SARS-CoV-2 Spike Protein Neutralization Assay

ADCD was adapted from methods described previously (76 (link)). Briefly, SARS-CoV-2 S-expressing expi293F cells were incubated with 10-fold diluted, heat-inactivated (56 °C for 30 min) plasma samples for 30 min at 37 °C. Cells were washed twice and resuspended in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% FBS (R10 media). During this time, lyophilized guinea pig complement (CL4051, Cedarlane) was reconstituted in 1 mL of cold water and centrifuged for 5 min at 4 °C to remove aggregates. Cells were washed with PBS and resuspended in 200 μL of guinea pig complement, which was prepared at a 1:50 dilution in Gelatin Veronal Buffer with Ca²⁺ and Mg²⁺ (IBB-300X, Boston BioProducts). After incubation at 37 °C for 20 min, cells were washed in PBS 15 mM (ethylenedinitrilo)tetraacetic acid (ThermoFisher Scientific) and stained with an anti-Guinea Pig Complement C3 FITC (polyclonal, ThermoFisher Scientific). Cells were then fixed with 4% formaldehyde solution, and fluorescence was evaluated on an LSRII flow cytometer (BD Bioscience).