Purification Of Duck Enteritis Virus And Development And Application Of An Indirect ELISA For Detection Anti-DEV Antibodies

Duck viral enteritis, or duck plague, was caused by duck enteritis virus. DVE was an acute, heat and septicaemic contagious viral disease that naturally affecting birds of the order Anseriformes such as ducks, geese and swans. It is characterized with wide epidemic, spread rapidly, the high level of morbidity and mortality. At present, it is urgent to establish a simple and rapid means for monitoring DEV infection and assessing vaccination in the field.After propagating in chicken embryo fibroblast, duck enteritis virus AV1222 strain was frozen and melt repeatedly and purified by differential velocity centrifugation. Then sucrose density gradient centrifugation, potassium tartrate density gradient centrifugation and cesium chloride density gradient centrifugation were used to purify DEV. By electron microscopic observation, testing concentration of virus and detection antigenicity by ELISA, sucrose density gradient centrifugation expressed better for purifying DEV. The virus purified by this method were tested by SDS-PAGE gel electrophoresis. Western-Blot results shows that 30%～40%, 40%～50% of the virus with better antigenicity, so they were mixed for ELISA kits antigen.Using the prepared highly purified DEV as coating antigen, an indirect ELISA for the detection of IgG antibody against DEV in serum was standardized. With the optimal working parameters, including 3.64μg/mL of the virus protein antigen for coating, testing sera dilution at 1:100, the standard of determining as positive sample S/P≥0.125 and others. The reaction between the antigen and the positive serum can be blocked by DEV and there is no any cross reaction to the positive sera of the other diseases above stated, demonstrating specificity of the diagnosis method is excellent. The variation coefficient of inter- and inner- batch antigen production testing quality control serum which can guarantee the quality of antigen and the accuracy of testing results is below 10 percents, demonstrating that repeatability of the antigen is good. Compared with the serum neutralization testing, the agreement ratio is 100%. Compared with the DEV-UL19 recombinant protein indirect ELISA kit, the agreement ratio is 95.35%. Some sera samples from ducks immunized with attenuated DEV were assayed with the ELISA kit, and the antibodies were detected first on 7 day post immunization in ducks vaccinated subcutaneously and and detected the highest level on 28 day PI. These results showed that ELISA kit could be applied to serological epidemiological investigation and monitoring of antibody level of immunized ducks.178 of 507 serum samples collected from Harbin Qiqihar and Daqing are positive tested by the diagnosis method and the positive rate is 35.10%. The assay was confirmed to be specific and sensitive, suggesting an applicable prospection.Therefore, the indirect ELISA assay based on purified virion antigen has good sensitivity specificity and repeatability, can be a technique support for further developing the commercial kit and afforded a simple and rapid means for assessment of vaccination in the field and investigation of DVE epidemiology.