Development And Application Of Monoclonal Antibodies Against Classical Swine Fever Virus

Classical swine fever(CSF) is a highly contagious,acute and multi-systemic hemorrhagic viral disease of pigs,which causes by classical swine fever virus(CSFV),a member of the genus Pestivirus of the family Flaviviridae.CSF can be pandemic and causes a severe economical loss worldwide.It is critical for development of an effective vaccine,alongside with accurate diagnostic methods,and plays a key role in the control and prevention of the disease.Currently,there are many methods to diagnose CSF, amongst which an inactivated virus-based ELISA is used to detect antibodies against CSFV.More importantly,it is a "gold standard" method to detect virus antigen for confirmation of CSFV infection.A monoclonal antibody-based ELISA for detection of CSFV is supposed to meet the requirement of sensitivity and specificity.In this study,a monoclonal antibody preparation,development of antigen captured ELISA was described.BALB/C mice were immunized with purified CSFV,followed by the fusion of spleen cells with myloma ceils,SP2/0,and screened by ELISA.One hybrodoma cell lines,designated as 2D1,which secreted antibodies against CSFV was obtained.The ELISA titers of culture supernatant and ascites were 2~4 and 100×2~8,respectively.The concentration was approximately 2 mg/ml after purification.The derivation of hybridoma was identified by analysis of the hybridoma chromosomes.The monoclonal antibodies can specifically recognize CSFV in infected cells by immunofluorescence assay.On this basis,the anti-CSFV monoclonal antibody 2D1 was chose to capture CSFV antigen and 3B9 was used to marker with HRP.Thus,a double-antibody sandwich ELISA was established by using the monoclonal antibodies 2D1 and 3B9.The capture ELISA was optimized by checkerboard assay and CSF antigen can be detected minimally.There were no cross reactions with bovine viral diarrhea virus,porcine respiratory and reproduction syndrome virus,pseudorabies virus,porcine parvavirus,Japanese encephalitis virus,and its reproducibility was also tested.It will provide a method for detection of CSF antigen.