Swine Fever Antibody Blocked The Establishment Of The Elisa Method For Detection And Evaluation Of The Classical Swine Fever Live Vaccine Effect

This research inoculated the recombinant baculovirus which contain the gene of envelope glucoprotein E2 of coding classical swain fever virus into SF9 cells of insects. To purify the glucoprotein E2, I collected the cells'supernatant after rejuvenation and drop cultrue, and then filtrated the supernatant and used the nickel pillar to do the affinity chromatography. Assayed by SDS-PAGE, there is an idio-expressive protein line at 53Kda which is almost same size to glucoprotein E2; Western blot shows that this protein line can idio-react to the classical swine fever positive serum. recovered hybridoma cell of classical swine fever monoclonal antibody WH303 from liquid nitrogen, inoculated into abdominal cavity of BALB/C mices after cultivating and proliferating. Collecting ascites containing monoclonal antibody and measuring the valency. Then using ProteinG affinity chromatography purifying monoclonal antibody, and marked through the way of sodium iodate after assayed by SDS-PAGE. Measure the concentration of enzyme labelled antibody and keep it at-20℃。Constrct the classical swine fever antibody blocking ELISA is based on purifying glucoprotein E2 and preparing HRP-monoclonal antibody. Through optimizing every condition of the reaction to definite the optimal reaction conditions:The optimal concentration of glucoprotein E2 for coating microplate was 0.09μg/ml;the optimal confining liquid is PBST+10% separated milk+Proclin300+5%trehalose and the blocking condition is 4μ16 hours; the optimal serum diluent is PBST+5% milk powder and the multiple of dilution is 2 and incubation condition for serum is 37℃1; the optimal concentration for HRP-monoclonal antibody diluent is 0.37μg/ml and the optimal HRP-monoclonal antibody diluent is Peroxidas Conjugate Stabilizer which reacting condition is 25℃30min. Using the definited ELISA reacting conditions to detect classical swine fever antibody serum panel. According the results of the experiments, the CUT-OFF determined by ROC is 36%;grey area is 32%-40%. The assay for the capacities of this classical swine fever antibody blocking ELISA is:this method doesn't cross react to frequent swine virus whose specificity and sensitivity is 94.2% and 98%;the results of using this method to detect laboratory quality control samples from European Union is consistent with the results using IDEXX CSFV Antibody Test Kit. Assayed by serum from immuned swine, the relevant of this method and IDEXX CSFV Antibody Test Kit is 0.8.We did the researches with this method to immunizing dose and immunizing programme of CSFV inferior vaccine. Detecting the change of antibody from 30-day-old weaning pigs immunized by 1200 RID,600 RID and 24000 RID and infecting experimental pigs with CSFV hadro-toxicum and observing the protection from antibody. The results showed that there were no significant deviation between different groups;the antibody level increased rapidly after 3 months of vaccination and keep stable at 70 days; the CSFV antibody level was still keeping in a high-level in 12 months and the blocking rate of CSFV antibody can protect from attacting of CSFV hadro-toxicum effectively.