Plaque Reduction Neutralization Test for SARS-CoV-2

PRNT was conducted as previously described [23 (link)]. A volume (300 µL) of each purified mAb under serial dilution starting from 10 μg/mL was incubated with 80 PFU of SARS-CoV-2 at the final volume of 600 µL at 4 °C overnight. The mixtures were added in triplicates to confluent monolayers of Vero E6 cells, grown in 12-well plates, and incubated at 37 °C in a humidified, 5% CO2 atmosphere for 60 min. Then, 4 mL/well of a medium containing 2% Gum Tragacanth (Sigma Aldrich) + MEM 2.5% FCS were added. Plates were left at 37 °C with 5% CO₂. After 3 days, the overlay was removed, and the cell monolayers were washed with PBS in order to completely remove the overlay medium. Cells were stained with a crystal violet 1.5% alcoholic solution. The presence of SARS-CoV-2 virus-infected cells was indicated by the formation of plaques. The inhibitory concentration (IC)50 was determined as the highest dilution of serum resulting in a 50% (PRNT50) reduction of plaques as compared to the virus control.