Pseudotyped virus neutralization assay

Fourfold serially diluted plasma from individuals convalescent from COVID-19, or monoclonal antibodies, were incubated with SARS-CoV-2 pseudotyped virus for 1 h at 37 °C. The mixture was subsequently incubated with 293T_ACE2 cells for 48 h, after which cells were washed with PBS and lysed with Luciferase Cell Culture Lysis 5× reagent (Promega). Nanoluc luciferase activity in lysates was measured using the Nano- Glo Luciferase Assay System (Promega) with the Glomax Navigator (Promega). The obtained relative luminescence units were normalized to those derived from cells infected with SARS-CoV-2 pseudotyped virus in the absence of plasma or monoclonal antibodies. The half-maximal inhibitory concentration for plasma (NT₅₀) or monoclonal antibodies (IC₅₀) was determined using four-parameter nonlinear regression (least squares regression method without weighting; constraints: top = 1, bottom = 0) (GraphPad Prism).