SARS-CoV-2 Neutralization Assay in VeroE6 Cells

The day before infection, VeroE6 cells were seeded at 10⁴ cells/well into 96-well plates. Plasma samples and antibodies were serial diluted in BA-1, consisting of medium 199 (Lonza) supplemented with 1% BSA and 1× penicillin/streptomycin. Next, the diluted samples were mixed with a constant amount of SARS-CoV-2 and incubated for 60 min at 37°C. The plasma/antibody/virus mix was then directly applied to VeroE6 cells (MOI of ∼0.1 PFU/cell; n = 3) and incubated for 18–20 h at 37°C. This amount of virus gave 30% to 50% infected cells at the end of the assay. Cells were subsequently fixed by adding an equal volume of 7% formaldehyde to the wells, followed by permeabilization with 0.1% Triton X-100 for 10 min. After extensive washing, cells were incubated for 1 h at RT with blocking solution of 5% goat serum in PBS (Jackson ImmunoResearch; catalog no. 005–000-121). A rabbit polyclonal anti-SARS-CoV-2 nucleocapsid antibody (GeneTex; catalog no. GTX135357) was added to the cells at 1:500 dilution in blocking solution and incubated at 4°C overnight. Alternatively, J2, a mouse monoclonal anti-dsRNA antibody (Scicons; catalog no. 10010500) was added to the cells under similar conditions to detect virus infected cells. Goat anti-rabbit Alexa Fluor 594 (Life Technologies; catalog no. A-11012) and goat anti-mouse Alexa Fluor 488 (Life Technologies; catalog no. A-11001) were used as a secondary antibodies at a dilution of 1:2,000. Nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific; catalog no. 62249) at a 1:1,000 dilution. Images were acquired with a fluorescence microscope and analyzed using ImageXpress Micro XLS (Molecular Devices). Using uninfected cells as a control, we estimated that infection could be reliably quantified if ∼0.7% of cells or more were infected.