Generation and application of monoclonal antibodies against Rift Valley fever virus nucleocapsid protein NP and glycoproteins Gn and Gc

Rift Valley fever virus (RVFV) is a vector-borne virus that causes high neonatal mortality in livestock and deadly haemorrhagic fever in humans. In this paper, we describe the generation of monoclonal antibodies (mabs) against all three structural proteins of RVFV (glycoproteins Gn and Gc and nucleocapsid protein NP). After immunization of BALB/c mice with individual recombinant proteins, a total of 45 clones secreting ELISA-reactive monoclonal antibodies against NP, Gn and Gc epitopes were obtained. Twelve clones were directed to NP, 28 to Gn, and 5 to Gc. Western blot analysis revealed that most of the mabs were reactive to linearized epitopes on recombinant as well as native virus proteins. Six mabs against NP, 21 against Gn and all mabs against Gc also detected conformational epitopes, as shown by indirect immunofluorescence on RVFV-infected cells. All of the mabs were evaluated for their use in a competition enzyme-linked immunosorbent assay (ELISA) for the detection of a RVFV infection. Several mabs were identified that competed with polyclonal rabbit serum, and one of them – mab Gn123, raised against Gn protein – was selected for a proof-of-principle study with field sera from a recent Rift Valley fever outbreak. The novel Gn-based competition ELISA demonstrated high performance, offering a promising alternative and addition to serological assays based on nucleocapsid protein.