Complement Deposition Assay for Antigen-Antibody Complexes

Complement deposition was performed as previously described [14 (link)]. Briefly, biotinylated antigens were bound to FluoSphere NeutrAvidin beads (Thermo Fisher Scientific; Waltham, MA, USA). To form immune-complexes, antigen-coated beads were incubated with 10 µL of 1:10 diluted serum samples. Non-specific antibodies were washed away, and immune complexes were incubated with guinea pig complement in GVB++ buffer (Boston BioProducts; Milford, MA, USA). Complement reaction was stopped using EDTA containing PBS (15 mM). Deposited C3 on beads were stained with anti-guinea pig C3-FITC antibody (MP Biomedicals; Irvine, California USA, 1:100, polyclonal) and analyzed on an anti-guinea pig C3-FITC antibody (MP Biomedicals, CA, USA, 1:100, polyclonal) and analyzed on an iQue analyzer (Intellicyt; Albuquerque, NM, USA).