Quantitative SARS-CoV-2 Neutralization Assay

The SARS-CoV-2 focus reduction neutralization test (FRNT) was performed in a certified Biosafety level 3 laboratory as previously described [21 (link)]. The tested antibodies were serially diluted, mixed with 50 μL of SARS-CoV-2 (10³ focus forming unit/mL, PFU/mL) in 96-well plates, and incubated for 1 h at 37°C. Mixtures were then transferred to 96-well plates pre-seeded with 2 × 10⁴/well Vero-E6-TMPRSS2 cells and incubated at 37°C for 24 h. The culture medium was then removed and then fixed with a 4% paraformaldehyde solution for 30 min and air-dried in the BSC again. Cells were further permeabilized with 0.2% Triton X-100 and incubated with cross-reactive rabbit sera anti-SARS-CoV-2-N (1:5000) for 1 h at RT before adding an Alexa Fluor 488 goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody (1:1000 Life Technologies). The fluorescence density of SARS-CoV-2 infected cells were scanned using a Sapphire Biomolecular Imager (Azure Biosystems) and the neutralization effects were then quantified using Fiji software (NIH).