ADCD Assay for Complement Activation

The ADCD assay was performed as previously described (Fischinger et al., 2019 (link)). Spike biotinylation, bead coupling, and immune complex formation was performed as described for ADCP, using red Neutravidin beads (Invitrogen) and 1:10 dilution of serum and 1:1 dilution of breastmilk. Following immune complex formation, plates were washed and guinea pig complement (Cedarlane) diluted in gelatin veronal buffer supplemented with calcium and magnesium (Boston BioProducts) was added. Plates were incubated for 20 minutes at 37°C. Plates were washed twice with 15mM EDTA in PBS and C3-deposition was detected by staining with anti-C3 FITC (MPbio). Fluorescence was acquired using an iQue (Intellicyt) and C3-deposition is reported as the median fluorescence intensity of FITC.