Development Of Monoclonal Antibodies Against Porcine Rotavirus And Double Antibodies Sandwich ELISA For The Virus Detection

Porcine rotavirus (PRV) belonging to reovirus family, rotavirus genus, is an important pathogen causing viral diarrhea in piglets. The disease mainly infects young piglets, with the age of1to10days. After infection, more than80%may develop disease, and the mortality rate can be as high as50%to100%. Severe diarrhea is the dominating syndrome. Death occurs in some cases due to severe dehydration, acid-base balance and secondary infection, especially while complicated by porcine transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV) co-infection, mortality rate can be extremely high. The disease is widely distributed in the world, causes serious damage in livestock industry.In this study, eight-week-old BALB/c mice were immunized with purified PRV virus. With lymphocyte hybridoma technology, the spleen cells from immunized mice were fused with myeloma SP/20cells. Indirect ELISA was followed to screen cells, screened hybridoma cells were cultured with limited dilution method, and then screened through three sub-cloning. As a result, two strains of stable hybridoma cell lines secreting anti-PRV monoclonal antibody were obtained signated2B3and1C11. Both strains were identified as IgM subclass by antibody subclass identification. Average chromosome number of2B3hybridoma is97, and chromosome number of1C11is102on average. Antibody titers in cell culture supernatant were measured by indirect ELISA,2B3supernatant titer was1:103, and1C11supernatant titer was1:102. Western-blot and indirect immunofluorescence assay showed that both these two monoclonal antibodies can specifically recognize PRV. These two monoclonal antibodies did not cross-react with TGEV, PEDV, or swine pseudorabies virus (PrV), indicating the good specificity. Adding ELISA results showed that two monoclonal antibodies identified different antigenic sites on PRV-VP6protein. Study results showed that these two strains of anti-PRV hybridoma monoclonal antibodies had outstanding detection specificity. These two monoclonal antibodiesestablished material foundation for PRV surveillance, rapid and accurate diagnosis, and differential diagnosis of pathogens.Double-antibody sandwich ELISA detecting porcine rotavirus was initially established with anti-PRV polyclonal antibody as coating antibody, and2B3monoclonal cell supernatant as a detection antibody. Concentration of caprylic acid saturated ammonium sulphate purified polyclonal antibody was7.161mg/mL. By SDS-PAGE analysis of the results, the purified IgG heavy and light chain bands were clear, and predominating, and purity was determined as100%. The optimal detection concentration of2B3cell supernatant and the best package polyclonal rabbit anti-PRV concentration were determined with the matrix method, which were (1:100) and (1:400), respectively. Using double antibody sandwich ELISA to detect PRV virus, it was found the8minimum detectable level was8.4×104TCID50, with the criteria of OD49onm>0-1528as positive. This test did not cross-react with PEDV, TGEV, or PrV, repetitive tests proved it had good stability. When20clinical samples were tested on the double antibody sandwich ELISA developed in this study, and tested on RT-PCR, correlation rate was95.7%. As indicated in this study, the double-antibody sandwich ELISA had good specificity and sensitivity, and can be used for rapid detection of PRV. The establishment of double antibody sandwich ELISA provided a simpler, more rapid and more sensitive method for PRV clinical detection.