Figure S4.

Additional Characterization of mAb Screening Assays, Related to Figure 4

A) Effects of GP antigen attachment strategy on ELISA results. ELISA plates were coated with GP_ec directly (left), or GP_ec was bound to plates coated with a capture antibody directed against the affinity purification tag on the C terminus of GP (StrepMAB-Classic, middle), or GP_ec was bound to plates coated with a mucin domain-specific capture mAb (h13F6; right). Plates were incubated with four biotinylated EBOV-specific mAbs and binding was detected with avidin-HRP. All four mAbs bound to GP_ec in the initial screening ELISA but mAbs 5.1.10B3 and 5.1.5E8 (blue) bound to native GP on Jurkat cells, whereas mAbs 5.1.5H5 and 5.1.5D7 did not. The two native GP-specific mAbs were relatively unaffected by the strategy used to attach GP_ec to the plate, while the two mAbs that do not bind native GP on Jurkat cells, showed reduced binding to GP_ec that was captured indirectly.

B) Representative neutralization screening assay using biologically-contained EBOV. Luciferase-expressing EBOV lacking the VP30 gene was incubated with the indicated mAbs at 10 μg/ml for 2 hours at 37°C, then added to VP30-expressing Vero cell cultures in duplicate wells. Luciferase activity was measured three days later. mAbs were considered neutralizing if the luciferase signal was reduced by > 90% compared to the average signal in six wells incubated with virus plus negative control antibody. Bars for neutralizing mAbs are colored red. Neg. control = VP35-specific mAb 5-69.3.2. Pos. control = mAb 226/8.1. The SD of three replicate assays is shown.