Establishment and preliminary application of an ELISA method for detecting serum anti-citrullinated aggregated rabbit IgG antibody

Objective To establish an ELISA method for detecting serum anti-citrullinated aggregated rabbit IgG( Cit-AgRIgG) antibody,and evaluate its value in the diagnosis of rheumatoid arthritis( RA). Methods First,the rabbit IgG solution was warmed up to 62 ℃ for aggregated denaturation to form AgRIgG,and then the arginine in AgRIgG was catalyzed into citrulline by peptidyl arginine deiminase( PAD) to form Cit-AgRIgG. Using Cit-AgRIgG as coated antigen,an ELISA method was established for detecting anti-CitAgRIgG antibody. Last,the established method was evaluated and used to detect clinical serum samples,and the obtained results were analyzed. Results The Cit-AgRIgG could bind highly with anti-cyclocitrulline peptide( CCP) antibody. The mean intra-assay and inter-assay coefficients of variation of the established ELISA method for detecting anti-Cit-AgRIgG antibody were 4. 5% and 9. 5%,respectively. The specific inhibitory test showed that the maximal inhibitory rates of Cit-AgRIgG,AgRIgG and CCP antigen on the antiCit-AgRIgG antibody were 80. 0%,65. 9% and 25. 0%,respectively. The cut off values for detecting anti-Cit-AgRIgG and antiAgRIgG antibodies were 0. 565 and 0. 459,respectively. When the established ELISA method was used to detect clinical serum samples from the patients with RA or other diseases and healthy controls,its sensitivity and specificity for the diagnosis of RA were 79. 3% and 96. 5%,respectively,its positive and negative predictive values were 84. 7% and 95. 1%,respectively,its positive and negative likelihood ratios were 22. 66 and 0. 215,respectively,and the Youden index and the area under the curve( AUCROC) were 0. 758 and 0. 890,respectively. Moreover,the positive rate of anti-Cit-AgRIgG antibody in RA patients was significantly higher than those in the patients with other diseases and healthy controls( P 0. 01). The total coincidence rates between anti-Cit-AgRIgG antibody and antiAgRIgG antibody and between anti-Cit-AgRIgG antibody and anti-CCP antibody were 97. 7% and 91. 7%,respectively. Conclusion The established ELISA method for detecting anti-Cit-AgRIgG antibody had good stability and specificity. The anti-Cit-AgRIgG antibody had the dual characteristics of RF and anti-CCP antibody,and it may be a new marker for the diagnosis and monitoring of RA.