SARS-CoV-2 conventional virus neutralization test

Plasma neutralization activity was evaluated using a cytopathic (CPE)-based assay as previously described [13]. Plasma samples were tested at an initial dilution of 1:8 and then diluted in eight two-fold steps. All samples were mixed with a SARS-CoV-2 Wuhan-1 Omicron viral solution that contained 100 TCID50 of the virus and incubated in 37°C and 5% CO₂ for 2 h. Next, the virus–plasma mixture was added to a 96-well plate containing 1.2 × 10⁴ Vero E6 cells. The plates were incubated for 4 days at 37°C in a humidified environment with 5% CO2 and examined for CPE by the Celigo Imaging Cytometer (Nexcelom Bioscience, Lawrence, MA, USA). The absence or presence of CPE was defined by comparing each well a positive control (plasma sample showing high SARS-CoV-2 neutralizing activity in infected Vero E6 cells) and a negative control (human serum sample negative for SARS-CoV-2 in ELISA and neutralization assay and Vero E6 cells alone). We defined neutralizing antibody titres less than the detection limit dilution as NAbs titre of 50% inhibitory dilution (EC50) = 4.