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

Marion Gransagne 1 Gabriel Aymé 2 Sébastien Brier 3 Gaëlle Chauveau-Le Friec 2 Véronique Meriaux 2 Mireille Nowakowski 4 François Déjardin 4 Sylvain Levallois 5 Guilherme Dias de Melo 6 Flora Donati 7, 8 Matthieu Prot 9 Sébastien Brûlé 10 Bertrand Raynal 10 Jacques Bellalou 4 Pedro Goncalves 11 Xavier Montagutelli 12 James P Di Santo 11 Françoise Lazarini 13 Patrick England 10 Stéphane Petres 4 Nicolas Escriou 1 Pierre Lafaye 2, * 
Abstract : 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.
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Submitted on : Thursday, January 13, 2022 - 3:39:56 PM
Last modification on : Friday, August 5, 2022 - 12:03:13 PM


VHH anti SARS-CoV2 nucleoprote...
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Marion Gransagne, Gabriel Aymé, Sébastien Brier, Gaëlle Chauveau-Le Friec, Véronique Meriaux, et al.. Development of a highly specific and sensitive VHH-based sandwich immunoassay for the detection of the SARS-CoV-2 nucleoprotein. Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2022, 298 (1), pp.101290. ⟨10.1016/j.jbc.2021.101290⟩. ⟨pasteur-03524979⟩



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