Assessing Functional SARS-CoV-2 Neutralizing Antibodies

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Pseudotype-based neutralization assay

NOTE: pMN is the Inhibition of pseudotype virus (PV) mediated transduction via human convalescent serum samples that contain antibodies directed against the SARS-CoV-2 Spike.

1. In a 96-well white plate with the aid of a multichannel pipette, add 50 µl of Dulbecco's Modified Eagle Medium (DMEM),10% fetaval bovine serum (FBS), 1% penicillin/streptomycin (Pen/Strep) to rows B to H, columns 1-12.
2. Add 5 µl convalescent sera into wells of row A, columns 2-10, in a total volume of 100 µl DMEM/10% FBS/1% P/S. Add a known amount (e.g., 5 µl) of positive (NIBSC 20/162 calibrant, HICC-pool 2 or HICC pool 3) and negative antisera (patient sera taken pre-2019) into wells A11 and A12 as controls.
3. Remove 50 µl from row A wells and perform two-fold serial dilutions down all the wells beneath it.
4. With each dilution step, use a multichannel pipette to mix 5-10 times by pipetting up and down and taking care not to produce air bubbles.
5. After completing the serial dilution, the final 50 µl from the final well of each column should be discarded.
   NOTE: At this point, each well should contain 50 µl of mixed DMEM and serial dilutions of patient sera or control sera.
6. Centrifuge plate for 1 min at 500 rpm to ensure no liquid is left on the walls of the well.
7. Using data obtained via pseudotype titration, calculate the amount of DMEM required to dilute your SARS-CoV-2 PV to obtain 5 x 10⁶ RLU in 50 µl, with a total volume of 5 ml.
8. Mix this diluted PV solution in a pipette basin using the multichannel pipette, and aliquot 50 µl into each well on the plate, except for wells A6-A12 (cell only control). A1-A6 will serve as PV-only control.
9. Centrifuge plate for 1 min at 500 rpm to ensure no virus is left on the walls of the well.
10. Incubate the plates for 1 h at 37 °C 5% carbon dioxide (CO₂), allowing time for the serum to bind the SARS-CoV-2 glycoprotein.
11. Prepare a plate of HEK 293T/17 target cells transfected 24 h before with angiotensin-converting enzyme 2 (ACE2) expression plasmid and transmembrane serine protease 2 (TMPRSS2) expression plasmid plasmids.
    1. Remove culture media from the plate.
    2. Wash the plate twice with 2 ml of PBS on one side of the dish to avoid cell detachment and discard.
    3. Add 2 ml of trypsin to the plate and put the plate into the incubator until the cells are detached.
    4. After cells have detached, add 6 ml of DMEM/10% FBS/1% penicillin/streptomycin to the plate to quench trypsin activity and resuspend cells gently.
    5. Count cells using TC20^TM Automated Cell Counter or counting-chamber slide and add 1 x 10⁴ cells in a total volume of 50 µl to each well.
12. Incubate the plate for 48-72 h, at 37 °C, 5% CO₂.
13. Read the plate using the Bright Glo^TM luciferase assay system on a GloMax® Navigator Microplate Luminometer by removing the culture medium from all wells and adding 25 µl of a 1:1 mix of PBS: Bright Glo^TM luciferase assay reagent.
14. From the raw data provided by the luminometer, calculate the half maximal inhibitory concentration (IC50) neutralizing antibody titers using established protocols. IC50 values below 1:40 dilution are considered negative. Data may alternatively be evaluated using AutoPlate.