Multiplex Assay for ACE2-Fc Binding

As described in (27 (link)), the selected ACE2-Fc variants at varied concentrations (0.02 to 5 μg/ml) were incubated with multiplex assay microspheres coated with SARS-CoV-2 RBD_wt, RBD_B1.1.529, or S-6P for 2 hours at RT. Lyophilized guinea pig complement was resuspended according to the 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 hour. Samples were washed, sonicated, and incubated with goat anti–guinea pig C3 Ab conjugated with biotin (Immunology Consultants Laboratory) at RT for 1 hour followed by incubation with streptavidin R–phycoerythrin (PE; Agilent Technologie) at RT for 30 min. 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 of PE from two independent replicates. Assays performed without ACE2-Fc and without complement serum were used as negative controls.