Antigen-Dependent Complement Deposition Assay

The ADCD assay was adapted from Fischinger et al. (57 (link)). Antigen was coupled to red fluorescent Neutravidin 1 μm beads (Invitrogen, Thermo Fisher Scientific, F8775) as described for ADCP. Immune complexes were formed by incubating 10 μL of coupled beads with 50 μL of antibody at concentrations of 50 μg/mL, 10 μg/mL, 2 μg/mL, and 0.4 μg/mL for 2 hours at 37°C. Plated were spun down, and immune complexes were washed with PBS. Lyophilized guinea pig complement (Cedarlane, CL4051) was resuspended in 1 mL of cold water, diluted 1:50 in GVB++ (gelatin veronal buffer and additional Ca2⁺ and Mg2⁺, Boston BioProducts, IBB-300X), and added to the immune complexes. The plates were incubated for 20 minutes 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 (MP Biomedicals, 0855385) for 15 minutes in the dark. Fluorescence was acquired with an Intellicyt iQue.