Development Of Immunogolloidal Gold Strip Assay For Detecting The PorcinE Reproductive & Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome virus, member of genus Arterivirus, the family Arteriviridae, was a causative agent of reproductive problems such as abortions, premature birth in sows, and pneumonia, growth retardation in weaning piglets. And it brought great economy loss in swine industry. So a new process with rapid and convenient to detect PRRSV in field test was necessary.The antigen of PRRSV was purified by differential centrifugation and sucrose density gradient centrifugation. The purified antigen was obtained and was used to immunize BALB/c mice. Hybridoma cells were prepared by fusing SP2/0 cells with spleen cells of BALB/c mice, and the fusing rate was 91.4%. The supernatant of hybridoma cells were detected by indirect ELISA, and the positive ratio was 20.6%. After three times cloning, five hybridoma cells secreting monoclonal antibody were established and named D5, D7, D10, E9, B5 respectively. The antibody titers of them were 1:25600, 1:12800, 1:6400, 1:12800, 1:6400 respectively. Rabbits were immunized with purified PRRSV N protein and the polyclonal antibody against N protein was obtained.Immunocolloidal gold strip combined with monoclonal antibody was rapid and convenient, and had good specificity and sensitivity. Colloidal gold with about 20 nm particle diameter was prepared by sodium citrate reduction. The monoclonal antibody D5 which had high titer and good bioactivity was conjugated with colloidal gold of 20 nm particle diameter which was uniform, without fragment or agglutination. Rabbit antibody against PRRSV N protein and goat antibody against mouse IgG were coated in turns on the nitrocellulose membrane as test line and control line. Assembled them and incised into immmocolloidal gold strip. Sensitivity testing showed that the strip could detect 6.25μg/mL purified PRRSV antigen. In the specificity testing, the strip could detected the PRRSV only, and had no cross reaction with other virus such as classical swine fever virus, pseudorabies virus, porcine circovirus, porcine parvovirus. 30 specimens were detected by immunocolloidal gold strip and RT-PCR, the results showed that 19 PRRSV positive specimens were detected by the strip and 21 PRRSV positive specimens were detected by the RT-PCR. And the main coincidence, positive coincidence and negative coicidence were 90.0, 90.5, and 88.9%, in respectively.