SARS-CoV-2 Neutralizing Antibody Quantification Assay

VN antibodies present in the sera were detected as described previously (22 (link), 23 (link)). In short, inactivated serum samples (at 56°C for 30 min) were first diluted 1:10 followed by two-fold serial dilutions. Vero cells (ATCC CCL-81) were seeded in a 96-well tissue culture plate. Diluted sera were mixed with 200 TCID₅₀ of SARS-CoV-2 Wuhan-Hu-1 and incubated for 1 h at 37°C. Serum–virus mix was added to a monolayer of Vero cells and further incubated at 37°C, 5% CO₂. After 8 h of incubation, cells were fixed using 4% Paraformaldehyde (PFA) and incubated for 30 min at room temperature. Next, PFA was removed, and cells were incubated for 15 min with 80% methanol. For staining, plates were blocked using 1% BSA in PBS–0.05% Tween 20 for 30 min at 37°C. SARS-CoV-2 was detected using a 1:1,000 dilution of rabbit polyclonal anti–SARS-CoV-2 nucleocapsid (Sino Biological). After 1 h of incubation at 37°C, cells were washed with PBS–0.05% Tween 20 and incubated with a 1:1m000 dilution of anti–rabbit–IgG–Alexa Flour 488 (Invitrogen). Finally, cells were washed twice with PBS–0.05% Tween 20. Fluorescent cells were counted using the C.T.L. S6 Ultimate-V Analyzer, and data were analyzed using CTL ImmunoSpot^® software. Neutralizing antibody titers are expressed as the dilution that gave a 50% reduction of stained cells (NT50).