96 well plate

Serum mediated neutralization of viral entry into Ace2 expressing cells were assayed using a pseudovirus consisting of spike protein (WA-1) and expressing luciferase reporter gene as described previously (Dickey et al., 2021 (link)). Mouse serum was diluted in a triplicate 5-fold series from 1:10 to 1:781,250. Serum was mixed 1:1 with pseudovirus expressing luciferase reporter gene in a 96-well plate (GenScript). After incubation at room temperature for 1 h, 20,000 HEK293 cells overexpressing Ace2 were added to each well. Cells were incubated with pseudovirus at 37°C, 5% CO₂ for 48 h, after which culture medium was removed and Fire-Lumi^TM luciferase substrate reagent was added to the wells. Luciferase signal was measured using an PHERAStar microplate reader and %inhibition was calculated using the following formula: $\%inhibition=100\times \left(1-\frac{X-\text{min}}{\text{max}-\text{min}}\right)$ where X is the luciferase signal, min is the average signal from duplicate wells without pseudovirus, and max is the average signal from duplicate wells without serum. Log IC₅₀ values were calculated in the same manner described for the RBD/Ace2 blocking assay.

Mouse serum was blinded and diluted in a duplicate fourfold series from 1:5 to 1:81,920. Serum was mixed 1:1 with pseudovirus containing the SARS-CoV-2 spike protein and a luciferase reporter in a 96-well plate (GenScript). After 1 hour of incubation at room temperature, 20,000 human embryonic kidney (HEK) 293 cells overexpressing Ace2 were added to each well. Cells and pseudovirus were incubated at 37°C, 5% CO₂ for 48 hours, after which culture medium was removed and Bio-Glo luciferase reagent (Promega) was added to the wells. Luciferase signal was measured using an EnVision plate reader, and % inhibition was calculated using the following formula $$\% \text{Inhibition}=100\times (1-\frac{X-\mathit{\text{min}}}{\mathit{\text{max}}-\mathit{\text{min}}})$$ where X is the luciferase signal, min is the average signal from duplicate wells without pseudovirus, and max is the average signal from duplicate wells without serum. Log ID₅₀ values were calculated in the same manner described for the RBD/Ace2-blocking assay.