Assessing Functional SARS-CoV-2 Neutralizing Antibodies

프로토콜

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.

자료

Name	Company	Catalog Number	Comments
MultiGuard Barrier pipette tips 1-20 μl	Sorenson BioScience	30550T
MultiGuard Barrier pipette tips 1-200 μl	Sorenson BioScience	30550T
NuncTM Cell Culture Dish Delta Surface Treated (10 cm sterile dishes)	Thermo Fisher Scientific	150350
Pipette basins (50 ml)	Generic
96-well white plate	Thermo FisherScientific	136101
HEK 293T/17 cells	ATCC	CRL-11268
Host cell entry receptor angiotensin-converting enzyme 2 (ACE2) expression plasmid: pCDNA3.1+-ACE2			Simmons 2004
Coronavirus Spike (S) protein priming transmembrane serine protease 2 (TMPRSS2) expression plasmid: pCAGGS-TMPRSS2			Bertram 2010
Dulbecco's modified Eagle medium (DMEM) with 4.5 g/L Glucose, supplemented with 10% foetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S)	Pan-Biotech	P04-04510 P40-37500 P06-07100
FuGENE® HD Transfection Reagent, 1ml	Promega	E2311
Trypsin-EDTA (0.05%), phenol red	Pan-Biotech	P10-040100
Positive control antibody (Research Reagent for Anti-SARS-CoV-2 Ab) that can neutralise the SARS-CoV-2 PV (NIBSC, code: 20/162, available internationally or HICC-pool 2/HICC pool 3)
COVID-19 human convalescent plasma to test
Bright Glo luciferase assay system	Promega	E2650