Complement Deposition Assay for Antibodies

ADCD was conducted as previously described [82 (link)]. Briefly, spike or RBD protein was biotinylated using EDC (Thermo Fisher Scientific) and EZ-link Sulfo-NHS-LC-LC (Thermo Fisher Scientific) and then coupled to red Neutravidin-conjugated microspheres (Thermo Fisher Scientific). Immune complexes were formed by incubating the bead+protein conjugates with diluted serum (ADCD 1:10 dilution) for 2 hours at 37°C and then washed to remove unbound antibody. The immune complexes were then incubated with lyophilized guinea pig complement (Cedarlane - Burlington, Ontario, Canada) and diluted in gelatin veronal buffer with calcium and magnesium (Boston BioProducts - Milford, Massachusetts, USA) for 30 minutes. C3 bound to immune complexes was detected by fluorescein-conjugated goat IgG fraction to guinea pig Complement C3 (MP Biomedicals - Irvine, California, USA). Flow cytometry was performed to identify the percentage of beads with bound C3 and a complement deposition score was calculated (% beads positive × median fluorescent intensity of positive beads). Flow cytometry was performed with an LSRII (BD) and analysis was performed using FlowJo V10.7.1.