Plaque Reduction Neutralization Tests (PRNT) Assay

A total of 0.1 million Vero-E6 cells were seeded into each well of a 24-well plate, at least 20 h before the experiment. Serum samples were heat-inactivated at 56°C for 1 h and subsequently serially diluted two-fold using DMEM supplemented with 2% FCS. SARS-CoV-2 virus stock was diluted to produce 30 plaque-forming units (Pfu) per well. Virus dilution was mixed with serial dilutions of sera and incubated at 37°C, for 1 h. Subsequently the mix of serum and virus were overlaid on a VeroE6 monolayer and the plates were incubated for 1 h at 37°C in 5% CO2 with intermittent rocking. At the end of incubation, the inoculum was discarded; the monolayer in each well was washed with serum-free media and DMEM-2%-FCS supplemented with 1.5% carboxymethyl cellulose was overlaid onto the monolayer. The plates were incubated for 72 h in 37°C and 5% CO₂. The monolayer was fixed using 4% PFA and the plaques were visualized by crystal violet staining. The number of plaques in the wells with no sera were counted and taken as control. The average number of plaques corresponding to each serum dilution was expressed as a percentage of that in the control wells. The inverse value of the dilution that reduced the plaque numbers to 90% of that in the control wells was taken as PRNT₉₀.