Generation Of Monoclonal Antibody Against PEDV S Protein And Detection Of PEDV Antigens Using The Developed Sandwich ELISA

Porcine epidemic diarrhea(PED), caused by porcine epidemic diarrhea virus, is a highly contagious disease characterized by severe watery diarrhea, vomiting, dehydration and high mortality in neonatal piglets, which caused a huge economic losses to the global swine industry. There are many reports related to the diagnostic method for PEDV such as immunofluorescence assay, ELISA, especially the recently most reported molecular method RT-PCR as a specific, sensitive method for PEDV diagnosis. However, RT-PCR is expensive and not suitable for detection of large-scale samples and epidemiological survey. Thus it is necessary to develop the methods with high specificity, sensitivity, low cost and suitability for large-scale detection PEDV antigens.In this study, a partial sequence of spike gene encoded by PEDV was obtained by PCR amplification and cloned to the prokaryotic vector p GEX-4T-1 to construct the expression plasmid p GEX- 4T-1-S. The recombinant protein was expressed under the IPTG induction and purified by urea. The Balb/c mice were immunized with the purified recombinant PEDV S protein emulsified with Freund’s complete adjuvant. The mouse spleen cells were collected and used to hybridize with myeloma cell after mouse antibody titer reached more than 10000 times measured by indirect ELISA. Hybridoma cell line(3F6) secreting monoclonal antibodies were successfully obtained, and its stability and specificity was further determined by indirect ELISA.A sandwich ELISA method to detect the porcine epidemic diarrhea virus antigen was established using the generated monoclonal antibody, and the reaction conditions were optimized. The optimal coating amount of capture antibody and enzyme-labeled antibody were 0.6μg and dilution of 1: 1000, respectively. The blocking buffer was 2% BSA. Detection of the positive PEDV samples using established ELISA and RT-PCR demonstrated a high congruence(100%) of those two methods. Detection of the suspicious PEDV samples collected from different pig farms in Jilin Province revealed a 68% of pigs as PEDV-positive.In summary, we have generated a monoclonal antibody against S protein of PEDV and established a sandwich ELISA for detection of PEDV antigens. This method was demonstrated to be specific, sensitive, and simple for detection of PEDV infection, which will play a significant role in the prevention and treatment of the porcine epidemic diarrhea.