Nanoluciferase-based cell fusion assay for rapid and high-throughput assessment of SARS-CoV-2-neutralizing antibodies in patient samples

Max Meyrath, Martyna Szpakowska, Jean Marc Plesseria, Olivia Domingues, Jérémie Langlet, Bernard Weber, Rejko Krüger, Markus Ollert, Andy Chevigné*

*Corresponding author for this work

Research output: Contribution to journalArticleResearchpeer-review

Abstract

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.

Original languageEnglish
Pages (from-to)351-381
Number of pages31
JournalMethods in Enzymology
Volume675
Early online date9 Sep 2022
DOIs
Publication statusPublished - 2022

Keywords

  • Cell fusion
  • COVID
  • HiBiT
  • High-throughput
  • Luciferase
  • NanoBiT
  • NanoLuc
  • Neutralizing antibodies
  • SARS-CoV-2
  • Syncytium
  • Virus neutralization assay

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