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Polypeptide combined with immune antibody and its application

An immune antibody, combined technology, applied to peptides combined with immune antibodies and their application fields, can solve the problem of waiting for further independent clinical research, rheumatoid arthritis sensitivity cannot be compared, and there is no data to support BiP rheumatoid arthritis And other issues

Active Publication Date: 2012-02-01
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)
It must be noted that, as RF is included in the ACR criteria, its sensitivity in the diagnosis of rheumatoid arthritis cannot be directly compared with those of other methods not included in the diagnostic criteria

Method used

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  • Polypeptide combined with immune antibody and its application
  • Polypeptide combined with immune antibody and its application
  • Polypeptide combined with immune antibody and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0119] The preparation of embodiment 1 polypeptide

[0120] Polypeptides are synthesized by solid-phase synthesis technology using Fmoc chemical method. 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 [M], Beijing: Science Press, 1985 .

[0121] The formation method and steps of disulfide bonds can be found in the literature: Huang Weide, Chen Changqing Peptide Synthesis [M], Beijing: Science Press, 1985, p85; Michael W.Pennington Peptide Synthesis Protocols (Methods in Molecular Biology), Humana Press, 1994, p91-169

[0122] Through the above steps, the specific sequence of the synthetic polypeptide is:

[0123] Polypeptide: His-Gln-Cys-His-Gln-Phe-Arg-Phe-Cit-Cit-Cit-Ser-Arg-Ala-Ala-Cys-Gly (SEQ ID No: 1), the third and the third Sixteen-position Cys forms a disulfide bond through a sulfhydryl group, and makes the polypeptide form a ring structure, and can simu...

Embodiment 2

[0144] Preparation of embodiment 2 biotin-labeled polypeptide

[0145] Biotin-polypeptide (SEQ ID No: 1) is carried out separately by linking with biotin and then purifying in the solid-phase synthesis process. For specific methods, see Deibel, M.R., Jr., Lobl, T.J.and Yem, A.W., A Technique for rapid purification of low yield products: Biotinylation of chemically synthesized proteins on-resin. Pept. Res. 1989, 2, 189-194.

[0146] When biotin is directly condensed by a condensing agent during polypeptide synthesis, the Kaiser test is used to detect whether the condensation reaction is complete. Please refer to the Sigma-Aldrich 60017 product description (60017 Data Sheet) for a detailed description of this reaction. This method can not only be applied to qualitative (Analytical Biochemistry 1970, 34, 595), but also quantitative (Analytical Biochemistry 1981, 117, 147) detection.

[0147]Biotin-labeled on the resin. First, the end of the protein or polypeptide is deprotected...

Embodiment 3

[0168] Example 3 In Vitro Detection of Rheumatoid Arthritis Antibodies

[0169] The ELISA method is used to detect the autoimmune antibodies in the serum of patients with rheumatoid arthritis. For specific experimental steps, please refer to the corresponding part of ELISA detection in US6,858,438.

[0170] The basic principle is: the polypeptide (antigen) is coated on the surface of the microtiter plate, and the diluted serum / plasma to be tested and the control substance are added to the wells of the reaction plate. If there is anti-CCP antibody in the serum / plasma to be tested, after incubation Afterwards, the specific antibody in the serum / plasma combines with the CCP antigen polypeptide in the well of the reaction plate to form a solid-phase antigen-antibody complex, washes away the unbound other components in the serum / plasma, and adds enzyme-labeled anti-human IgG antibody , incubate, the anti-CCP antibody bound to the solid-phase CCP antigen (polypeptide) is combined wi...

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Abstract

A polypeptide that binds to immune antibodies, with three arginine side chains modified to be electrically neutral or electronegative amino acids in its sequence. Two non-adjacent cysteine ​​side chain sulfhydryl groups on these polypeptide sequences form a disulfide bond to form a ring. This kind of polypeptide can not only combine with rheumatoid arthritis autoimmune antibody, but also have high affinity to HLA-DR. It is verified by experiments that the specificity of combining the polypeptide with the autoimmune antibody of rheumatoid arthritis is greater than 95%, and the detection sensitivity of the autoimmune antibody of rheumatoid arthritis is greater than 75%. Compared with similar products sold in the market, its sensitivity is significantly improved, which is more conducive to the in vitro detection of rheumatoid arthritis autoimmune antibodies.

Description

technical field [0001] The present invention relates to a polypeptide that binds to immune antibodies, in particular to a polypeptide that binds to rheumatoid arthritis immune antibodies, and more specifically relates to a polypeptide that has three arginines modified and binds to rheumatoid arthritis autoimmune antibodies peptide. 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 mostl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08G01N33/564
Inventor 朱绍荣
Owner SHANGHAI RONGSHENG BIOLOGICAL PHARM CO LTD
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