Generation And Epitope Analysis Of Monoclonal Antibody Against CSFV Envelope Glycoprotein E2

Generation and Epitope Analysis of MonoclonalAntibodies against CSFV Envelope Glycoprotein E2Postgraduate:Ma gangAdvisor:Tu ChangchunThe Military Veterinary Institute, The Quartermaster Universty of P.L.A.,Changchunl30062, ChinaA panel of monoclonal antibodies(All~ B2~ E3~ H6) against E2 protein of classical swine fever virus (CSFV) were generated by immunization of Balb/C with: a. E. coil梕xpressed one third part of N terminal of E2, which includes predominant neutralizing antigenic determinants, b. DNA vaccine based on entire E2 gene. All 4 hybridomas were characterized for their potency of secreting monoclonal antibody , cell supertenant and ascites titer respectively is: 1: 32? 640 and 1: 800?2.l>( l0~ . these monoclonal antibodies could react with CSFV preparated by PK?5 cell infected with Shimen strains, and the result of western梑lotting with purified prokaryote梕xpressed E2 protein is positive, which indicates these monoclonal antibodies all aim directly at CSFV. McAb All had strong reactivity with strain Shimen in indirect imxnuflofluorescent assay and indicate the epitope of All is on the surface of virus or be presented to the surface of cell In indirect ELISA, All could cross reacted with bovine viral diarrhea virus (BVDV), while other three not, indicating All probably recognizes a common antigenic epitope of pestvirus, and the others are specific to CSFV. These antibodies?including previously four strains hybridomas: D5,D8,GB,GE) classes have IgG(B2, GB) and IgM(the rCst).These monoclonal antibodies were purified according to their classes, which enhancd the ability of binding antigen. Two peptides (P1, P2)were synthesized on the basis of prediction of antigen epitope of E2 protein and the sequence of HCLV and preliminary mapping was done with purifying McAb and two peptides. The results showed that McAb secreted by All hybridoma cell strains reacted with two peptides and McAb secreted by D5, D8 hybridoma cell strains only reacted with P2 peptide. These61antibodies will be used to delineate the relationship between E2 gene variation and the antigenic change of the virus.To search for a improved way to produce McAb, we made a try to compare ~ree梡rotein medium(CD hybridoma medium) and 15% New Calf Serum RPMI164O medium, the results showed that CD medium is suitable to harvest McAb, especiafully for low titer hybridomas in RPMI164O medium, which not only to enhance the titer, but to be purified unnecessarily when McAb used.