Development And Clinical Application Of Capture ELISA For Detecting Antibody Against Porcine Circovirus Type2

Porcine circovirus type2(PCV2) is the primary pathogen of postweaning multisystemic wasting syndrome (PMWS) and other associated diseases. The most common clinical signs are depression, dyspnea, unthriftines wasting, anorexia, jaundice and Anemia. PCV2present a trend all of the world along with the development of international trade exchange. PMWS is an economically important disesae in virtually all regions of the world and has made a great loss in swine industry, so a rapidly and accurately diagnosis method is very important to the diease prevention and control. This study prepared the antibody against PCV2, developed a capture ELISA for detecting antibody against PCV2, and made a comparative study of clinical application with indirect ELISA.Preparation of antibody against PCV2:ORF2of PCV2is encoding a major capsid protein (Cap) with good immunogentcity and the imporptant area to distinguish PCV1and PCV2. Cap was used as immunogen to immune6-8weeks Balb/C mice. The spleen cells from the immunized mice with highly antibody levels were fused with the SP2/0myeloma cells. Five positive clones were selected with indrect ELISA after subcloning:2D3,3E10,1C5,3H11,4F9. Ascitic of the monoclonal antibodies reacted with Cap protein in indirect ELISA at a titer of1:51200,1:51200,1:25600,1:25600and1:25600, and sublimated by the caprylic acid ammonium sulfate method.2D3and3E10reacted with Cap protein specifically in Western Blot.Development of capture ELISA:The higly sublimated antibody were used as coating matters, and the cap react with it after sealed by0.5%BSA, then detecting the antibody in serum is possiable. The reaction conditions of ELISA were optimized. The dilution of antidy for coating was1:3200, the concentration of cap that reacting is1:2000, as3.52μg/mL, the dilution of the serum that will be detected is1:40, and the dilution of goat anti-swine IgG is1:1600. The threshold of capture ELISA was0.2, the figure of OD630nm is greater than0.2, confirm as positive, the figure of OD630nm is smaller than0.2, confirm as negative. And made the studys of its sensibility, spectificity, repeatability, all of the results were perfect.Clinical application of capture ELISA:1928serum samples were used in this study which comes from about20pig farms. They need to be tested by the capture ELISA and the indirect ELISA kits produced by Wuhankeqian Animal Biological Products Co.,Ltd. The proportion of positive is62.97%(1214/1928) tested by capture ELISA, the proportion of positive is72.67%(1401/1928) tested by indirect ELISA, and there were1589(1164positives,425negatives) serum samples that had the same results tested by all the two methods, the coincidence rate is82.42%(1589/1928). Comparative analysis through specificity experiments, the capture ELISA we developed has better specificity than indirect ELISA.The results show that the capture ELISA can detect the antibody against PCV2. The success of developing capture ELISA laid the base for the development of capture ELISA antibody test kit fof PCV2. It also provides an useful diagnotic method for postweaning multisystemic wasting syndrome.