ADCC Receptor Engagement Assay

Using the Promega ADCC reporter bioassay kit and a published protocol, in vitro engagement of the ADCC receptor was assessed [35 ]. MDCK cells were infected at a multiplicity of infection of 1 overnight with each respective virus, as mentioned earlier in the IF assay section. The next morning, antibody dilutions were added onto the cells in addition to 75,000 effector cells per well. Cells were then left in the 37°C incubator for six hours. The luciferase substrate was added in the dark and the luminescence activity was read after 5 min using Synergy Hybrid Reader (BioTek). Anti-stalk mAb CR9114, which has known ADCC reporter activity and binding to H4 HA, was used as a positive control [29 (link),57 (link),58 (link)]. Fold induction over an irrelevant antibody (anti-Lassa GPC) was calculated and data were analysed in Prism 7.