High-throughput screening (HTS) neutralization assay.

A total of 400 PFU of recombinant EBOV expressing eGFP or EBOV/BDBV-GP were incubated with various concentrations of MAbs in black polystyrene 96-well plates with clear bottoms (Corning, NY) for 1 h at 37°C in MEM containing 10% fetal bovine serum and 0.1% gentamicin sulfate. Next, 4 × 10⁴ Vero-E6 cells in the same medium were added to the virus-antibody mixtures, followed by incubation at 37°C for 4 days. The fluorescence intensity of infected cells at a 488-nm wavelength was measured in triplicate using a 2104 EnVision multilabel reader (Perkin-Elmer, Waltham, MA). The signal readout was normalized to virus control aliquots with no MAb added and is presented as the percentage of neutralization.