Development of ELISA method for anti-typeIIcollagen peptide(260-272) antibody and its preliminary application

Objective To understand serum levels of anti-type Ⅱ collagen peptide,namely CⅡ(260-272) antibody in rheumatoid arthritis(RA),other autoimmune diseases and healthy control,and explore the value of this antibody in diagnosis and monitoring of RA disease.Methods Synthetic peptide of CⅡ(260-272) was coupled to bovine serum albumin(CⅡ(260-272)-BSA),using this conjugate as coated antigen,ELISA method was established for anti-CⅡ(260-272) antibody.After its methodology assessed,results of clinical cases tested by this method were evaluated.Results The establishment of ELISA method for anti-CⅡ(260-272) antibody has the better results of stability and specificity,and its intra-and inter-assay coefficients of variation were 8.4% and 9.6% respectively.The specific inhibitory rate of this method was up to 76.3%.Clinical test results found that the positive rate of anti-CⅡ(260-272) antibody was 75.0%(30/40) in RA,this result was significantly higher than that of other autoimmune diseases and healthy control group(P0.01).When absorbance of 0.213 was considered cut-off value,the anti-CⅡ(260-272) antibody diagnostic specificity,positive/negative predictive value,positive/negative likelihood ratio and Youden's index was 91.0%,63.8%/94.5%,8.33/0.275 and 0.66 in RA patients,respectively.ROC curve analysis revealed that this antibody on the accuracy of RA diagnosis was at middle level(AUCROC value= 0.890).No correlations was found between level of anti-CⅡ(260-272) antibody and rheumatoid factor(RF)(in 13 cases of RF-negative RA patients,10 cases of anti-CⅡ(260-272) antibody were positive,positive rate was 76.9%),but significantly correlated with anti-cyclic citrullinated peptide(CCP) antibody(P0.01),furthermore,serum levels of anti-CⅡ(260-272) antibody decreased with the improvement of clinical symptoms of RA cases.Conclusion ELISA method for anti-CⅡ(260-272) antibody was successful established,serum levels of this antibody to be tested could be used as a new reference for diagnosis and condition monitoring of RA disease.