We determined neutralizing antibody titers in serum samples by using a plaque reduction neutralization test (PRNT) against SARS-CoV-2. Serial 5-fold dilutions of heat inactivated (30 min at 56°C) serum samples were incubated with virus for 1 h at 37°C. Each virus–serum mixture was then added to duplicate wells of Vero E6 cells in a 48-well format, incubated for 1 h at 37°C, and overlaid with 500 μL of 2.0% carboxymethylcellulose in DMEM per well. Plates were then incubated at 37°C for 72 h, fixed with 10% buffered formalin, and stained with 0.5% crystal violet. Serum dilutions with >70% reduction of plaque counts compared with virus controls were considered positive for virus neutralization. We used negative serum samples plus virus controls to estimate the percent reduction.

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