Recognition of the SARS-CoV-2 nucleoprotein by VHHs and development of a sandwich immunoassay

Coronaviruses are enveloped viruses with a positive-sense RNA genome and with a nucleocapsid of helical symmetry. Coronavirus nucleoproteins (N) localize to the cytoplasm and the nucleolus and is required for viral RNA synthesis. During virion assembly, N protein binds to the viral RNA and leads to a helical nucleocapsid formation. The current situation of the Covid-19 pandemic shows the importance of obtaining reliable solutions for the rapid detection of SARS-CoV-2 and the determination of the immunity of the population for the virus. Several solutions have been developed in record time for diagnosis. Regarding diagnosis, the reference method remains the RT-PCR (Reverse Transcriptase Polymerase Chain Reaction) which allows to detect the RNA of the SARS-Cov-2. This is an expensive method, requiring the transport of the sample to well-equipped laboratory and qualified personnel to carry out the analyzes. It is therefore necessary for diagnostic tests to be widely available and accessible highlighting the importance to develop an antibody-based assay. Nucleocapsid is the most abundant coronavirus protein and it is of upmost importance to develop specific antibodies to detect this protein in a diagnostic test. We immunized one alpaca with the recombinantNprotein of SARS-CoV2. After phage display selection, we identified 6 VHHs recognizing the C terminal domain (CTD) and 2 VHHs the N terminal domain. All VHHs have an affinity in the nanomolar range and can bind on ELISA the N extracted from infected cells. These VHHs are also able to recognize SARS-CoV-2 virus in infected cells or on infected hamster tissues. An ELISA sandwich assay has been set up using one anti NTD and one anti-CTD VHHs to detect the nucleoprotein in solution. This assay has allowed us to detect as little as 20 ng/ml of N.