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
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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Assessing Functional SARS-CoV-2 Neutralizing Antibodies. J. Vis. Exp. (Pending Publication), e22211, doi: (2024).

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