral chromatography
  • Composition for detecting rheumatoid arthritis immune antibody by colloidal gold lateral chromatography

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101] The synthesis of embodiment 1CCP peptide I and CCP peptide II

[0102] 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.

[0103] 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 .

[0104] In this example, various CCP peptides were synthesized by Jill Biochemical (Shanghai) Co., Ltd., and the specific sequences are as follows:

[0105] 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

[0107] Example 2 The Combination of CCP Peptide I and CCP Peptide II with High Molecular Polymers

[0108] 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

[0109] Example 3 IgG labeled colloidal gold

[0110] Heat 99ml ultrapure water to 80-90°C, add 1% (w / v) HAuCl 4 1ml, continue heating and stirring until boiling. Fully 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.

[0111] In 100ml prepared colloidal gold sol that has been cooled to room temperature, use 1% (w / v) K 2 CO 3 Adjust the pH to 8.2.

[0112] 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.

[0113] Add BSA solution to block for 30min (5% (w / v) solution, add 25ml). Centrifuge at 10,000rpm for 25min...

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Abstract

The invention discloses a composition for detecting a rheumatoid arthritis immune antibody by colloidal gold lateral chromatography. The composition comprises a cyclic citrullinated peptide (CCP) peptide composition, a macromoleclar polymer and a lateral chromatography detection medium, wherein the macromoleclar polymer is combined with the lateral chromatography detection medium in a covalent ornon-covalent form; the CCP peptide composition comprises CCP peptide I and CCP peptide II and is covalently or non-covalently combined with the macromoleclar polymer; and the molar ratio of the CCP peptide I to the CCP peptide composition is 0 to 1. The composition can realize rapid in-vitro detection of the rheumatoid arthritis immune antibody in 15 minutes, bring convenience to self-diagnosis of a patient and provide reference for immediately learning disease progression.

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 colloidal gold lateral flow chromatography, so as to 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 betw...

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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