Protocol for multiplexed detection of antibody binding to cell-surface-expressed hemagglutinin by flow cytometry

Measuring antibody binding titers to viral proteins is essential to assess population immunity and monitor viral escape. We present a multiplexable flow cytometry protocol to quantify antibody binding to native-like, cell-surface-expressed antigens and demonstrate the method using influenza A virus hemagglutinin. We describe steps for the transfection of human suspension cells, cell staining and fixation, data acquisition, and analysis. In a single run, up to 500 samples can be measured simultaneously against three antigens per well within three days. For complete details on the use and execution of this protocol, please refer to Daniel et al.¹.