SARS-CoV-2 Antibody Detection by Flow Cytometry

The assay to detect patient serum antibodies against SARS-CoV-2 antigens using flow cytometry has been previously described in detail (35 (link)). Briefly, HEK293 cells were transfected to transiently express SARS-CoV-2 full-length Spike (Wuhan-1 D614), Membrane and Envelope proteins. Diluted patient serum was added to the cells, followed by adding AlexaFluor 647-conjugated anti-human IgG (H+L) (Thermo Fisher Scientific) or anti-human IgM (A21249, Thermo Fisher Scientific). The LSRII flow cytometer (BD Biosciences, USA) was used to acquire the cells and patients were confirmed SARS-CoV-2 antibody positive if, in at least two of three quality-controlled experiments, their median fluorescence intensity (ΔMFI   =   MFI transfected cells − MFI untransfected cells) was above the positive threshold (mean ΔMFI + 4SD of 24 pre-pandemic controls). Data were analysed using FlowJo 10.4.1 (BD Biosciences), Excel (Microsoft, USA), and GraphPad Prism (GraphPad Software, USA).