ADCD Assay for Complement Deposition

The ADCD assay was adapted from Fischinger et al.^{84 (link)}. Antigen was coupled to non-fluorescent Neutravidin 1 μm beads (Invitrogen, #F8777) as described for ADCP. Immune complexes were formed by incubating 10 μL of coupled beads with 10 μL of diluted sample for 2 h at 37 ˚C. Plates were spun down, and immune complexes were washed with PBS. Lyophilized guinea pig complement (Cedarlane, #CL4051) was resuspended in cold water, diluted in Gelatin Veronal Buffer, Boston BioProducts, IBB-290X) and added to the immune complexes. The plates were incubated for 50 min at 37 ˚C and the reaction was stopped by washing the plates twice with 15 mM EDTA in PBS. To detect complement deposition, plates were incubated with Fluorescein-conjugated goat anti-guinea pig complement C3 (1:50 diluion) (MP Biomedicals, #0855385) for 15 min in the dark. Fluorescence was acquired with a Stratedigm 1300EXi cytometer.