Development Of Monoclonal Antibody Of Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea (PED) is an viral infection diseases, which is caused by PEDV. Its main shows are diarrhea and vomiting in general clinical, and can cause acute enteritis for almost pig groups. Especially for lactation piglets,death rate can reach more than90%. Monoclonal antibody has an important significance in the structure, function and variation of the viral antigen. In this study, had accomplished amplification and purification of PEDV, and the preparation and identification of monoclonal antibodies.1. Amplification and purification of PEDVIn the research, vero cells was elected to amplify PEDV. After the CPE was above90%, gathered and cryopreserved the cells. Then purified the sample, and did the following tests:TCID50, RT-PCR,SDS-PAGE and Western blot. The results show, the time of collecting cells was about72h later, TCID50=10-48/100μl, and had amplified the gene M by RT-PCR. The sample still had immunoreactivity by SDS-PAGE and Western blot. The protein level of the sample was4.4mg/ml.2. The preparation of monoclonal antibody against PEDVTaked the purified antigen to immune BALB/c mice. Then detected the serum titer of immuned mice by ELISA. When the titer is more than104-105, the splenocyte and SP2/0cells could be fused.The positive hybridomas were selected to subclone for4times by limiting dilution assay and ELISA in the study. And accomplish the amplification culture.The results suggested that, the serum positive rate was100%,the fusion rate closed to100%,and the hybridomas positive rate reached41.76%.3series of postive hybridomas finally were getted, which were named PEDV.1, PEDV.2and PEDV.3.The above results suggested that, the monoclonal antibody against PEDV had preparated.3. Identification of monoclonal antibodies against PEDVThrough large-scale expansion of ascitic fluid, produced lots of monoclonal antibodies against PEDV.Then detected the titer of ascitic fluid by ELISA. By following appraisal,identified the monoclonal antibodies against PEDV:antibody btypes,stability,adding test,specificity and affinity.The results suggested that,the immunoglobulin isotypes of the McAb were identified with the Immunoglobulin Isotype Kit as IgGl/K. It had better stabilization and specificity.Consequently, the preliminary evaluation of monoclonal antibodies against PEDV were achieved in the study.