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.