Isotyping and FcR Profiling of Spike Antigens

Isotyping and FcR profiling was conducted as previously described^{60 (link),61 (link)}. Briefly, antigens (NVX-CoV2373 Spike, SARS-CoV-2 Spike, S1, RBD, S2, HKU-1 RBD, or OC43 RBD) were carboxyl coupled to magnetic Luminex microplex carboxylated beads (Luminex Corporation) using NHS-ester linkages with Sulfo-NHS and EDC (Thermo Fisher), and then incubated with serum for 2 hours at 37°C. Isotyping was performed by incubating the immune complexes with secondary mouse-anti-rhesus antibody detectors for each isotype (IgG1, IgG2, IgG3, IgG4, IgA), then detected with tertiary anti-mouse-IgG antibodies conjugated to PE. FcR binding was quantified by incubating immune complexes with biotinylated FcRs (FcγR2A-1, FcγR2A-2, FcγR3A, courtesy of Duke Protein Production Facility) conjugated to Steptavidin-PE (Prozyme). Flow cytometry was performed with an IQue (Intellicyt) and analysis was performed on IntelliCyt ForeCyt (v8.1).