Preparation and identification of monoclonal antibodies against rabbit hemorrhagic disease virus.

To develop a detection method for rabbit hemorrhagic disease virus(RHDV) and its variant strains,the spleen cells of BALB/c mice,immunized with RHDV purified from rabbits livers by sucrose density gradient centrifugation,were fused with SP2/0 cells.Hybridoma cells secreting monoclonal antibodies(McAbs) against RHDV were screened by indirect ELISA based on recombinant VP60 protein expressed in insect cells baculovirus system and the major characteristics of the McAbs were examined.Six strains of hybridoma cells secreting McAbs of AD4,AG10,DE2,BC9,BE8,and BH3 were obtained after 3 cycles of cell fusion,and the subtypes of the McAbs belonged to IgG1(AD4,AG10,DE2),IgG2b(BC9,BE8) and IgM(BH3) respectively.The hybridoma cells had the average chromosome numbers of 87 to 89 and had the stable antibody titer during 20 generations culture in vitro.Analyses using Western-blot and immunofluorescence assays indicated that McAbs DE2 and AG10 could recognize linearity epitopes and others recognize conformation epitopes of VP60 protein.Analysis using competitive ELISA assay revealed that the 6 strains of McAbs could recognize 5 types of RHDV antigen epitopes.