Development Of Monoclonal Antibodies Against Porcine Parvovirus And Their Preliminary Application

Porcine parvovirus (PPV) is one of major causative agents, resulted in a sydrome of reproductive failure in swine, which includes stillbirths, mummified failure in early embryonic death, and infertility. There is no obvious sydromes in pregnant sows. PPV is a small DNA virus was first found in cultured cells of classical swine fever virus, and then been found that it is one of the main reasons of embryonic and fetal death, and is distributed worldwide. PPV was isolated in different area in our country at the early 80's 20th century, and found that it is one of the the main pathogens cause sow reproductive problems. Porcine parvovirus infection becomes a world-wide problem and causes a great economic loss in pig industry in China. At present, a lot of diagnostic methods for porcine parvovirus infection have been developed based on its aetiology and serology. The BALB/c mice were immunized subcutaneously with PPV, which was purified with polyethylene glycol. After being immunized three times, the mice with high serum antibody titer were immunized intraveinally with purified PPV for the final immunization. Three days later, spleen cells from immunized mice were fused with SP2/0-Ag-14 myeloma cells. Indirect enzyme linked immunosorbent assay (iELISA) was used to screen hybridoma cells, and limiting dilution method was applied to subclone positive hybridoma cells for three times, and three positive clones, designated as 3C5, 3C6 and 3B6 respectively, were obtained from hybridoma cell lines. The titers of their ascitic fluids were 10×29, 10×28, 10×28 in indirect ELISA, respectively. All of them were subtyped to be IgM and reacted with PPV specifically in indirect immunofluorecent assay (IFA), 3C5 and 3C6 all have hemagglutination inhibition. The ascites was prepared and purified, two of them were labeled with HRP and a preliminary application was carried out.Based on these monoclonal antibodies, Dot-ELISA was developed to detect PPV antigens in clinical samples. In Dot-ELISA, the sensitivity was 2.43 TCID50s for PPV, PCV1,PCV2,CPV did not reacted in the developed Dot-ELISA. Eleven of 50 clinical samples were detected to be positive in Dot-ELISA, and the result was confirmed by PCR.