Systems serology

ADCP and ADCD assays were performed essentially as described (10, 27). SARS-CoV-2 spike protein and RBD were biotinylated (Thermo Fisher Scientific) and coupled to 1-μm yellow (ADCP) and red (ADCD) fluorescent beads for 2 hours at 37°C. Excess antigen was removed by washing twice with 0.1% bovine serum albumin in PBS. Next, 1.82 × 10⁸ antigen-coated beads were added to each well of a 96-well plate and incubated with diluted samples (ADCP, 1:100; ADCD, 1:10) at 37°C for 2 hours to facilitate immune complex formation. After the incubation, complexed beads were washed, and for ADCP assays, 2.5 × 10⁴ THP-1 cells (American Type Culture Collection) were added per well and incubated for 16 hours at 37°C. For ADCD assays, 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 (GVB) containing Mg²⁺ and Ca²⁺ (GVB++, Boston BioProducts) to the immune complexes for 20 min at 37°C. After washing twice with 15 mM EDTA in PBS, immune complexes were stained with a fluorescein-conjugated goat IgG fraction to guinea pig complement C3 (MpBio). After incubation with THP-1 cells or staining of cells for ADCD, cell 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. For ADCP assays, a phagocytosis score was calculated as the percentage of bead-positive cells × GMFI/1000, in which GMFI denotes geometric mean fluorescence intensity. For ADCD assays, the median of C3-positive events is reported. All samples were run in duplicate on separate days.