Characterization Of Conformation-specific Monoclonal Antibodies Against Rabies Virus Nucleoprotein

Rabies is an ancient infectious disease that affects human and animal central nervous system. Once the symptoms are apparent, it causes fatal encephalomyelitis, which is not able to be cured. Vaccination is the only way to control this disease. In order to enhance the detection, diagnosis, and prevention of rabies, and to study the structure, antigen variation, strength, function in infection, transcription and replication of rabies virus proteins, we prepared monoclonal antibodies against purified L16 rabies virus proteins, and we determined the epitopes of some of these antibodies. This research falls into three parts:Part I:The cultivation and purification of rabies virus. In this part, BSR cells were infected with Rabies virus strain L16 for 4 hours, and then the media was collected and followed by mild centrifugation to get rid of cell debris. The supernatant containing virus proteins was ultracentrafuged with sucrose gradient, and the purified virus proteins were separted by 10% SDS-PAGE. The results indicates that rabies viruses in the cell culture media were enriched and purified well so that the viral protein content reached to 1.0 miligram per mL. This provided the basis for the preparation of monoclonal antibodies.Part II:The preparation and characterization of monoclonal antibodies against rabies virus strain L16. In this part, we immunized Balb/c mice with propiolactone inactivated rabies virus made in partâ… . After the first injection and a boost injection, the spleen cells of the mice were fused with myeloma (SP/20) cells to form cells clones. By screening with ELISA and immunofluorescence, we found many positive cell clones producing monoclonal antibodies against different viral proteins. We further explored the corresponding antigen of four of those monoclonal antibodies (mAb/N40,mAb/N42,mAb/N46 and mAb/P49). First we transfected viral protein including N and P in BSR cells with the aid of viccinia virus, then we examined the reactivity of these four monoclonal antibodies with the tranfected cell via immunofluorescence. We found that mAb/N40, mAb/N42, and mAb/N46 all recognize N protein, while mAb/P49 recognizes P protein. Western blotting experiments of these antibodies revealed that mAb/N42 and mAb/P49 react with denatured corresponding antigen, indicating they recognize linear epitopes, while mAb/N40 and mAb/N46 do not react with denatured N protein, indicating they recognize conformational epitopes.Part III:the determination of epitopes recognized by anti-N monoclonal antibodies and the application of these antibodies. In order to determine the location of epitopes of the three anti-N monoclonal antibodies, we constructed plasmids containing various N and C-terminal truncation and in-vitro transcribed and translated the corresponding proteins. These mutant proteins were immunoprecipitated with the three anti-N antibodies. The analysis of the immunoprecipitation found that mAb/N40 recognizes epitope amino acid 126-284. while mAb/N42 and mAb/N46 recognize epitope amino acid 284-325. These findings indicate although both mAb/N40 and mAb/N46 recognize conformational epitopes, the regions they recognize are different, which means they are not identical antibodies. We tested these three antibodies against neutral formalin-fixed mouse brain tissue infected with street virus DRV and laboratory virus B2C in immunohistochemistry experiment, and we found that only mAb/N42 showed positive response, while mAb/N40 and mAb/N46 showed negative response. This finding further confirmed the latter two recognize conformational epitopes presented in natural folded N protein, in accordance with the Western Blot results. These monoclonal antibodies will be widely applied in the rabies diagnosis and the study of rabies virus protein structure.