Integrated Laboratory Evaluation of Rift Valley Fever Virus Antibodies Using the Competitive ELISA and Virus Neutralization Test

<b>Background:</b> Rift Valley fever virus (RVFV) is a significant mosquito-borne zoonotic virus with high public health and veterinary importance in Africa and the Middle East. Reliable diagnostic assays for detecting antibodies and assessing their functional neutralizing capacity are essential for surveillance programs, vaccine monitoring, and outbreak preparedness. <b>Objective:</b> This study evaluates and compares the analytical performance of a competitive enzyme-linked immunosorbent assay (cELISA) and a virus neutralization test (VNT) for detecting RVFV antibodies in vaccinated sheep sera, establishing an integrated laboratory workflow for virus titration, serological detection, and functional neutralization. <b>Methods:</b> Twenty serum samples were collected from sheep pre-vaccination and one month post-vaccination with Smithburn live attenuated RVFV vaccine. Sera were tested using a commercial multispecies RVFV competitive ELISA to detect antibodies specific to the viral nucleocapsid protein. Viral titration was conducted in Vero cells, and 50% tissue culture infective dose (<i>TCID</i>₅₀<i>/</i>0.1 mL) was calculated using the Reed and Muench method. VNT was performed at 24, 48, 72, and 96 h after infection with different viral doses (10² to 10⁵<i>TCID</i>₅₀<i>/</i>0.1 mL), and the neutralizing ability of serial serum dilutions (1:2 to 1:1024) was tested. Compared with the control, protection was determined by cytopathic effect (CPE) inhibition. <b>Results:</b> ELISA revealed robust antibody signals up to a 1:32 dilution, with signal-to-noise (<i>S/N</i>) < 40%, whereas for higher dilutions, antibody detection became inconclusive or negative. Virus titration was performed to verify a stock concentration of 10^6.5<i>TCID</i>₅₀/0.1 mL. The VNT exhibited time- and dose-dependent kinetics; high protection rates (≥97) were observed at 1:2-1:8 dilutions against 10²-10³<i>TCID</i>₅₀<i>/</i>0.1 mL challenge doses; however, neutralizing efficacy decreased significantly at higher viral loads and higher serum dilutions. While cELISA and VNT results correlated strongly at low serum dilutions, the cELISA showed decreased sensitivity at dilutions ≥ 1:64, where the VNT remained capable of detecting functional neutralizing activity. <b>Conclusions/Discussion</b>: The results demonstrate that while both assays correlate well at high antibody concentrations, they diverge at lower concentrations. This discrepancy highlights the functional difference between binding antibodies (N-protein) and neutralizing antibodies (Gn/Gc glycoproteins). Consequently, the cELISA is ideal for rapid screening, whereas the VNT is indispensable for confirming functional immunity. Integrating both assays provides a more accurate immunological profile for RVFV surveillance and vaccine evaluation.