Preparation Of Monoclonal Anitbody Against Feline Calicivirus And Establishment Of The Sandwich ELISA For The Deteciton Of Feline Calidvirus

Feline Calicivirus（FCV） also named as Feline Infectious nose-conjunctivitis virus, whichcan induce upper respiratory tract infection, chronic gastroenteritis and mouth disease, was calledFeline Infectious nose–conjunctivitis. This is an important virus disease in feline, which hashigh incidence and worldwide distribution, it can easily infect felines within one-year-old andlead to death. The FCV and FHV always infected with each other because FCV can cause similarclinical symptoms to FHV. In view that FCV has already brought about a great threat to felines, itis of significance to carry out immunization and epidemiological research on FCV actively,which also could promote the national standardization process of feline as experimental animals.Three anti-FCV monoclonal antibodies named D8, E5and H4were successfully preparedand a double-antibody sandwich ELISA was established, which could detect FCV rapidly,sensitively and specifically. The three hybridoma cell lines were obtained through positivehybridoma selection,,and the sub-type of D8, E5were IgM while the H4was IgG2b The titersof hybridoma culture and ascites were2⁷and1000×2⁷respectively. Three monoclonal antibodiesagainst FCV were all positive and had good specificity. An indirect ELISA was used to detectthe supernatant titers of the10^th,20^thpassaged hybridoma cells and hybridoma cellscryopreserved for1,3and6months, there were no differences between these supernatant titers.The results showed that D8, E5and H4hybridoma could secrete anti-FCV monoclonalantibody stably.As the two monoclonal antibodies（E5, H4） recognized different epitopes, they were used ascoating antigen and enzyme-labeled secondary antibody respectively. A double-antibodysandwich ELISA for the detection of FCV was established and the reaction conditions were alsooptimized, the optimal dilution of peridium for E5McAb was1:4000, the optimal dilution of FCV antigen was1:40,2%of BSA was selected as blocking solution and the bloking time was1h at37℃. The optimal dilution of peroxidase-labeled H4McAb （IgG II） was1:1000and thereaction time was1h at37℃. Finally, the substrate reaction time was10¹5min.