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.
【저자키워드】 COVID-19, diagnostic, Immunochemistry, nanobodies, phage display, nucleoprotein, antibody engineering, hydrogen-deuterium exchange, single domain antibodies, CTD, C-terminal domain of the nucleoprotein, HDX-MS, hydrogen deuterium exchange–mass spectrometry, N, nucleoprotein, NTD, N-terminal domain of the nucleoprotein, 【초록키워드】 SARS-CoV-2, antibody, COVID-19 pandemic, variant, ELISA, immunoassay, N protein, SARS-CoV-2 N protein, epitope, human coronaviruses, Analysis, cytoplasm, hydrogen, tissues, domains, domain, viral RNA synthesis, nasal swabs, infected cell, N-terminal, Nucleolus, immunized, Seven, coronavirus protein, human coronavirus, detect, develop, specific antibody, required, recognize, conformational, recognizing, C-terminal, Spectrometry, the SARS-CoV-2 virus, 【제목키워드】 immunoassay, development, the SARS-CoV-2,