Luminex Bead-Based Complement Deposition Assay

Complement deposition was performed as described previously^{49 (link)}, using Luminex beads (Luminex Corp). Briefly, Luminex microspheres were coupled to biotinylated antigens, and immune complexes were formed using 1:30 diluted plasma samples. After a 2-h incubation at 37 °C, guinea pig complement in GVB++ buffer (Boston BioProducts) was added to immune complexes for 20 min at 37 °C. To stop the complement reaction, EDTA-containing phosphate-buffered saline (15 mM) was used, then C3 deposition on beads was detected using a 1:100 diluted anti-guinea pig complement C3 antibody (MP Biomedicals). MFI values were analyzed by flow cytometry on an iQue analyzer (Intellicyt) (Fig. S6).