NanoLuc-based microneutralization assay for high-throughput detection of neutralizing antibodies against rift valley fever virus.

Plaque reduction neutralization tests (PRNTs) are the gold standard for measuring neutralizing antibodies but are labor-intensive, low-throughput, and subject to variability from manual plaque counting. We developed a NanoLuc-based microneutralization (mNT) assay using rMP12-NanoLuc that quantifies luminescence in culture supernatants as a surrogate for infection. The assay runs in 96-well plates without overlay, fixation, or staining, using 35 focus-forming unit (FFU) per well and a 40 hour readout with back-titration to ensure accuracy. mNT titers from vaccinated animal sera showed strong correlation with conventional PRNTs. This approach enables rapid, reproducible, high-throughput detection of RVFV neutralizing antibodies, with the main limitations being substrate cost and the requirement for a luminometer.•The assay enables high-throughput, objective neutralization testing in 96-well plates.•Optimized conditions yield mNT titers that strongly correlate with conventional PRNTs.•Limitations include NanoLuc substrate cost and the need for a luminometer.