Development And Application Of An Indirect Enzyme-Linked Immunosorbent Assay Detecting Antibody Against Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of porcine reproductive and respiratory syndrome in swine, characterized by reproductive failure in sows and pneumonia in neonatal pigs. PRRSV is an enveloped, single stranded, positive sense RNA virus, classified in the order of Nidovirales, family Arteriviridae, genus Arterivirus. Nucleocapsid protein (N protein), coded by open reading frame 7 (ORF 7) of PRRSV, is of immunogenicity and lasts for as long as one year in swine. In this study, affinity chromatography purified PRRSV N protein was used to establish an indirect ELISA detecting serum antibody against PRRSV, providing support to research in diagnosis and pathogenicity mechanisms of PRRSV infection.Recombinant bacteria pGEX-6p-1-N/BL21 was induced at its optimal conditions. Harvested cells were ultrasonicated and purified using GST-Agarose resin affinity chromatography, recombinant N protein was obtained at concentration of 0.557mg/mL.Purified N protein was used as the coating antigen in our experiment. Concentration, temperature and time of every reagent in the ELISA was determined though a series of optimization steps, then the indirect ELISA detecting PRRSV antibody was established. Briefly, N protein was coated at concentration of 174ng/100μL over night at 4℃, then blocked with 10% NCS-PBS for 2h at 37℃; serum was 40 fold diluted and added to each well and incubated for 30min at 37℃; 50μL 640 fold diluted HRP-conjugated rabbit-anti-swine IgG was added to each well for another reaction time of 30min at 37℃; Color development of TMB was stopped with sulphuric acid and evaluated at OD450.A total number of 55 clinically confirmed positive sera and 127 negative control sera were tested with the ELISA. The data was submitted to the Two-graph receiver operator characteristic (TG-ROC) analysis and the cut-off and intermediate range were determined at 90% confidence level.A set of 220 clinical sera were tested by our ELISA and IDEXX-ELISA kit. According to test results comparison, coincident ratio of 94.8%, 81.5% and 84.6% for positive, negative and total samples were attained, respectively, indicating high sensitivity and specificity of the established ELISA method.