SARS-CoV-2 Antibody Detection by S-Flow Assay

The S-Flow assay was performed as previously described (39 (link)). Briefly, plasma samples were diluted (1:200) in 1xPBS with 2mM EDTA and 0.5% BSA (PBS/BSA/EDTA) and incubated with 293T-spike cells (80000 cells/100µl) for 30 minutes on ice. The cells were washed with PBS/BSA/EDTA and stained either with anti-IgM PE (dilution 1:100, Biolegend) and anti-IgG Alexa Fluor™ 647 (dilution 1:600, Thermo Fisher) or anti-IgA Alexa Fluor 647 (dilution 1:800, Jakson ImmunoResearch) for 30 minutes on ice. The cells were washed with 1xPBS and fixed using buffer of the True-Nuclear Transcription Factor Staining kit (Biolegend). After fixing, the cells were washed and resuspended in 1xPBS. The results were acquired using FACS Canto II, BD Biosciences. The gating strategy for anti-IgM, anti-IgG or anti-IgA positive cells was based on the 293T control cells incubated with negative SARS-CoV-2 reference plasma. The data were reported as percentage of positive cells for anti-IgM, anti-IgG or anti-IgA. The NIBSC Research Reagent (20/130) and panel (20/118) (WHO Solidarity II) was utilized to set the cutoff for positivity based on the background staining of the negative SARS-CoV-2 plasma and calculated following formula: cut-off= % positive cells + 2x standard deviation.