The use of nanobodies in a sensitive ELISA test for SARS-CoV-2 Spike 1 protein

Georgina C. Girt, Abirami Lakshminarayanan, Jiandong Huo, Joshua Dormon, Chelsea Norman, Babak Afrough, Adam Harding, William James, Raymond J. Owens*, James H. Naismith

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

1 Citation (Scopus)

Abstract

Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens in the fluid has important uses in biotechnology, and is integral to many point-of-care SARS-CoV-2 diagnostics. Sandwich enzyme-linked immunosorbent assays (ELISAs) are a sensitive, well-established method of measuring antigens in solutions. They use one ligand to capture and the other ligand to detect the target analyte. Detection is commonly achieved using colorimetric readout obtained upon the reaction of a substrate with HRP-conjugated secondary ligand. Nanobodies, the V H H domain of camelid antibodies, have expanded the repertoire of molecules used in antigen detection. Nanobodies' high affinity for target antigens, their compact structure, their high stability and ease of production has driven research into their use as diagnostic reagents. Guided by a structural understanding of epitopes on the receptor-binding domain of the SARS-CoV-2 Spike protein, we investigated various combinations of engineered nanobodies in a sandwich ELISA to detect the Spike protein of SARS-CoV-2. We have identified an optimal combination of nanobodies. These were selectively functionalized to further improve antigen capture, enabling the measurement of sub-picomolar amounts of SARS-CoV-2 Spike protein in solution. With this combination, the routine detection limit in samples inactivated by heat and detergent corresponded to less than seven focus-forming units of infectious SARS-CoV-2.

Original languageEnglish
Article number211016
JournalRoyal Society Open Science
Volume8
Issue number9
DOIs
Publication statusPublished - 30 Sep 2021

Bibliographical note

Funding Information: This work was support by the Rosalind Franklin Institute funding delivery partner EPSRC, Wellcome Trust (grant no. 100209/Z/12/Z) and Biological Sciences Research Council (BBSRC; BB/T006161/1). Containment level 3 experiments were funded through the generous support of philanthropic donors to the University of Oxford's COVID-19 Research Response Fund.

Open Access: Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits
unrestricted use, provided the original author and source are credited.

Publisher Copyright: © 2021 The Authors.

Citation: Girt GC et al. 2021 The use of nanobodies in a sensitive ELISA test for SARS-CoV-2 Spike 1 protein. R. Soc. Open Sci. 8: 211016.

DOI: https://doi.org/10.1098/rsos.211016

Keywords

  • ELISA
  • nanobody
  • SARS-CoV-2
  • site-specific conjugation
  • Spike

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