As described in (59 (link)), the selected ACE2-Fc variants at varied concentrations (0.02–5 μg/mL) were incubated with multiplex assay microspheres coated with SARS-CoV-2 RBD or S-6P for 2hr at RT. Lyophilized guinea pig complement was resuspended according to manufacturer’s instructions (Cedarlane), and 2 μL per well was added in veronal buffer with 0.1% gelatin (Boston BioProducts). After washing, the mixtures of ACE2-Fc/microspheres were incubated with guinea pig complement serum at RT with shaking for 1 h. Samples were washed, sonicated, and incubated with goat anti-guinea pig C3 antibody conjugated with biotin (Immunology Consultants Laboratory) at RT for 1 h followed by incubation with streptavidin R-Phycoerythrin (PE, Agilent Technologie) at RT for 30min. After a final wash and sonication, samples were resuspended in Luminex sheath fluid and complement deposition was determined on a MAGPIX (Luminex Corp) instrument to define the median fluorescence intensity (MFI) of PE from two independent replicates. Assays performed without ACE2-Fc and without complement serum were used as negative controls.