Preparation Of Monoclonal Antibodies And Establishment Of Monoclonal Antibodies Double Sandwich Elisa For Detection Of Goose Parvovirus

Gosling pavovirusis is a highly contagious disease caused by goose parvovirus (Goose Parvovirus, GPV).The morbidity and mortality of goslings are up to 95%-100%. Due to the short duration, fast spread and high mortality of the disease, it has been one of the most serious diseases in goose industry and causes huge economic losses to the goose industry. It has aroused great attention of domestic and foreign scholars. Past diagnosis of the disease is usually based on epidemiology, clinical symptoms, pathological changes to the initial diagnosis. But in the study of the goose parvovirus clinical cases and human experimental infection cases, we find that a considerable portion of patients lack obvious clinical and pathological features and it is difficult to make a preliminary'diagnosis. Supporting agar diffusion test, agar diffusion inhibition test, neutralization tests and other serological diagnostic methods and pathogen detection methods are commonly used, but these methods lack sensitivity or specificity and are not suitable for mass detection. There are no other effective way besides the injection of anti-serum or egg yolk antibody to treat the disease. Given the disadvantages the disease in diagnosis, prevention and treatment, it is urgently needed to find a good biological agents in clinical treatment of the disease and a simple, rapid, sensitive, specific, and mass detectable diagnostic methods.BALB/c mice were immunized by concentrated GPV virus and anti-GPV hybridoma cells (4B4) were obtained by hybridoma technique, indirect ELISA and limiting dilution assay. After identification, the heavy chain of MAb is IgG1 subtype and the light chain isκchain. The results of ELISA and western blots showed that GPV virus and GPV VP3 protein could be specially recognized by 4B4 MAb. It indicated that the epitope recognized by 4B4 Mab should be the conservated region of GPV VP3 protein. This research has settled the foundation for the characterization of GPV epitope and the investigation of GPV diagnostic reagent.In order to establish the method to detect GPV, in this research rabbit anti-GPV IgG was used to be capture antibody and anti-GPV monoclonal antibody was used to be detection of antibody. Based on chess-board experiment, the optimal coating concentration of rabbit anti-GPV IgG is 16μg/mL, the optimal working concentration of anti-GPV monoclonal antibody is 10.8μg/mL, The optimization of dilution for enzyme-labeled second antibody is 1:4000, and double sandwich ELISA whose sensitivity of detecting GPV is 0.312μg/mL. was established. Dead gooses in a goose ranch in Yanbian were detected by this method. The positive rate is 75.79%. The concordant rate with virus neutralization test is 91.11%.This research has settled the foundation for the characterization of GPV epitope and the investigation of GPV diagnostic reagent. Double sandwich ELISA established by this research provides a convenient and fast serodiagnosis for detecting GPV and epidemiologic survey in district.