SARS-CoV-2 MA pseudotyped virus neutralization assay

A 40-µg/ml initial dilution of mAbs was fourfold serially diluted over 11 dilutions. 55 µl of antibody dilutions was incubated with a 55-µl aliquot of SARS-CoV-2 MA pseudotyped virus (containing ∼10³ infectious units) for 1 h at 37°C. 100 µl of the mixture was subsequently incubated with HT1080_muAce2 cells for 48 h. Consequently, the resulting antibody starting dilution was 10 µg/ml, and each well received 50 µl virus. Following incubation, cells were washed with PBS and lysed with Luciferase Cell Culture 5× reagent (Promega), and nanoluc luciferase activity in cell lysates was measured using the Nano-Glo Luciferase Assay System (Promega). RLUs obtained were normalized to those derived from cells infected with SARS-CoV-2 MA pseudovirus in the absence of mAbs (equivalent to 0% neutralization). The half-maximal and 90% ICs (IC₅₀ and IC₉₀) for mAbs were determined using four-parameter nonlinear regression (GraphPad Prism). In detail, we applied the least squares regression method without weighting, with top and bottom values constrained to 0 (% neutralization) and 100 (% neutralization), respectively, while allowing for a variable HillSlope.