ADCC Assay for Antibody Activity

An ADCC reporter bioassay kit (Promega) was used to assess whether any of the MAbs elicit ADCC activity. The protocol has been described previously but was modified as needed (14 (link), 23 (link)). Vero.E6 cells (50,000 per well) were added onto white round-bottom 96-well cell culture plates (Corning Costar) and infected with VSV-LASV at a multiplicity of infection (MOI) of 1.0. Virus was prepared in MEM, and this medium was removed from the cells after 16 h. Twofold serial dilutions of each antibody were added to the cells in duplicate starting from a concentration of 100 µg/ml. Seventy-five thousand effector cells were added to the cells with the antibody dilutions, and cells were incubated at 37°C for 6 h. Luciferase substrate was added, and luminescence was measured 2 to 5 min later using a Synergy Hybrid Reader (BioTek). Human monoclonal antibody CR9114 was used on influenza virus (A/duck/Czechoslovakia/1956 H4N6)-infected Vero.E6 cells as a positive control (42 (link), 43 (link)).