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A kind of cyclic citrulline chimeric peptide antigen and its application

The technology of citrulline peptide antibody and citrulline peptide is applied in the fields of clinical immunity and medical laboratory diagnosis, which can solve the problems of limited detection sensitivity and technical difficulties in sensitivity, so as to improve the detection sensitivity and facilitate the exposure of antigenic epitopes. , to avoid the effect of steric hindrance

Inactive Publication Date: 2017-01-11
陈仁奋
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In this way, if only synthetic peptides containing a single epitope are used like the current commercial kits, a considerable part of the citrullinated peptide antibody subtypes will be missed, which objectively limits the improvement of detection sensitivity
While coating the above three synthetic peptides on the ELISA plate at the same time, due to the competition between the short peptides for the limited coating sites, it also causes technical difficulties in improving the sensitivity.

Method used

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  • A kind of cyclic citrulline chimeric peptide antigen and its application
  • A kind of cyclic citrulline chimeric peptide antigen and its application
  • A kind of cyclic citrulline chimeric peptide antigen and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] 1. Synthesis and application of citrulline peptide sequence

[0044] Firstly, three citrulline-rich dominant antigenic epitopes (citrulline peptide 1, citrulline peptide 2, and Amino acid peptide 3), then chimerized by glycine (G) connection, and a disulfide bond formed by inserting a cysteine ​​(C) at the N-terminal and C-terminal of the chimeric peptide, and finally designed and synthesized A cyclic chimeric peptide antigen (CCCP) containing the above three epitopes and having a protein β-turn similar structure was obtained.

[0045] The polypeptide is automatically or artificially synthesized by Polypeptide Biochemical Company using FMOC solid-phase synthesis method. After HPLC analysis and purification, the purity of the antigen reaches more than 90%. Its related amino acid sequence is as follows (X is citrulline):

[0046] (1) Amino acid sequence of citrulline chimeric peptide containing multiple epitopes

[0047] Cyclic citrulline chimeric peptide (CCCP):

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Embodiment 2

[0065] Circular citrulline chimeric peptide (CCCP) and linear citrulline chimeric peptide (LCCP) containing multiple antigenic epitopes and citrulline peptide 1 (CP1) containing a single antigenic epitope, respectively Peptide 2 (CP2), citrullinated peptide 3 (CP3) short peptides were used in the ELISA plate, and the sera of 50 cases of rheumatoid arthritis were detected at the same time. Such as figure 1 As shown, with an ELISA optical density (OD) of 0.2 as the positive cut-off value, the detection sensitivity of cyclic citrulline chimeric peptides to RA serum is 83%, which is much better than that of linear citrulline chimeric peptides (72%) , citrulline peptide 1 (45%), citrulline peptide 2 (29%) and citrulline peptide 3 (31%).

Embodiment 3

[0067] ELISA kit of the present invention is carried out clinical verification, detected 150 routine rheumatoid arthritis (RA) patients respectively, 168 examples of disease control groups: wherein scleroderma (Scleroderma, Scl) 15 examples, mixed connective tissue disease (MCTD) 15 cases, 27 cases of systemic lupus erythematosus (SLE), 10 cases of psoriasis (Psoriasis), 13 cases of Sjogren's syndrome (SS), 88 cases of other diseases (other diseases), and 60 cases of healthy controls (Healthy).

[0068] see results figure 2 . Taking 24AU / ml as the positive cut-off value (dotted line in the figure), it was found that most (82.7%) RA showed positive results, while the positive rate of disease controls was very low (7.1%), and 100% of healthy controls were negative. Statistics show that the detection sensitivity of the kit of the present invention to patients with rheumatoid arthritis reaches 82.7%, and the specificity reaches 94.7% (Table 1). However, the two commercial ELISA...

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Abstract

The invention discloses a cyclic chimeric citrullinated peptide antigen and an application thereof. The preparation of the cyclic chimeric citrullinated peptide antigen comprises the following steps: firstly connecting and jogging three small-molecular antigen peptides, namely a citrullinated peptide1, a citrullinated peptide 2 and a citrullinated peptide 3 derived from a silk polymerizing protein / an intermediate filament protein, and then synthetizing a cyclic polypeptide with a similar protein beta-corner structure by forming a disulfide bond through two cysteines inserted into the end N and the end C of a chimeric peptide. The cyclic chimeric citrullinated peptide antigen coats a solid-phase vector to prepare an indirect enzyme linked immunosorbent assay kit used for detecting the hypotype of multiple anti-citrullinated protein antibodies contained in RA (Rheumatoid Arthritis) serum. The cyclic chimeric citrullinated peptide antigen and the ELISA kit thereof which are disclosed by the invention have the advantages of simple preparation and experimental operation process, good result repeatability, qualification or quantification and wide clinical application and scientific research value and are outstandingly enhanced in detection sensibility and diagnosis value on RA compared with an international similar kit.

Description

technical field [0001] The invention belongs to the field of clinical immunity and medical experiment diagnosis, and specifically relates to a cyclic citrulline chimeric peptide antigen and its application. Cyclic chimeric citrullinated peptide (CCCP) and its enzyme-linked immunosorbent assay kit for detecting related antibodies. Background technique [0002] Rheumatoid arthritis (RA) is a common chronic autoimmune disease. About 1% of people worldwide suffer from this disease. RA can cause pain, stiffness, and swelling in the joints. Irreversible bone and joint damage may occur within 1-2 years of onset, and may cause various complications. Early diagnosis of RA and drug treatment to relieve the disease are very important to control the disease and improve the prognosis (Gabriel SE. The epidemiology of rheumatoidarthritis. Rheum Dis Clin North Am 27 (2001) 269-281). [0003] The current diagnosis of rheumatoid arthritis is based on the classification criteria of the Ame...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00G01N33/68G01N33/543
Inventor 陈仁奋魏秀妹
Owner 陈仁奋
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