Fig 1. Representative influenza HA-specific ADP assay.
The ADP assay is performed as describe in (A). In brief, FITC fluorescent beads coated with hemagglutinin (HA) and fluorescent internalization probe (FIPCy5) are opsonized with 10μg/ml purified IgG from blood plasma or sera. HA-coated beads are incubated 16 hr with monocyte-like cell line, THP-1. Cy5 fluorescence from surface bound beads is quenched. Cells are fixed and analysed by flow cytometry. (B) Gating strategy of single cells. (C) Gating strategy for internalized beads+ (FITC+, Cy5+) and surface associate beads+ (FITC+. Cy5-) cells. Uptake of H3N2 A/Wyoming/03/2003 HA-coated beads in presence of influenza sero-negative human serum IgG or healthy human adult plasma IgG (top 2 panels). Uptake of H1N1 A/Puerto Rico/08/1939 HA-coated beads opsonized with IgG from macaque plasma either pre-influenza infection or post-influenza infection (bottom 2 panels). Data are representative of 14 human donors (Fig 2) and 6 macaques (Fig 5).