Rheumatoid arthritis autoantibody conjugated antigen and applications thereof

A technology for arthritis and rheumatoids, applied in the field of bioengineering, can solve problems such as insufficient sensitivity

Active Publication Date: 2015-10-21
SHANGHAI EAST HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the specificity of CCP antibody detection reagents commonly used in clinical practice for auxiliary diagnosis of RA is as high as 95%, but the sensitivity is not enough, between 50-75% (Fan LY, et al. ClinicaChimicaActa2010, 411: 2049-2053)

Method used

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  • Rheumatoid arthritis autoantibody conjugated antigen and applications thereof
  • Rheumatoid arthritis autoantibody conjugated antigen and applications thereof
  • Rheumatoid arthritis autoantibody conjugated antigen and applications thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0076] Preparation method of polypeptide

[0077] The polypeptides of the present invention may be recombinant polypeptides or synthetic polypeptides. The polypeptides of the invention may be chemically synthesized, or recombinant. Correspondingly, the polypeptide of the present invention can be artificially synthesized by conventional methods, and can also be produced by recombinant methods.

[0078]A preferred method is to use liquid-phase synthesis technology or solid-phase synthesis technology, such as Boc solid-phase method, Fmoc solid-phase method or a combination of the two methods. Solid-phase synthesis can quickly obtain samples, and the appropriate resin carrier and synthesis system can be selected according to the sequence characteristics of the target peptide. For example, the preferred solid phase carrier in the Fmoc system is Wang resin connected with the C-terminal amino acid in the peptide, the Wang resin structure is polystyrene, and the arm between the amin...

Embodiment 1

[0093] Example 1 Preparation, identification and storage of CCP5F-H polypeptide and its BSA coupling protein

[0094] The CCP5F, CCP5G and CCP5H polypeptides of the present invention were prepared according to conventional polypeptide synthesis methods, and their physicochemical properties were identified as follows:

[0095] The CCP5F polypeptide of the present invention is a cyclic polypeptide with a length of 18 amino acids ( Figure 4 ), a molecular weight of 2100.85 Daltons (Dal), before coupling with BSA, the amino group (-NH 2 ) end is modified as a hydrogen chain (H), and the modification at the end of a hydroxyl group (COOH) is a hydroxyl group (OH). In the Discovery HS C-18 column of HPLC, its shifting ultraviolet absorption peak at 220um wavelength is 95% A peak (main peak) in 0-2 minutes, and 5% B peak after 2-10 minutes. After the polypeptide is coupled with BSA, the concentration is still calculated according to the concentration of the CCP5F polypeptide (BSA i...

Embodiment 2

[0098] Example 2 Comparison of CCP5F, CCP5G and CCP5H polypeptide characteristics and antigen antibodies

[0099] CCP5F, CCP5G and CCP5H polypeptide BSA-coupled protein were used to immunize purebred goats in Texas, USA, respectively, in order to produce higher titer antiserum. Figure 5 To identify the reactivity (reaction titer) of the anti-CCP5F-H antibody with the antigen, the anti-CCP5F-H antiserum reacted with the corresponding antigen CCP5G polypeptide-BSA antigen protein after three-fold serial dilution, and the signal was amplified by the secondary antibody-HRP reaction Finally, different absorbance (OD) and gradient curves were shown on 96-well ELISA plates to represent the titers of goat anti-CCP5F, CCP5G and CCP5H antibodies.

[0100] The identification of goat anti-CCP5F-H antiserum ELISA titer reaction, the method and steps are: CCP5F-H is coated with 100 microliters / hole (ul / well) at a concentration of 0.5 micrograms / microliter (ug / ml) in PBS On a 96-well ELISA...

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Abstract

The present invention relates to a rheumatoid arthritis autoantibody conjugated antigen and applications thereof. The present invention discloses a novel detection antigen of rheumatoid arthritis antibody, wherein particularly the detection antigen can be used as the RA diagnosis and early diagnosis indicator, and is superior to the clinically developed detections of CCP, CRP, IgG-RF, IgM-RF and the like, such that the detection antigen can be used as the valuable reference indicator for clinical early treatment or prevention of RA, and can further be used as the RA pathogenesis development indicator.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a new protein for diagnosis and treatment of rheumatoid arthritis. Background technique [0002] Rheumatoid arthritis (RA) is a chronic, systemic, autoimmune disease characterized by non-specific, symmetrical chronic inflammation of the peripheral joints, chronic inflammation and hyperplasia of the synovial membrane, and the formation of pannus. Invasion of articular cartilage, subchondral bone, ligaments and tendons, etc., resulting in destruction of articular cartilage, bone and joint capsule, eventually leading to joint deformity and loss of function, some patients with varying degrees of systemic manifestations. RA is a common multiple autoimmune disease in China, with a prevalence rate of about 0.3%-0.4%. In the United States, patients with this disease account for about 1% of the population, and the incidence rate of women is 2-3 times higher than that of men. All age groups ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/64C07K14/765C07K14/795G01N33/68
Inventor 范列英宗明
Owner SHANGHAI EAST HOSPITAL
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