Pe conjugated goat f ab 2 anti human igg

Cell surface-expressed SARS-CoV-2 ancestral and variant S were obtained by transfecting 8 μg of SARS-CoV-2 full-length DNA plasmid with 25 μl PEImax in 400 μl OptiMEM onto 12 to 15 ml HEK293T cells in a petri dish (seeded the day before at a density of 3.0x10⁶). After 48 h, cells were harvested and frozen until further use. After thawing, HEK293T cells expressing the S of interest were seeded at 20.000 to 30.000 cells per 96-well in FACS buffer (PBS/0.5% FCS) and were incubated 1:1 with non-purified supernatants from small scale IgG production or a dilution of purified HEK293F-produced mAbs for 30 min at 4°C. Subsequently, cells were washed twice with FACS buffer and incubated for 30 min at 4°C with 1:1000 diluted PE-conjugated goat F(ab)’2 anti-human IgG (Southern Biotech 2042-09) in the dark. Cells were washed and analysed on the FACS Canto II analyser. Samples were analysed by FlowJo X software (BD Biosciences) and percentage of cells that showed binding were plotted correspondingly.