Plaque reduction neutralization assay
Serum samples (non-denatured) were diluted in DMEM from 1:10 to 1:320.
SARS-CoV-2 virus was diluted in DMEM to a final concentration of
10-3 pfu/ml. 300 µl serum and 300 µl diluted virus
were mixed and incubated in37 °C incubator at 5% (v/v)
CO2 for 1 hr. Then, the serum-virus mixture was added to
Vero E6 cells and incubated in, 37 °C incubator at 5% (v/v)
CO2 for 1 hr. Serum-virus mixture was discarded from the
plate and the plate was covered with 2% methylcellulose and DMEM at 1:2
ratio and incubated for 4 days. Methylcellulose was discarded and cells
are washed with 1xPBS. Then, 4% PFA was used for cell fixation at room
temperature for 20 minutes. After fixation, cells were stained with
crystal violet solution for 30 minutes at room temperature. Plaques were
counted after washing with PBS to remove the excessive stain. The number
of plaques were used to calculate virus neutralization titers.