SARS-CoV-2 Antibody Binding Assay

ADCD assays were performed as previously described^{26 (link)}. Briefly, SARS-CoV-2 S and RBD were biotinylated (Thermo Fisher Scientific) and coupled to 1 μm red fluorescent neutravidin beads (Thermo Fisher Scientific) for 2 h at 37 °C, and excess antigen was washed away afterwards. For the formation of immune complexes, 1.82 × 10⁸ antigen-coated beads were added to each well of a 96-well round bottom plate and incubated with 1:10 diluted samples at 37 °C for 2 h. Lyophilized guinea pig complement was reconstituted according to the manufacturer’s instructions (Cedarlane) with water, and 4 μl per well was added in gelatin veronal buffer containing Mg²⁺ and Ca²⁺ (GVB⁺⁺, Boston BioProducts) to the immune complexes for 20 min at 37 °C. Immune complexes were washed with 15 mM ethylenediaminetetraacetic acid in PBS, and fluorescein-conjugated goat IgG fraction to guinea pig complement C3 (MP Biomedicals) was added. After staining, samples were fixed with 4% paraformaldehyde, and sample acquisition was performed via flow cytometry (IntelliCyt, iQue Screener Plus) using a robot arm (PAA). All events were gated on single cells and bead-positive events; the median of C3-positive events is reported. All samples were run in duplicate on separate days.