Measuring PfRH5 Antibody Binding to FcR and C1q

The binding of PfRH5 antigen-specific antibodies to human Fc receptors (FcR) and complement C1q was measured using a previously-described assay.⁷⁴ (link)^,75 (link) Briefly, avi-tagged FCGR2A, FCGR2B, FCGR3A, and FCGR3B proteins were produced and purified by the Duke Human Vaccine Institute Protein Production Facility. These proteins were then biotinylated with BirA ligase using a commercially available kit (Avidity, #BirA500). Purified human C1q protein (Sigma, #C1740) was biotinylated using EZ-Link Sulfo-NHS-LC-LC-Biotin (Pierce, #A35358) according to the manufacturer’s instructions. These biotinylated Fc domain-binding proteins were then incubated with streptavidin-PE (Prozyme, #PJ31S) to generate the assay detection reagents. Magplex-C microspheres were coupled to biotinylated PfRH5 antigen as described above, blocked with PBSA, and added to 384-well plates so that each well contained 1500 RH5-coupled beads. Plasma from test subjects was diluted in PBSA, added to the beads, and incubated for 2 h at RT on a plate shaker (800 rpm). The beads were then washed, incubated with one of the PE/FcR conjugates for 1 h at RT on a plate shaker (800 rpm), washed again, and acquired on an Intellicyt iQue Screener PLUS flow cytometer. Results were reported as the median PE fluorescence intensity.