Flow Cytometry-based Cell-ELISA for HCoV Spike Binding

Flow cytometry-based Cell-ELISA (CELISA) binding of mAbs with HCoV spikes was performed as described previously (43 , 81 (link)). A total of 4×10⁶ HEK293T cells were seeded into 10cm round cell culture dishes and incubated at 37°C. After 24h, HEK293T cells were transfected with plasmids encoding full-length HCoV spikes and were incubated for 36–48h at 37°C. The cells were harvested and distributed into 96-well round-bottom tissue culture plates for individual staining reactions. For each staining reaction, cells were washed three times with 200μl FACS buffer (1xPBS, 2%FBS, 1mM EDTA). The cells were stained for 1h on ice in 50μl staining buffer with 10μg/ml of primary antibody. After washing three times with 200μl FACS buffer, the cells were stained with 50μl/well of 1:200 diluted R-phycoerythrin (PE)-conjugated mouse anti-human IgG Fc antibody (SouthernBiotech cat.# 9040–09) and 1:1000 dilution of Zombie-NIR viability dye (BioLegend cat.# 423105) on ice in dark for 45min. Following three washes with FACS buffer, the cells were resuspended and analyzed by flow cytometry (BD Lyrics cytometer), and the binding data were generated by calculating the Mean Fluorescence Intensity using FlowJo 10 software. Mock-transfected 293T cells were used as a negative control.