The Study On Monoclonalantibodies Double Sandwich Elisa Detecting Pseudorabies Virus

Rabbits and Kunming mice were immunized with the purified PRV of Min A respectively, then the positive serum was obtained and the IgG was purified.For the rabbit-anti-PRV serum, it's AGP titer was 1:32 ,and the protein concentration of IgG was 7.34 mg/ml.For the mice-anti-PRV serum, it's AGP titer was 1:16 ,and the protein concentration of IgG was 5.74 mg/ml.The cell line named OH6 which could secrete McAb stably was anabiosised, then the ascites were produced and the IgG was purified by the common methods. The hydridoma cell line OH6 and the ascites were re-evaluated. The average number of chromosome of the hybridoma cell was 94.Through detection by the indirect ELISA having been established, the ELISA titer of cell supernate and ascite were 1:1000 and 1:200000.The McAb had the activity of neutralization with or without complement,the titer was 1:64 and 1:16 respectively.The McAb did not crossly react with PRRSV, HCV, PPV, which demonstrated good specificity.Based on the rabbit-anti-PRV IgG and McAb, the double sandwich enzyme-linked immunosorbent assay (ELISA) for detection of Pseudorabies virus (PRV) was developed. The best coating concentration of the rabbit-anti-PRV IgG was 4.59ug/ml. The best working concentration of McAb was 6.45ug/ml. The assay was specific, sensitive, reliable, convenient, and had a high coincidence rate with animal experiment, virus isolation and neutralization experiment. The sensitivity of the double sandwich ELISA was 200 ng/ml. The whole operation time only need 4.5h(not including coating). The scientific criterion of determination had been made. The assay did not require specific equipment and technique so that it can be extended easily.The detection of the artificially infected rabbits and naturally infected swine showed that the best tissues for the diagnosis of PRV were tonsil and brain.