SARS-CoV-2 Microneutralization Assay Protocol

Live virus microneutralization assay was performed as we described previously 58 (link). Briefly, serum specimens were serially diluted in 3-folds from 1:10. Equal volumes of serially diluted sera and the desired SARS-CoV-2 strains (1 × 10³ PFU/mL) were mixed, added onto 96-well plates and incubated for 1 h at 37^oC. Mixtures were then transferred to 96-well plates pre-seeded with 2 × 10⁴/well Vero E6 cells and incubated at 37^oC overnight. The culture medium of the plates was removed, air-dried in a biosafety cabinet (BSC) for 20 min. Cells were then fixed with 4% paraformaldehyde solution. To further permeabilize the cells, 100 μL of 0.2% Triton X-100 in PBS was added to each well and the plates were incubated at RT for 15 min. After three washes in PBS, the plates were incubated with cross-reactive rabbit anti-SARS-CoV-2 N IgG (diluted 1:4000 in 0.2% Triton X-100 in PBS) for 1 h, followed by the addition of Alexa Fluor 488 goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (diluted 1:1000 in 0.2% Triton X-100 in PBS, Life Technologies) and incubation at RT for 1 h. The plates were analyzed using Sapphire Biomolecular Imager (Azure Biosystems). The numbers of SARS-CoV-2 foci were quantified using custom-built ReadPlate 3.0 plugin in ImageJ 59 (link).