Development Of Monoclonal Antibody Against Gosling Plague Virus And Colloidal Gold Immunochromatographic Strip For The Detection

Goose parvovirus (GPV) infection known as goose plaque, belongs to Parvovirinae Dependovirus, and causes high-contagius and septico-infection diseases in domestic goslings and Muscovy ducklings. It caused90%mortality in susceptible goslings under the age of4days to20days. It can cause acute enteritis and also liver, kidney, heart and other organs septic lesions, particularly the fibrous and embolism of the small intestine. At present, GPV infection spreads every year in different degree that hinder the healthy development of breeding industry. Because GPV mainly infects young goslings and Muscovy ducklings,early diagnosis of the disease is extremely important.In the experiment,goose embryo with no antibodies was inoculated with GPV isolated from Nanjing.In order to remove the other proteins.the allantoic fluid containing GPV was mixed with equal amount of chloroform.At the concentration of sodium chloride is2.2%,a final concentration of6%PEG-6000could settle down the vast majority of the virus which was purified by sucrose density gradient ultracentrifugation.Using PCR and electron microscopy,we could find that the virus is mainly distributed between35%and50%of sucrose concentrations, and the virus protein content was216μg/ml.SDS-PAGE electrophoresis analysis showed that the purity of virus was more than90%purity, to achieve the request of the experiment.Three hybridomas cell lines secreting monoclonal antibodies (MAbs) against GPV were established by fusing SP2/0with spleen cells from BALB/c mice immunised with formaldehyde-killed purified GPV, naming1E12、2H11and3F1. The indirect ELISA titers of the ascites were1:32000、1:80000and1:16000,respectively.Indirect immunofluorescence test proved that the three hybridomas cell lines have good specificity; western blot test proved that all three monoclonal antibodies were against structural protein VP3of goose parvovirus.The gold particle in colloidal solution was coupled with purified monoclonal antibody2H11, and then sprayed on the glass fibers. The purified polyclonal antibody from rabbit against gosling plague virus served as the capture-antibody was coated on the nitrocellulose membrane. Then the sandwich-based gold immunochromatographic (IC) strip for the detection of GPV was assembled in regular order. The detection results indicated that the IC strip was specific to GPV and the sensitivity was104.1GELD50/0.2ml for detecting sample.We can obtain the results in fifteen minute.The IC strip is specific, reproducible, and can be used for rapid clinical diagnosis of GP.