Overview
This video showcases a neutralization assay measuring serum efficacy in neutralizing SARS-CoV-2 viruses. It involves pseudotyped viruses, serum dilution, and luminescence-based quantification, revealing the neutralization titer.
Protocol
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.
- 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.
- 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.
- Remove 50 µl from row A wells and perform two-fold serial dilutions down all the wells beneath it.
- 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.
- 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. - Centrifuge plate for 1 min at 500 rpm to ensure no liquid is left on the walls of the well.
- 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.
- 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.
- Centrifuge plate for 1 min at 500 rpm to ensure no virus is left on the walls of the well.
- 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.
- 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.
- Remove culture media from the plate.
- Wash the plate twice with 2 ml of PBS on one side of the dish to avoid cell detachment and discard.
- Add 2 ml of trypsin to the plate and put the plate into the incubator until the cells are detached.
- 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.
- Count cells using TC20TM Automated Cell Counter or counting-chamber slide and add 1 x 104 cells in a total volume of 50 µl to each well.
- Incubate the plate for 48-72 h, at 37 °C, 5% CO2.
- Read the plate using the Bright GloTM 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 GloTM luciferase assay reagent.
- 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.
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Materials
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 |