SARS-CoV-2 Neutralization Assay Development

We developed a sensitive SARS-CoV-2 neutralization assay^{15 (link)} by incorporating WT SARS-CoV-2 spike protein or with N501Y mutation, representing the current prevalent B.1.1.7 (alpha variant) mutation in the United States, onto lentiviruses and measuring pseudoviral entry into angiotensin converting enzyme 2 (ACE2) overexpressing 293 cells (293-ACE2). To determine the half-maximal inhibitory concentration (NT50), 3-fold serially diluted serum samples from different patients were incubated with red fluorescent protein-encoding WT SARS-CoV-2 or alpha variant pseudotyped virus at 0.2 multiplicity of infection (MOI) for 1 hour at 37°C. The mixture was subsequently incubated with 293-ACE2 cells for 72 hours, after which cells were collected, washed with FACS buffer (1 × phosphate buffered saline + 2% fetal bovine serum) and analyzed by flow cytometry using BD FACSymphony A5 analyzer. Percent infection obtained was normalized for samples derived from cells infected with WT SARS-CoV-2 or alpha variant pseudotyped virus in the absence of serum. The NT50 was determined using 4-parameter nonlinear regression (GraphPad Prism 8.0).