Establishment Of A Early Warning Specificity Detection Method For Rheumatoid Arthritis

Objectives: S ecreted by B cell from the rheumatoid arthritis(RA) sufferer, anti-CCP antibody has specificity for early diagnosis of RA, and at the same time it is of significant value in predicting bone fracture degree and in RA clinical prognosis evaluation.This study intends to establish a kind of quantitative detection method, which is to detectthe content of anti-CCP antibody colloidal gold in humans’ serum, and this will provide a kind of technical means for the early warning and diagnosis of the disease and prognosis assessment. Meanwhile, it also intends to promote the progress of the disease specific detection method.Methods:1. Through the synthetic technology of the artificial synthesis of CCP peptides,The carbon imine coupling technology makes the CCP polypeptide and bovine serum albumin(BSA) couple with each other; to optimize the condition for coupling; to identify the coupling products by applyingthe method of ultraviolet(UV) and the SDS-PAGE; using indirect ELISA, it intendsto detect the biological activity, then toconfirm the optimal CCP peptide coupling.2.The development ofthe colloidal gold strip which is used to detect the level of anti-CCP antibody in serum.Based on colloid gold particles, p H optimization, marking the lowest amount of antibodies to optimize, immune protection solution p H value optimization, strip material selection and processing, the aspects of the best quantity of package is studied. Performance of strip from the linear range, precision, stability, recovery, and the evaluation of the clinical test samples, etc. To establishhuman serum anti-CCP antibody colloidal gold quantitative detection method.Results: 1.When the CCP- BSA in conjunction coupling 12 h is confirmed as the best,CCP- BSA coupling products which can be combined with anti CCP antibody are gained.Once the coupling is confirmed to be successful and the coupling products are tested to be effective, it can be used toprepare and developthe colloidal gold strip. 2. This study establishes the indirect colloidal gold technique conditions for quantitative detection methods:Gold chloride acid and sodium citrate volume ratio is 1:1; the best preparation of colloidal gold; Colloidal gold solution to save the best conditions for adding 0.05% sodium azide; Mark 1 m L colloidal gold solution to join the 3μL 0.2mol/m L K2CO3, minimum amount of labeled antibody was 8.8 μg; Immune gold protection fluid optimum p H of 8.8; Best quality control line package is sheep anti rat Ig G concentration of 1.0 mg/m L; Lines of package is CCP-BSA coupling antigen optimal concentration of 1.5mg/m L; Package is 0.4% sucrose content in the buffer; Gold mat gold shop quantity is 400μL/10 cm, jinbiao quantitative meter reading time as reagent reaction after 14 min.3.The quantitative detection method of colloidal gold performance indicators are: Linear range 50 ~ 500 RU/m L, R2=0.993; the recovery rate of high, medium and low serum respectively100.96%、97.17%、91.46%; with 40 clinical samples and foreign kit parallel test; the results show that both correlation; the regression equation: Y = 1.004 + 7.968 X, R2 = 0.955.Conclusion: 1.By the coupling method ofcarbodiimide, the CCP peptides and the BSA macro-molecular can be coupled successfully, and the coupling productscan be combinedwith anti-CCP antibody specifically.2. It sets up a kind of quantitative detection methodwhich is used to detect the anti-CCP antibody colloidal gold in humans’ serum. This methodcan be applied in the clinical quantitative detection of anti-CCP antibodies, and it also provides technical supports for early clinical warning diagnosis and prognosis assessment of RA.