Generation of monoclonal antibody against Rift Valley fever virus and evaluation of its diagnostic potential

Abstract

Rift Valley fever virus (RVFV) belongs to the family Bunyaviridae and genus Phlebovirus and it causes Rift Valley fever (RVF) which is one of the major viral hemorrhagic fevers in Kenya and Africa as a continent. RVFV is transmitted by Aedes and Culex mosquito bites and contact with infected body fluids including blood and milk. Due to the presence of other Bunya viruses in Kenya that have the same symptoms as RVFV (like Crimean-Congo Hemorrhagic Fever Virus and Nairobi Sheep Disease Virus), there is a need for RVFV specific diagnostic tests. This may be achieved by use of monoclonal antibodies (mAbs) against RVFV to set up diagnostic systems, considering that mAbs are currently widely used not only for research purposes, but also for diagnostics and therapeutic purposes. This study was aimed at developing mAbs against RVFV and their diagnostic potential validated by use of the mAbs in ELISA systems. BALB/c mice were immunized with purified, inactivated RVFV (attenuated live vaccine strain: Smithburn) and the rise in IgG titers was tested by IgG indirect ELISA. When the IgG titers were high enough, the mouse spleen was harvested and splenocytes were fused with mouse myeloma SP2 cells. Four clones of anti-RVFV mAbs were successfully established. Among them, clone “P1E5” was further cultured in large scale and purified by protein G column. The purified mAb was characterized by western blot, focus reduction neutralization test (FRNT), and isotyping. For determining its diagnostic potential, the purified mAb was applied in antigen detection ELISA and antigen capture IgG indirect ELISA as the capture antibody. The antigen detection ELISA was used to test the cross reactivity of the anti-RVFV P1E5 mAb to other arboviruses; dengue virus serotypes 1-4 (DENV 1-4), yellow fever virus (YFV), West Nile virus (WNV) and chikungunya virus (CHIKV). At the same time, the antigen capture IgG indirect ELISA was also used to test 22 human serum samples. The results obtained were compared to two other similar ELISA systems that used mAb against the nucleocapsid (N) protein of RVFV and anti-RVFV polyclonal antibodies (pAbs) as the capture antibodies. When applied in western blot, the mAb could detect RVFV and specifically reacted with L segment derived protein (250kDa). The mAb did not show any neutralizing activity in the FRNT. Upon isotyping, the mAb was found to be IgG1 and carrying the Kappa light chain. When the P1E5 mAb was applied in antigen detection ELISA it specifically detected RVFV but not DENV 1-4, YFV, WNV and CHIKV. In the antigen capture IgG indirect ELISA, the mAb could detect IgG from patient sera that were known to have anti-RVFV IgG. The results obtained were in agreement with two other ELISA systems that used mAb against the N protein of RVFV and anti-RVFV pAbs as the capture antibodies. The anti-RVFV mAb was successfully applied in antigen detection ELISA and antigen-capture IgG indirect ELISA. Results from this project demonstrate the potential for development of a platform for RVF diagnosis in Kenya, using immunoreagents that can be produced locally. At the same time, this project has contributed in setting up the technology of mAb development in a local laboratory; hence the technology can be transferred and applied for other infectious agents, thus enhancing diagnosis of infections in Kenya.