SARS-CoV-2 Neutralization Assay with Pseudoviruses

Virus neutralization assays were performed to test plasma and MAb neutralization of PSVs. Plasma samples were prepared by heat inactivation at 56°C for 45 min, followed by 5-min centrifugation at 2,000 rpm to remove particulates. Plasma samples were used at a starting dilution of 1:20 in supplemented DMEM; MAbs were used at a starting concentration of 25 μg/mL in supplemented DMEM. Viruses were diluted to a concentration to yield luminescence at least three times above the cell-only controls, as determined by the PSV titration. Neutralization reagents were serially diluted 1:4 in independent duplicates per plate and incubated with equal volumes of virus for 1 h at 37°C before adding TZM-bl cells with 40 μg/mL of DEAE-dextran (Sigma, St. Louis, MO, USA). The plates were incubated at 37°C for 48 h before reading the luminescence. Nonspecific neutralization by plasma was evaluated using MuLV env DNA pseudotyped with the pSG3Δenv backbone DNA. Uninfected normal human serum was also tested against all the PSVs as a negative control.