Production, Characterization And Application Of Monoclonal Antibodies Against Soft-shelled Turtle Iridovirus (STIV)

In this study, the purified antigen of soft-shelled turtle iridovirus (STIV) was used to immunize the Balb/C mice. After fusion and selection, the hybridoma lines secreting monoclonal antibodies against STIV were produced. And the immune biological characteristics were analyzed by the indirect enzyme linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA) and western blot. The antigen-detection ELISA and IFA systems were developed. Moreover, these monoclonal antibodies were preliminarily used to compare and analyze the antigen of three strains STIV, RGV and FJIV, which belongs to Ranavirus genus. The results were as following.1. Extraction and purification of soft-shelled turtle iridovirus:Three different purification methods were used and compared. The results indicated that a number of cell-associated viral particles were discarded with cell debris during centrifugation if freezing-thawing was used only before differential purification. In order to maximize the yield, the method of the magnetic force stir and sonication was used to purify the viral suspensions, but it will easily scathe and destroy the full virion. A abundant highly purified virion could be isolated using differential and sucrose gradient centrifugation after the treatment with the combination of freezing-thawing,the magnetic force stirring, and homogenization. And the virus particles were observed by transmission electron microscope after differential and sucrose grading, and the virions were intact and in shape. The purified virus proteins were analyzed by SDS-PAGE and confirmed to be the better immunogen to immunize Balb/C mice.2. Production of monoclonal antibody to the soft-shelled turtle iridovirus:The Balb/C mice were immunized with purified soft-shelled turtle iridovirus antigen. And then the mouse spleen cells were fused with SP₂/O myeloma cells using standard procedures. After primary and secondary screening, the positive hybridomas were selected and cloned using three cycles of limiting dilution. Eight stable monoclonal hybridomas designated 1D3, 2A4, 2H1, 3A1, 4B5, 5E1, 6F2 and 8E1 were produced. 3. Characterization of monoclonal antibody to the soft-shelled turtle iridovirus:The Mabs obtained were three kinds of isotype. Mab 2A4 was subclass IgA, Mab8El was subclass IgG2a, and the other Mabs 1D3, 2H1, 3A1, 4B5, 5E1 and 6F2 were subclass IgG1. ELISA assays showed that eight Mabs could specifically recognize the antigen of STIV, and had no cross reaction with the cell lines EPC, Co, FHM. The ELISA titers of ascites were between 10^-4 and 10^-6. Immunofluorescent studies showed that all Mabs (except for Mab5El) had fluorescence characteristics, and the specific fluorescence signals appeared virus assembly sites in the cytoplasm of STIV-infected (EPC) cells. None of the Mabs possessed the ability to neutralize SGIV in vitro cell cultures. Western blot analysis demonstrated that Mabs 1D3 and 2A4 reacted specifically to a single linear protein with an approximately molecular weight of 50kDa and 25kDa respectively, Mab3A1 reacted with three STIV proteins at molecular mass of about 14-16kDa. The identity of the target antigen of the other five Mabs could not be determined by western blot analysis. These Mabs against STIV might be highly specific and sensitive, and they might be used to detect soft-shelled turtle iridovirus and analyze its structure proteins.4. Application of monoclonal antibody in iridovirus:The eight monoclonal antibodies against STIV were used to analyze three different virus strains STIV, FJIV and RGV. ELISA assay revealed that all of the monoclonal antibodies produced specifically positive reactions with the three strains. Detected by IFA, the seven Mabs (except for Mab5E1) showed specific fluorescence signals. The results showed that the antigenicity of STIV, RGV and FJIV is significantly similar, which suggest that Ranavirus sp. had highly conservative antigenic epitopes.The iridovirus structure is very complicated, so it was difficult to elucidate the pathogenesis and immune mechanisms. The successfully production of STIV-specific Mabs will provide an important basis to analyze the structure and function proteins of Ranavirus sp., virus replication and assembly. The Mabs may prove to be a very useful tool in the study of the distribution and fluctuation of virions after invasion and rehabilitation processes.