Development Of Monoclonal Antibodies Against PCV2and The Potential Application Competitive ELISA For PCV2Antibody Determination

The procine circovirus disease with the characteristic of physical deterioration, diarrhea, weight loss, difficult breathing is caused by procine circovirus type2(PCV2). Pigs are the main host of procine circovirus. Pigs have strong susceptibility to PCV2, pigs of all ages can be infected, result in immunosuppressed. PCV2has caused huge economic losses to the pig industry worldwide, and how can we diagnosis pigs antibody levels fast and accurately has became an important step for prevention and treatment of this disease.In this study, the gene encoding the capsid protein free of nuclear localization signal was coloned into the prokaryotic expression vector pET-32a, resulting in a fusion protein with immunogenicity induced by IPTG. By optimizing the expression conditions, the protein was also present in the form of inclusion body. Through the steps of denatured by Urea, dialysis renaturation and purificated with Ni column, the protein was used to immune6-8weeks BALB/c mice as an immunogen, after fusion, hybridoma were selected by indirect enzyme-linked immuneosorbent assay (ELISA), and the posive clones were subcloned.For hybridoma designated as3E7,3C7,5A7and6D6which stably secreted of monoclonal antibodies against the capsid protein of PCV2were obtained, in indirect ELISA their supernatant titers were1:1280,1:1280,1:2560and1:10240respectively, and their ascites titers were1:12800,1:102400,1:12800and1:204800respectively. Of these monoclonal antibodies, the monoclonal antibody3C7had fluorescence reaction activity with PCV2and the chimeric PCV1-2virus built in our laboratory, its supernatant and ascites fluorescent titers were1:80,1:3200respectively. Subclass of all four monoclonal antibodies are IgM.Using the expressed and purified fusion protein as the coating antigen, the monoclonal antibody3C7was selected as the competitive antibody, a competitive ELISA was developed for determing PCV2antibody according to the characteristics of the four monoclonal antibodies. After optimizing serum dilution,selection of blocking reagents, competitive time, et al, a specific, sensitive competitive ELISA method was established eventually, and by the method there has no cross reactions with classical swine fever, porcine reproduction and respiratory syndrome specific antibody.By this method, we detected189pig serum samples, the results show that the detection result of this method had a93.7%coincidence rate with a commercial PCV2antibody detection kit, and85.2%with that of IFA.The positive coincidence rate and negative coincidence rates with the former were94.9%and87.9%, which were87.4%and76.3%with the later, respectively.The competitive ELISA method established was simple, fast and high sensitive, which can be widely used in the determination of serum antibody of pigs against PCV2.