Neutralization of SARS-CoV-2 Pseudoviruses

Neutralization activity of IgG1 Fc domain variants was measured as previously described^{35 (link)}. Briefly, HT1080_ACE2 cells were seeded in 96 U-well black plates 24 h prior to infection with SARS-CoV-2_WT, SARS-CoV-2_MA10 or SARS-CoV-2_B.1.351 pseudoviruses. Pseudovirus particles were pre-incubated with mAbs (four-fold serially diluted starting at 10 μg/ml) for 1 h at 37°C and then added to a monolayer of HT1080_ACE2 cells. Following a 48-h incubation at 37°C, cells were carefully washed with PBS and lysed with Luciferase Cell Culture Lysis reagent (Promega) for 15 min. Nano luciferase activity was detected by adding Nano-Glo Luciferase Assay System (Promega) and measured by SpectraMax Plus spectrophotometer (Molecular Devices), using 0.5 s integration time. Data were collected and analyzed using SoftMax Pro v.7.0.2 software (Molecular Devices). Relative luciferase units were normalized to those derived from cells infected with the relevant SARS-CoV-2 pseudoviruses in the absence of mAbs.