Rift Valley fever virus (RVFV) is a mosquito-borne, zoonotic virus that infects 
ruminants, including cattle, sheep, goats, camels, and buffalo. Multiplexing 
diagnostic assays that can simultaneously detect antibodies against multiple 
RVFV antigens offer a high-throughput test for disease surveillance and vaccine 
evaluations. We describe the improvement and evaluation of a previously 
developed fluorescence microsphere immunoassay (FMIA) for the detection of IgG 
and IgM antibodies against the RVFV glycoprotein (Gn) and the immunogenic 
nucleocapsid protein (Np). Well-characterized vaccinated and experimentally 
infected ruminant sera were used for the evaluation of the assay. Recombinant 
viral proteins were produced and then coupled to polystyrene magnetic beads for 
analysis using the Luminex MAGPIX system with xMAP technology. The FMIA was 
performed in parallel with virus neutralization tests. Our results revealed the 
highest median fluorescence intensity (MFI) values for the detection of IgG 
antibodies against RVFV Np, indicating that this antigen would be a good 
candidate for a screening assay. The Np and Gn targets could differentiate 
infected animals from animals vaccinated with a candidate subunit vaccine 
formulation based on the RVFV Gn and Gc proteins. The results presented in this 
report demonstrate that FMIA provides a rapid and robust serological diagnostic 
tool for the detection of antibodies against RVFV. The targets developed in this 
assay provide the basis for the development of a companion diagnostic test for 
an RVFV Gn/Gc subunit vaccine that is capable of differentiating infected from 
vaccinated animals (DIVA), as well as a multiplex serodiagnostic assay that can 
simultaneously screen for several ruminant diseases.