Assessing Functional SARS-CoV-2 Neutralizing Antibodies
Published: February 29, 2024
Abstract
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 …
Divulgaciones
The authors have nothing to disclose.
Materials
| 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 |
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| 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 |
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Citar este artículo
Assessing Functional SARS-CoV-2 Neutralizing Antibodies. J. Vis. Exp. (Pending Publication), e22211, doi: (2024).