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A CCP antigen for detecting anti-citrullinated peptide antibody and its preparation method

A citrulline peptide antibody, citrulline technology, applied in the field of biological detection, can solve the problems of steric hindrance, difficult to coat with nitrocellulose membrane, etc., to avoid steric hindrance, meet the needs of clinical testing, Effects of high specificity and sensitivity

Active Publication Date: 2022-05-17
青岛硕景生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] To sum up, the current CCP antigens on the market mainly have the following disadvantages: in the prior art, most of the CCP antigens are simple combinations of citrulline peptides with different antigenic sites. It is also easy to cause steric hindrance; in addition, because the molecular weight is too small and it is a cyclic structure, it is not easy to be coated on the nitrocellulose membrane with a relatively large pore size

Method used

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  • A CCP antigen for detecting anti-citrullinated peptide antibody and its preparation method

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Effect test

Embodiment 1

[0038] A CCP antigen for detecting anti-citrullinated peptide antibodies. The CCP antigen is obtained by coupling a polypeptide and a carrier protein at a molar ratio of 80:1 to 20:1; the polypeptide part has the following structure:

[0039] NH 2 -S1-S2-(GGGS)5GGG-S3-S4-COOH; wherein, S1, S2, S3 and S4 are all small citrulline-containing peptides; the middle is chimerized with a linker (GGGS)5GGG. S1 and S4 include cyclized cysteines.

[0040] The sequence of S1 is shown in SEQ ID NO: 1, which is HSCDSERHX; the sequence of S2 is shown in SEQ ID NO: 2, which is GXSRNHHGS; the sequence of S3 is shown in SEQ ID NO: 3, which is HQCHQKRAX; the sequence of S4 is shown in SEQ ID NO: 4, it is HXRAACGRSGS; X is citrulline.

Embodiment 2

[0042] Preparation of a CCP antigen for detecting anti-citrullinated peptide antibody

[0043] 1. Synthesis of CCP antigen cyclized polypeptide

[0044] Four antigenic sites S1, S2, S3 and S4 targeting rheumatoid arthritis were screened out by using the serum of patients with rheumatoid arthritis, and then connected through the linker (GGGS) 5GGG. The cysteines in S1 and S4 form disulfide bonds. Ultimately the polypeptide portion of the CCP antigen is formed. The polypeptide is synthesized by the Polypeptide Synthesis Company using the FMOC solid-phase synthesis method. After HPLC analysis and purification, the purity of the antigen reaches more than 90%. The specific sequence is as follows: NH 2 -S1-S2-(GGGS)5GGG-S3-S4-COOH.

[0045] 2. Preparation steps of CCP antigen

[0046] (1) Prepare the main solution and store it at -20°C for later use.

[0047] MES buffer: weigh 3.90g MES, then dilute to 200ml with pure water, the final concentration is 0.1M;

[0048] EDC solut...

Embodiment 3

[0053] Preparation of a CCP antigen for detecting anti-citrullinated peptide antibody

[0054] 1. Synthesis of CCP antigen cyclized polypeptide

[0055] Four antigenic sites S1, S2, S3 and S4 targeting rheumatoid arthritis were screened out by using the serum of patients with rheumatoid arthritis, and then connected through the linker (GGGS) 5GGG. The cysteines in S1 and S4 form disulfide bonds. Ultimately the polypeptide portion of the CCP antigen is formed. The polypeptide is synthesized by the Polypeptide Synthesis Company using the FMOC solid-phase synthesis method. After HPLC analysis and purification, the purity of the antigen reaches more than 90%. The specific sequence is as follows: NH 2 -S1-S2-(GGGS)5GGG-S3-S4-COOH.

[0056] 2. Preparation steps of CCP antigen

[0057] (1) Prepare the main solution and store it at -20°C for later use.

[0058] MES buffer: weigh 3.90g MES, then dilute to 200ml with pure water, the final concentration is 0.1M;

[0059] EDC solut...

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Abstract

The invention belongs to the technical field of biological detection, and specifically relates to a CCP antigen for detecting anti-citrullinated peptide antibodies and a preparation method thereof. The CCP antigen is obtained by coupling a polypeptide and a carrier protein according to a certain ratio; the polypeptide part has the following formula: Structure: NH 2 ‑S1‑S2‑(GGGS)5GGG‑S3‑S4‑COOH; wherein, S1, S2, S3 and S4 are all citrulline-containing small peptides; S1 and S4 include cyclized cysteine. The polypeptide of this antigen contains multiple antigenic sites, and a disulfide bond is formed through the cysteine ​​side chain sulfhydryl groups at the N and C terminals of the sequence to form a ring structure, and at the same time, four short peptides containing citrulline are connected through a linker Carry out tandem chimerism to make the structure more stable and avoid steric hindrance; the cyclized polypeptide sequence is coupled with the carrier protein, which can be better coated on the nitrocellulose membrane and increase the binding capacity of the anti-citrullinated peptide antibody. Ability to meet the needs of clinical testing.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a CCP antigen for detecting anti-citrullinated peptide antibodies and a preparation method thereof. Background technique [0002] Rheumatoid arthritis (RA) is a systemic autoimmune disease that often involves peripheral arthritis and multiple systems, causing irreversible damage to bones and joints and causing great harm to patients. At present, the incidence rate of rheumatoid arthritis in my country can reach 0.33%, and there is an increasing trend year by year. Therefore, early diagnosis and active treatment of rheumatoid arthritis are of great significance to improve the quality of prognosis. The method of diagnosing rheumatoid arthritis mainly includes relying on clinical symptoms, laboratory examination and X-ray detection to confirm the diagnosis. Among them, the clinical diagnosis is greatly affected by subjective factors, coupled with the divers...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C07K1/04C07K1/10G01N33/531G01N33/543G01N33/68
CPCC07K7/06C07K14/77C07K14/765C07K14/795G01N33/531G01N33/543G01N33/6854G01N33/6893C07K2319/00G01N2800/102Y02P20/55
Inventor 刘万建杨帆杜金芳王婷李林李文
Owner 青岛硕景生物科技有限公司
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