SARS-CoV-2 Pseudotyped Virus Neutralization Assay

SARS-CoV-2 pseudotyped particles were produced by cotransfection of pSARS-CoV-2 S_trunc and pNL4-3ΔEnv-nanoluc in 293T cells (11 (link), 34 (link)). Fourfold serially diluted purified plasma IgG/IgA from COVID-19 convalescent individuals and healthy donors or monoclonal antibodies were incubated with the SARS-CoV-2 pseudotyped virus for 1 hour at 37°C. Subsequently, the mixture was incubated with Ace2-expressing cells for 48 hours. HT1080_Ace2 cl. 14 cells (34 (link)) were used for plasma-derived IgG or IgA assays, and 293T_Ace2 cells (11 (link)) were used for monoclonal antibody assays. After incubation, cells were washed twice with PBS and lysed with Luciferase Cell Culture Lysis 5× Reagent (E1531; Promega). NanoLuc Luciferase activity in lysates was measured using the Nano-Glo Luciferase Assay System (N1150; Promega) with the GloMax Navigator Microplate Luminometer (Promega). Relative luminescence units obtained were normalized to those derived from cells infected with SARS-CoV-2 pseudotyped virus in the absence of plasma-derived or monoclonal antibodies. The half-maximal and 90% inhibitory concentrations for purified plasma IgG or IgA or monoclonal antibodies (IC₅₀ and IC₉₀) were determined using four-parameter nonlinear regression (GraphPad Prism).