Assessing Functional SARS-CoV-2 Neutralizing Antibodies

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 …

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