Development of ELISA Kit Using Recombinant Nucleoprotein as Antigen of PRRS Virus I.The Optimal Reaction Conditions of ELISA

The purified N protein was used to be coated on the well of 96-well plate, each following step was optimized. As a result, an indirect ELISA was constructed to detect antibody against PRRSV. The various factors and conditions of ELISA were explored, and the optimal reaction conditions of ELISA were determined. It was shown that the optimal concentration of recombinant N protein for coating of plate was1 mg/L, the optimal coating condition of recombinant N protein for ELISA was 4℃ overnight, the dilution of serum sample was 1∶40, serum sample for detecting and HRP-labeled rabbit anti-porcine IgG should be incubated at 37℃ for 30 min and 40 min respectively, the substrate for ELISA was incubated at 37℃ for 10 min before terminated with the stopping solution.The blocking test for the reaction between the PRRSV positive serum and the recombinant N protein was carried out by using the PRRSV antigen.The results showed that PRRS virus could block the reaction completely. Meanwhile, it was confirmed that there was no cross reaction between the antibodies against other porcine infectious diseases.The results revealed that the indirect ELISA by the purified recombinant nucleoprotein had good specificity for the detection of PRRSV antibody in serum. The difference value among wells in a plate and among plates for ELISA was both less than 7%, which showed the assay had a good retrievality.