Plaque reduction neutralization test

The neutralizing antibodies to live SARS-CoV-2 were quantified using a plaque reduction neutralization test (PRNT); serum samples were first heat-inactivated for 30 min at 56°C and then diluted 1:2 in 96-well plates. After the samples were incubated for 1 h at 37°C together with 50% tissue culture infectious dose (TCID₅₀) SARS-CoV-2, the serum/virus mixtures were added to VeroE6 cells in another 96-well plate and incubated for 1 h at 37°C. The cytopathic effect (CPE) on VeroE6 cells was analyzed at 3 days post-infection. Neutralization was defined as the absence of 50% CPE compared with virus controls, and the results were calculated by using the Reed-Muench (16) or Spearman-Kärber method (17). The positive cutoff for defining seropositivity for neutralizing antibodies to live SARS-CoV-2 was 1/4, and the neutralizing antibody detection data that were below the cutoff value were included in the analysis as half of the cutoff value.