SARS-CoV-2 Spike Antibody Assay

The S-Flow assay was performed as previously described with minor modifications^{23 (link)}. Briefly, 293 T cells were transiently transfected with an expression vectors for SARS-CoV-2 Spike or control vector. Two days after transfection, cells were harvested and incubated with patient sera diluted 1:625 in FACS buffer (PBS with 0.5% BSA and 2 mM EDTA) for 20 min at 4 °C. After washing, cells were stained with goat anti-human IgG Alexa Fluor 647 (1:200; Thermo Fisher Scientific) and goat anti-human IgM Alexa Fluor 488 (1:200; Thermo Fisher Scientific) for 20 min at 4 °C. Cells were washed, fixed in 4% formaldehyde and analyzed on a BD LSRFortessa flow cytometer for antibody binding. Specific antibody binding was determined based on control-vector transfected cells. Serum was considered test-positive if the percentage of IgG-positive cells of Spike-transfected cells exceeded the percentage of control-vector transfected cells by more than 10%.