Preparation Of Monoclonal Antibodies Against Duck Enteritis Virus And Preparation Of Standard Antigen And Standard Antisera Of Duck Plague

Duck viral enteritis(DVE),also known as duck plague,is an acute,contagious,septicemic and lethal disease of ducks and geese,caused by duck enteritis virus(DEV).Duck plague in China belong to the third category of animal disease,is one of the important diseases currently threatening the duck farming industry development.The disease is characterized by rapid spread,morbidity and mortality,and widespread.In this study,three monoclonal antibodies against duck plague virus were prepared,and antigen detection method was established.Duck serum against DEV VAC strain was prepared,the standard of serum and antigen of DEV were prepared after a series of calibration.1.Preparation of Monoclonal Antibodies against Duck Enteritis Virus and Development of Indirect Immunofluorescence Assay using Monoclonal Antibody of DEVIn this study,DEV VAC strain was amplified and purified by SPF duck embryo,and the monoclonal antibody was prepared by immunizing BABL/c mice with purified virus.The antibodies were obtained by the optimized indirect immunofluorescence method after screening and subcloning them.Three hybridoma cells capable of secreting antibodies against DEV were identified as DEV-5H10,DEV-4H11,DEV-6D8,DEV-5H10 and DEV-6D8 were identified as IgG2b monoclonal antibody,DEV-4H11 was IgM antibody,DEV-5H10 and DEV-6D8 were able to react with DEV VAC strain with specific bands of 69KDa by Western-blot analysis and their ascites.titers were 1:1600,1:3200,respectively.Monoclonal antibody DEV-6D8 was a primary antibody,FITC-labeled goat anti-mouse IgG was secondary antibody.The incubation time of the primary and secondary antibodies were 30 min.The indirect immunofluorescence assay was used to detect viral antigens,which could detect 1-10 TCID50 infected cell viruses,which is of great significance in the diagnosis of clinical duck plague virus.2.Development of standard antigen on Duck Enteritis VirusIn this study,DEV VAC strain was amplified by SPF duck embryo,and was measured for viral content.The results showed that TCID50=10-4.8/0.1mL.The results of Western-blot,hemagglutination test,neutralization test and PCR showed that the standard antigen has no DHV(Duck hepatitis virus),GPV(Goose parvovirus),EDS(Egg drop syndrome),IBDV(Infectious bursal disease virus),REV(Reticuloendotheliosis virus),REOV(Reovirus),NDV(Newcastle disease virus),AIV(Avian influenza)and other exogenous virus contamination.Through the aseptic test and mycoplasma detection,proved that the virus without bacteria,mycoplasma contamination.Adding the 1%BSA cryoprotectant to the detected virus solution,then packaging and freeze-drying virus,through the physical character inspection,CV value calculation,homogeneity determination,stability testing,shelf life test and other experiments to obtain standard antigen which is of great significance to the quality control and effectiveness evaluation of poultry biological products.3.Development of standard antiserum Duck Enteritis VirusDEV VAC strain was used to immunize SPF ducks to prepare DEV multi-antiserum.The serum was tested by indirect immunofluorescence antibody assay established in this study.The results showed that the IFA titer was 1:27 and the neutralization titer was 1:58.This multivitamin was tested by hemagglutination inhibition test,indirect immunofluorescence assay,demonstrating that the serum only reacts with DEV and did not correlate with GPV(Goose parvovirus),EDS(Egg drop syndrome),IBDV(Infectious bursal disease virus),REV(Reticuloendotheliosis virus),NDV(Newcastle disease virus),AIV(Avian influenza)and other viral reactions.Through the aseptic test and mycoplasma detection,proved that the serum without bacteria,mycoplasma contamination.Adding the one thousandth of thiomersal preservatives to the detected serum solution,then packaging and freeze-drying serum,through the physical character inspection,CV value calculation,homogeneity determination,stability testing,shelf life test and other experiments to obtain standard antiserum which is of great significance to the quality control and effectiveness evaluation of poultry biological products.