Standardization Research Of Blocking ELISA Kit For Detection Of Antibody To PCV2 Based On Cap Protein

Porcine circovirus (PCV) is the smalliest animal virus which have two open reading frame ORF1 to encode replication associated protein and ORF2 to encode capsid protein. It has two serotype 1 (PCV1) and 2 (PCV2). Only PCV2 has pathogenicity, and can damage immune organ inducing immunosuppression which make secondary mixed infection facility to result porcine circovirus associate diseases (PCVAD), such as postweaning multisystemic wasting syndrome (PMWS), porcine dermatitis and nepHropathy syndrome (PDNS), porcine respiratory and reproductive syndrome (PRRSV), arthritis and so on, which PMWS can induce serious immunosuppression to cause economy loss in swine industry.Capsid is the major structure protein of PCV2 and has many B cells epitopes and neutralisation epitopes. The antibody detection techniques of PCV2 include ELISA, IPMA, and IFA. Among them, ELISA could be used to detect mass clinical serum since and result assessment can be standardization. In our country, commercial ELISA antibody detection kit were all based on indirect ELISA which had high request for the antigen purity otherwise had the possibility of false positive result. Our laboratories had established blocking ELISA antibody detection method bu using HRP labeled 5H7 monoclonal antibody to the conservation epitopes of Cap protein. In this study, we assembled the ELISA kit successfully and evaluated its property in many aspects.The main contents are as followings:1. Preparation and quality detection of components for PCV2 Blocking ELISA KitBased on the blocking ELISA antibody detection method, the research prepared components for the ELISA kit, including recombinant Cap protein, ELISA plates coating with Cap antigen, positive and negative control serum,5117HRP, TMB, stop solution, sample diluents,10-diplo washing solution. The results showed that the purified Cap protein had accurate molecular weight at 41kDa and excellent antigenicity by SDS-PAGE analysis. The concerntation of three batches Cap were all more than 0.8mg/mL having high repeatability. The ELISA plates coated with the Cap were slick having no crack and the vacuum pack had no air leakage. The positive control serum had reaction strap with Cap protein at 41kDa but negative control serum had no strap by Western-Blot assessment. And the control sera are all neative in the steriling tests.5H7HRP had reaction strap with Cap protein at 41kDa by Western-Blot analysis. The titer of 5H7HRP was higher than 1:3000 with steriling test fiting the criterion TMB can reacte with 5H7 RP normally. The results of trait test and steriling test for TMB, stop solution, sample diluents and 10-diplo washing solution were conformity. The results illustrated that the components could be used to assemble the ELISA kit.2. Assembly and preservation of PCV2 Blocking ELISA KitThe important components were dealed with homologous protectants to assemble the ELISA kit. The titers of 30 anti-PCV2 sera were 1:32-256 detected by the kit. Meanwhile, th kit could not react with the antiboies to CSFV, PRRSV, PRV, SIV、FMDV, PCV1 and Ecoli. It indicated that this kit had better sensitivity and specificity. Moreover the results demonstrated that the components and the ELISA kit can keep its property stability after 7 days at 37℃ or 12 months at 4℃. The all indicated that the ELISA kit was suited for detecting mass clinical serum on account of its well stabilization.3. Application of PCV2 Blocking ELISA KitDetected serum from pigs with different PCV2 vaccines, PMWS and healthed pigs or pigs at different ages from different nurserys to evaluate the effectiveness of vaccine immunity procedure and antibody distribution in swinery using the blocking ELISA kit and compare the coincidence between the commercial kit. The results showed that the anti-PCV2 antibodies levels in pigs with PCV2 oil adjuvant vaccine, PCV2 infection or finishing pigs were higher than those in others groups. It illustrated that the ELISA kit could distinguish the level of antibody from different pigs and evaluat the effectiveness of vaccine immunity. The coincidence between the commercial kit and blocking ELISA kit were 90.91%. It indicated that the ELISA kit had perfect reliability for detecting the serum from clinal.In summary, the blocking ELISA kit was assembled successful. The results indicated that it has better sensitivity, specificity and reliability. The kit could be used for detecting the serum antibody to PCV2.