Pseudovirus entry and Serum Neutralization assays

Adapted from Walls et al. 2020²⁴. HEK-hACE2 cells were cultured in DMEM with 10% FBS (Hyclone) and 1% PenStrep with 8% CO₂ in a 37 °C incubator (ThermoFisher). One day prior to infection, 40 μL of poly-lysine (Sigma) was placed into 96-well plates and incubated with rotation for 5 min. Poly-lysine was removed, plates were dried for 5 min then washed 1× with water prior to plating cells. The following day, cells were checked to be at 80% confluence. In a half-area 96-well plate a 1:3 serial dilution of sera was made in DMEM in 22 μL final volume. 22 μL of pseudovirus was then added to the serial dilution and incubated at room temperature for 30–60 min at room temperature. HEK-hACE2 plate media was removed and 40 μL of the sera/virus mixture was added to the cells and incubated for 2 h at 37 °C with 8% CO₂. Following incubation, 40 μL 20% FBS, and 2% PenStrep containing DMEM was added to the cells for 48 h. Following the 48–72 h infection, One-Glo-EX (Promega) was added to the cells in half culturing volume (40 μL added) and incubated in the dark for 5 min prior to reading on a Varioskan LUX plate reader (ThermoFisher). Measurements were done on all sera samples from each group in at least duplicates. Relative luciferase units were plotted and normalized in Prism (GraphPad) using a zero value of cells alone and a 100% value of 1:2 virus alone. Nonlinear regression of log(inhibitor) vs. normalized response was used to determine IC₅₀ values from curve fits.