Preparation Of Monoclonal Antibodies Against Bovine Viral Diarrhea Virus And Establishment Of A Double Antibody Sandwich ELISA For Virus Detection

Bovine viral diarrhea(BVD),also known as mucosal disease(MD),is caused by bovine viral diarrhea virus(BVDV).The clinical symptoms are mainly mental depression and massive salivation,milk production decrease or even stopped milk,diarrhea,and conjunctivitis,etc.The pregnant cattle showed miscarriage,abnormal fetal output or birth of persisitently infected(PI)cattle.The newborn calf died due to severe diarrhea.The PI cattle will continue to shed BVDV to susceptible animals,and is the main source of BVDV infection.Bovine viral diarrhea has a worldwide prevalence and has caused huge losses to the global cattle economy.E0 protein is one of the most conserved proteins of the bovine viral diarrhea virus structural protein and can be secreted from infected cells.The E0 can be detected with an antigen capture ELISA(ACE)without any treatment and has important clinical diagnostic value.E2 is the main protective antigen of BVDV.The p80 protein is the most conserved nonstructural protein and can be used for differential diagnosis.An antigen capture ELISA was established and had good sensitivity,specificity,and repeatability for detecting the viral antigens of BVDV.It is now a simple and economical method to identify PI cattle in the world.However,no monoclonal antibodies against BVDV p80,E0 and E2 proteins are available at present,and there is no sensitive and effective ACE for detecting BVDV antigens in China.There is an urgency to carry out relevant research in China.In this study,BVDVs cytopathic strain VEDEVAC and non-cytopathic strain 3877 were propagated in MDBK cells,and purified by PEG concentration and sucrose density gradient centrifugation.The purified BVDVs were used as an immunogen to immunize the Balb/c mice.The positive hybridomas were screened with indirect ELISA and indirect immunofluorecent assay(IFA).After three subclonings,7 well-defined hybridomas stably secreting monoclonal antibodies were obtained,and named 1E3,2C3,3F5,1B7,2B10,3E10,and 1B12.IFA results showed that the antibodies secreted by these 7 hybridomas had good specificity.Western blotting showed that the 4 monoclonal antibodies secreted by 4 hybridomas(2B10,1B7,3F5 and 3E10)had conformational epitopes,and the other 3 monoclonal antibodies secreted by other 3 hybridomas(1E3,2C3 and 1B12)had linear epitopes.IFA showed that the hybridoma 3E10 secreted a monoclonal antibody against E2 protein of BVDV,and hybridoma 3F5 secreted a monoclonal antibody against p80.Meanwhile,the monoclonal antibodies 3E10 and 3F5 were used as capture antibodies,and HRP-labeled 1B12 was used as a detection antibody,a double antibody sandwich ELISA was established for detection of BVDV in the established method.The specific reaction experiments showed that this method had no reaction with MDBK cells and other bovine respiratory disease pathogens,such as infectious bovine rhinotracheitis virus(IBRV)and bovine parainfluenza virus type 3(BPIV-3),and only had reaction with BVDV.In conclusion,7 hybridomas secreting monoclonal antibodies were obtained in this study.The epitops recognized by the 4 monoclonal antibodies(2B10,1B7,3F5 and 3E10)were conformational,and those recognized by other 3 monoclonal antibodies(1E3,2C3 and 1B12)were linear.And the hybridoma 3E10 secreted a monoclonal antibody against E2 protein of BVDV,and hybridoma 3F5 secreted a monoclonal antibody against p80.Meanwhile,a double antibody sandwich ELISA was established by selecting monoclonal antibodies 3E10 and 3F5 as capture antibody and HRP-labelled monoclonal antibody 1B12 as detecting antibody,respectively.The method might be used to detect BVDV and provided the technical support for the detection of BVDV in China.