Neutralization titers to SARS-CoV-2 were determined as previously described (15 (link), 29 (link)). Briefly, 100 plaque-forming units of SARS-CoV-2 (2019-nCoV/USA_WA1/2020), Alpha, Beta, Gamma, Delta, and Omicron (BA.1, BA.2, BA.2.12.1, BA.4, and BA.5) was used on Vero-TMPRSS2 cells. The GISAID (global initiative on sharing all influenza data) IDs of Omicron live viral isolates used in neutralization assays are EPI_ISL_7171744 (BA.1), EPI_ISL_8818457 (BA.2), EPI_ISL_11685455 (BA.2.12.1), EPI_ISL_12416220 (BA.4), and EPI_ISL_13512579 (BA.5). Purified mAb was serially diluted threefold in duplicate starting at 10 μg/ml concentration in a 96-well round-bottom plate and incubated for 1 hour at 37°C. This antibody-virus mixture was transferred into the wells of a 96-well plate that had been seeded with Vero-TMPRSS2 cells the previous day at a concentration of 2.5 × 104 cells per well. After 1 hour, the antibody-virus inoculum was removed, and 0.85% methylcellulose in 2% fetal bovine serum (FBS) containing Dulbecco’s minimal essential medium was overlaid onto the cell monolayer. Cells were incubated at 37°C for 16 to 40 hours. Cells were washed three times with 1× PBS (Corning cellgro) and fixed with 125 μl of 2% paraformaldehyde in PBS (Electron Microscopy Sciences) for 30 min. Following fixation, plates were washed twice with PBS, and 100 μl of permeabilization buffer was added to the fixed cells for 20 min. Cells were incubated with an anti–SARS-CoV spike primary antibody directly conjugated with Alexa Fluor 647 (CR3022-AF647) for up to 4 hours at room temperature.
Plates were then washed twice with 1× PBS and imaged on an ELISPOT reader (CTL Analyzer). Foci were counted using Viridot (counted first under the “green light” setting followed by background subtraction under the “red light” setting). IC50 titers were calculated by nonlinear regression analysis using the 4PL (four parameter logic) sigmoidal dose curve equation on Prism 9 (GraphPad Software). Neutralization titers were calculated as 100% × [1 − (average foci in duplicate wells incubated with the specimen) ÷ (average number of foci in the duplicate wells incubated at the highest dilution of the respective specimen)].
Plates were then washed twice with 1× PBS and imaged on an ELISPOT reader (CTL Analyzer). Foci were counted using Viridot (counted first under the “green light” setting followed by background subtraction under the “red light” setting). IC50 titers were calculated by nonlinear regression analysis using the 4PL (four parameter logic) sigmoidal dose curve equation on Prism 9 (GraphPad Software). Neutralization titers were calculated as 100% × [1 − (average foci in duplicate wells incubated with the specimen) ÷ (average number of foci in the duplicate wells incubated at the highest dilution of the respective specimen)].
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