Assessing Functional Neutralizing Antibody Responses Against a Panel of SARS-CoV-2 Variants of Concern Using Standardized, Quantitative Pseudotype Neutralization Assays

To begin, add 50 microliters of DMEM with 10 percent FBS and 1 percent Penicillin-Streptomycin to rows B to H, columns 1 to 12 of a white 96-well plate.

Add 5 microliters of convalescent sera into wells of row A, columns 2 to 10, in a total volume of 100 microliters DMEM with 10 percent FBS and 1 percent Penicillin-Streptomycin. Add 5 microliters of positive and negative antisera into wells A11 and A12 as controls.

Remove 50 microliters of the solution from the wells of row A and perform two-fold serial dilutions of all the wells beneath it. For each dilution, use a multichannel pipette to mix the solutions 5-10 times by pipetting up and down without creating air bubbles.

After completing the serial dilution, discard the final 50 microliters from the last well of each column.

Centrifuge the plate for 1 minute at 500 rotations per minute to ensure no liquid is left on the walls of the well. Calculate the amount of DMEM required to dilute the SARS-CoV-2 PV as described in the text manuscript.

Mix the diluted PV solution in a pipette basin and aliquot 50 microliters into each well, except for wells A6 to A12 which comprise the cell-only control. The A1 to A6 wells serve as PV-only control.

Centrifuge plate for 1 minute at 500 rotations per minute to ensure no virus is left on the walls of the well. Then, incubate the plates at 37 degrees Celsius with 5 percent carbon dioxide for 1 hour to allow the serum to bind with the SARS-CoV-2 glycoprotein.

Next, prepare a plate of HEK 293T/17 target cells transfected 24 hours earlier with ACE2 and TMPRSS2 plasmids. Remove the culture media from the plate, and wash it twice with 2 milliliters of PBS, by adding it to one side of the dish to avoid cell detachment.

Discard the PBS and add 2 milliliters of trypsin to the plate. Place the plate in an incubator until the cells are detached.

After cells have detached, add 6 milliliters of DMEM with 10 percent FBS and 1 percent Penicillin-Streptomycin to the plate to quench trypsin activity, and resuspend cells gently.

Count the cells using an automated cell counter or by counting the chamber slide. Add 1 x 10⁴ cells aliquoted in a total volume of 50 microliters to each well and incubate the plate at 37 degrees Celsius with 5 percent carbon dioxide for 48 to 72 hours.

After incubation, remove the culture media from all wells, and add 25 microliters of a 1:1 mix of PBS and a commercial luciferase assay reagent. Read the plate using a commercial luciferase assay system with a microplate luminometer.

Using the raw data generated by the luminometer, calculate the IC50 neutralizing antibody titers as described in the text manuscript.