A DirectView focus reduction assay for high-throughput quantification of neutralizing antibodies against influenza A...

The focus reduction assay (FRA) is a widely used method in influenza virus research and surveillance to evaluate neutralizing antibodies. However, traditional FRA relies on time-consuming, labor-intensive immunostaining workflows that constrain throughput. Here, we used a fluorogenic neuraminidase (NA) substrate, BTP3-Neu5Ac, to directly visualize viral foci without immunostaining. Foci were captured with a fluorescence plate reader, and neutralizing antibody titers were determined from foci counts across antibody dilutions. This assay, termed DirectView-FRA, was applied to measure the neutralizing activity of antisera and monoclonal antibodies against seasonal A(H1N1)pdm09, A(H3N2), B/Victoria, and B/Yamagata viruses, as well as high pathogenicity avian influenza A(H5N1) viruses. Results were consistent with those of the traditional FRA. By simplifying the workflow, DirectView-FRA enables rapid, high-throughput testing of neutralizing antibodies to support timely public health responses during influenza epidemics and to assess viruses with pandemic potential.