Establishment And Preliminary Application Of Competitive ELISA For Detection Of PRRSV Antibody

Porcine Reproductive and Respiratory Syndrome(PRRS)is an acute,highly infectious and high mortality viral infectious disease,which mainly causes fever and reproductive disorders in sows,and piglets are characterized by dyspnea,which seriously affects the healthy development of pig industry Monitoring the occurrence,development and epidemic dynamics of the disease,and detecting the immune status of the pig herd are essential for formulating correct and effective disease prevention and control measures.PRRS serological survey can quickly and effectively detect the immune status of individuals and pigs,and play an important role in understanding the distribution and epidemic dynamics of diseases in pigs,and early warning the development of diseases.PRRS serological survey can quickly and effectively detect the immune status of individuals and pigs,and play an important role in understanding the distribution and epidemic dynamics of diseases in pigs,and early warning the development of diseases.In order to establish a highly sensitive and specific serological detection method to better monitor the epidemic dynamics of diseases and the immune status of pigs,this study cloned the N protein gene sequence of Hu N4 strain onto the p ET-24 a vector,successfully constructed the recombinant cloning vector p ET-24a-N,and transformed it into the Escherichia coli receptive cell Rosetta(DE3).Through optimizing protein purification conditions,high-purity N protein was obtained.Two nanobody(Nb)with high affinity to N protein screened in the VHH library were expressed in prokaryotic and purified.Coupling Nbs with peroxidase(HRP)by sodium periodate method The labeled nanoantibodies,Nb12-HRP,and Nb1-HRP,were tested for their labeling efficiency by direct ELISA,and their affinity was optimized.Finally,Nb1-HRP was chosed as a competitive antibody.Using purified N protein as the coating antigen and nanoantibody Nb1-HRP coupled to HRP as the competitive antibody,a competitive ELISA(c ELISA)detection method was successfully established by optimizing reaction conditions.By testing 76 negative serum samples,use the formula(Cut-off value = PI(average value)+ 3 × Variance(SD))calculation obtained a critical value of 50.78%.The established c ELISA method was used to detect porcine serum at different times after PRRSV immunization,and compared with IDEXX commercial kit to evaluate the sensitivity of the established method.The results indicate that the established c ELISA method has high sensitivity.The results of detecting specific sera such as BL21,PRV,TGEV,ASFV,and PCV2 were all negative,indicating that this method has strong high specificity;PRRSV specific pig serum was tested for different subtypes and epidemic strains,and all antibodies were positive,indicating that the established c ELISA detection method has a broad spectrum;The coefficient of variation of serum PI values in both inter and intra batch tests is within 10%,indicating that the established method has good repeatability.Finally,using the c ELISA method established in this experiment and IDEXX PRRSV ELISA antibody detection kit to simultaneously detect 333 clinical serum samples,the results showed that the coincidence rate between the two was as high as 96.40%.In conclusion,the c ELISA antibody detection method established in this study has high specificity,sensitivity,and reproducibility.It can be used to detect antibodies to PRRSV N protein in pig serum,monitor the prevalence of PRRS.