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Composition for externally detecting rheumatoid arthritis antibody and application thereof

An in vitro detection and composition technology, which is applied in the field of detecting the antigen of rheumatoid arthritis autoimmune antibodies and the antigen composition of rheumatoid arthritis autoimmune antibodies, which can solve the difference of antibodies, and there is no data to support BiP in predicting rheumatoid arthritis. Arthritis, detection sensitivity is not significantly improved, etc.

Active Publication Date: 2010-06-09
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. There is currently no data to support the role of BiP in predicting rheumatoid arthritis, and these reports BiP antibodies need to wait for further confirmation through independent clinical research (Clinica ChimicaActa 2004, 350, 17-34)
Although these cyclic or linear polypeptides derived from filaggrin or artificially designed showed high specificity (>90%) in the detection of rheumatoid arthritis, but due to the presence of numerous HLA- DR alleles, the resulting antibodies are also correspondingly different, which makes the detection sensitivity not significantly improved (<70%)

Method used

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  • Composition for externally detecting rheumatoid arthritis antibody and application thereof
  • Composition for externally detecting rheumatoid arthritis antibody and application thereof
  • Composition for externally detecting rheumatoid arthritis antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0140] Example 1 Synthesis of Polypeptides

[0141] 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, Beijing: Science Press, 1985.

[0142] For the formation method and steps of disulfide bonds, please refer to 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

[0143] Through the above steps, the specific sequence of the synthetic polypeptide is:

[0144] Polypeptide 1: His-Glu-Cys-His-Glu-Phe-Arg-Phe-Cit-Gly-Cit-Ser-Arg-Ala-Ala-Cys-Glu (SEQ ID No 1), on which the third and the first Sixteen-position Cys forms a disulfide bond through a sulfhydryl group, and makes the polypeptide have a ring structure, and can simula...

Embodiment 2

[0146] Example 2 Purification of polypeptides

[0147] First, the protecting group at the end of the protein or polypeptide is deprotected and neutralized in dimethylamide (DMF) solvent. The polypeptide is cut off from the synthetic resin with hydrogen fluoride, and the obtained crude product is passed through a reversed-phase chromatography C18 or C8 column (such as: 5 μm, 250×4.6 mm), and the mobile phase A (0.1% (v / v) trifluoroacetic acid Acetonitrile solution) and mobile phase B (0.1% (v / v) trifluoroacetic acid in water) were gradient elution solvents. Within 45 minutes, the mobile phase A accounted for the total volume of the two phases A and B changed from 0% (v / v) to 100% (v / v) to collect the target polypeptide, and the organic phase was removed by desalting chromatographic column (GE Healthcare) or rotary evaporation. The solvent and the target polypeptide can be determined by combining LC-MS or directly injecting the collected polypeptide through the molecular weight...

Embodiment 3

[0148] Example 3 The purity and molecular weight detection of polypeptide

[0149] The above synthesized polypeptide 1 and polypeptide 2 were determined by reverse phase chromatography (RP-HPLC) respectively, and the specific method is as follows:

[0150] 4.6×250mm 5μm C18 analytical column (Kromasil);

[0151] Mobile phase A is trifluoroacetic acid (trifluoroacetic, TFA) added to 100% acetonitrile (acetonitrile, ACN), so that the concentration of TFA is 0.1% (v / v);

[0152] Mobile phase B is TFA added to 100% water, so that the concentration of TFA is 0.1% (v / v);

[0153] The flow rate is 1.0ml / min;

[0154] The detection wavelength is 220nm;

[0155] Elution gradient: The ratio of mobile phase A to the total volume of A and B is 15% (v / v). After injection, linear gradient elution (Gradient Elution) is used, and mobile phase A accounts for A within 25 minutes. The ratio of the total volume of the two items of B and B was changed from 15% (v / v) to 50% (v / v), and then equi...

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Abstract

The invention discloses an antigenic composition which comprises a polypeptide I and a polypeptide II, wherein the molar ratio of the polypeptide I to the sum of the polypeptide I and the polypeptide II is 0-1. An annular polypeptide is generated in a mode of forming a disulfide bond from two nonadjacent cysteine side chain hydrosulphonyls on a sequence. The polypeptide not only can be combined with a rheumatoid arthritis autoimmune antibody, but also can simultaneously have high affinity for HLA-DR. Through experimental verification, the specificity of the polypeptide combined with the rheumatoid arthritis autoimmune antibody is larger than 95 percent, and the detection sensitivity for the rheumatoid arthritis autoimmune antibody is larger than 75 percent. Compared with like products sold on the market, the sensitivity of the composition is markedly improved, thereby being more beneficial to externally detecting the rheumatoid arthritis autoimmune antibody.

Description

technical field [0001] The invention relates to an antigen for detecting autoimmune antibodies, in particular to an antigen for detecting autoimmune antibodies in rheumatoid arthritis, and more specifically relates to an antigen composition for detecting autoimmune antibodies in rheumatoid arthritis. 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. Great pain; 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 and 50 years old, and t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53
Inventor 王绍成朱绍荣
Owner SHANGHAI RONGSHENG BIOLOGICAL PHARM CO LTD
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