Article (Scientific journals)
Nanoluciferase-based cell fusion assay for rapid and high-throughput assessment of SARS-CoV-2-neutralizing antibodies in patient samples.
Meyrath, Max; Szpakowska, Martyna; Plesseria, Jean-Marc et al.
2022In Methods in Enzymology, 675, p. 351-381
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Keywords :
Angiotensin-Converting Enzyme 2; Antibodies, Neutralizing; Antibodies, Viral; COVID-19; Cell Fusion; Humans; Luciferases; Neutralization Tests/methods; Peptidyl-Dipeptidase A/metabolism; SARS-CoV-2; Spike Glycoprotein, Coronavirus/metabolism; COVID; HiBiT; High-throughput; Luciferase; NanoBiT; NanoLuc; Neutralizing antibodies; Syncytium; Virus neutralization assay
Abstract :
[en] After more than two years, COVID-19 still represents a global health burden of unprecedented extent and assessing the degree of immunity of individuals against SARS-CoV-2 remains a challenge. Virus neutralization assays represent the gold standard for assessing antibody-mediated protection against SARS-CoV-2 in sera from recovered and/or vaccinated individuals. Neutralizing antibodies block the interaction of viral spike protein with human angiotensin-converting enzyme 2 (ACE2) receptor in vitro and prevent viral entry into host cells. Classical viral neutralization assays using full replication-competent viruses are restricted to specific biosafety level 3-certified laboratories, limiting their utility for routine and large-scale applications. We developed therefore a cell-fusion-based assay building on the interaction between viral spike and ACE2 receptor expressed on two different cell lines, substantially reducing biosafety risks associated with classical viral neutralization assays. This chapter describes this simple, sensitive, safe and cost-effective approach for rapid and high-throughput evaluation of SARS-CoV-2 neutralizing antibodies relying on high-affinity NanoLuc® luciferase complementation technology (HiBiT). When applied to a variety of standards and patient samples, this method yields highly reproducible results in 96-well, as well as in 384-well format. The use of novel NanoLuc® substrates with increased signal stability like Nano-Glo® Endurazine™ furthermore allows for high flexibility in assay set-up and full automatization of all reading processes. Lastly, the assay is suitable to evaluate the neutralizing capacity of sera against the existing spike variants, and potentially variants that will emerge in the future.
Disciplines :
Author, co-author :
Meyrath, Max
Szpakowska, Martyna ;  University of Luxembourg
Plesseria, Jean-Marc
Domingues, Olivia
Langlet, Jérémie
Weber, Bernard
Krüger, Rejko ;  University of Luxembourg > Luxembourg Centre for Systems Biomedicine (LCSB) > Translational Neuroscience
Ollert, Markus ;  University of Luxembourg
Chevigné, Andy ;  University of Luxembourg > Faculty of Science, Technology and Communication (FSTC)
External co-authors :
Language :
Title :
Nanoluciferase-based cell fusion assay for rapid and high-throughput assessment of SARS-CoV-2-neutralizing antibodies in patient samples.
Publication date :
09 September 2022
Journal title :
Methods in Enzymology
Publisher :
Elsevier, United States
Volume :
Pages :
Peer reviewed :
Peer Reviewed verified by ORBi
Focus Area :
Systems Biomedicine
Funders :
Commentary :
Copyright © 2022 Elsevier Inc. All rights reserved.
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