Development of a highly specific and sensitive VHH-based sandwich immunoassay for the detection of the SARS-CoV-2 nucleoprotein

The current COVID-19 pandemic illustrates the importance of obtaining reliable methods for the rapid detection of SARS-CoV-2. A highly specific and sensitive diagnostic test able to differentiate the SARS-CoV-2 virus from common human coronaviruses is therefore needed. Coronavirus nucleoprotein (N)...

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Published in:The Journal of biological chemistry 2022-01, Vol.298 (1), p.101290-101290, Article 101290
Main Authors: Gransagne, Marion, Aymé, Gabriel, Brier, Sébastien, Chauveau-Le Friec, Gaëlle, Meriaux, Véronique, Nowakowski, Mireille, Dejardin, François, Levallois, Sylvain, Dias de Melo, Guilherme, Donati, Flora, Prot, Matthieu, Brûlé, Sébastien, Raynal, Bertrand, Bellalou, Jacques, Goncalves, Pedro, Montagutelli, Xavier, Di Santo, James P., Lazarini, Françoise, England, Patrick, Petres, Stéphane, Escriou, Nicolas, Lafaye, Pierre
Format: Article
Language:English
Subjects:
Animals
antibody engineering
Biochemistry
Biochemistry, Molecular Biology
COVID-19
Cricetinae
diagnostic
Electrophoresis, Polyacrylamide Gel
Enzyme-Linked Immunosorbent Assay - methods
Humans
hydrogen-deuterium exchange
immunochemistry
Life Sciences
Limit of Detection
nanobodies
Nucleocapsid Proteins - analysis
Nucleocapsid Proteins - immunology
nucleoprotein
phage display
SARS-CoV-2 - immunology
single domain antibodies
Single-Domain Antibodies - immunology
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Summary:The current COVID-19 pandemic illustrates the importance of obtaining reliable methods for the rapid detection of SARS-CoV-2. A highly specific and sensitive diagnostic test able to differentiate the SARS-CoV-2 virus from common human coronaviruses is therefore needed. Coronavirus nucleoprotein (N) localizes to the cytoplasm and the nucleolus and is required for viral RNA synthesis. N is the most abundant coronavirus protein, so it is of utmost importance to develop specific antibodies for its detection. In this study, we developed a sandwich immunoassay to recognize the SARS-CoV-2 N protein. We immunized one alpaca with recombinant SARS-CoV-2 N and constructed a large single variable domain on heavy chain (VHH) antibody library. After phage display selection, seven VHHs recognizing the full N protein were identified by ELISA. These VHHs did not recognize the nucleoproteins of the four common human coronaviruses. Hydrogen Deuterium eXchange–Mass Spectrometry (HDX-MS) analysis also showed that these VHHs mainly targeted conformational epitopes in either the C-terminal or the N-terminal domains. All VHHs were able to recognize SARS-CoV-2 in infected cells or on infected hamster tissues. Moreover, the VHHs could detect the SARS variants B.1.17/alpha, B.1.351/beta, and P1/gamma. We propose that this sandwich immunoassay could be applied to specifically detect the SARS-CoV-2 N in human nasal swabs.
ISSN:0021-9258
1083-351X
DOI:10.1016/j.jbc.2021.101290