One-Step Immunoassay for the Detection of SARS-CoV-2 Nucleocapsid Protein Using Screened Fv-Antibodies

Abstract

Fv-antibodies against the nucleocapsid protein (NP) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were screened from an Fv-antibody library, and a one-step immunoassay was performed to detect SARS-CoV-2 using real viral samples. The Fv-antibody library was prepared using site-directed mutagenesis of the CDR3 region, which was composed of 11 amino acids. To screen the target Escherichia coli from the Fv-antibody library, the expressed probes [N-terminal domain (NTD) labeled with GFP and C-terminal domain (CTD) labeled with GFP] were reacted separately with the Fv-antibody library. After oligonucleotide sequencing, two clones for each probe were selected as the final clones. The screened Fv-antibodies with the binding affinity to NTD (or CTD) were expressed as soluble proteins, and the affinity constant (KD) was calculated to be 25.4 nM for NTD and 26.9 nM for CTD. The expressed Fv-antibodies were used for the one-step immunoassay based on switching-peptides, which were bound to the expressed Fv-antibodies. The one-step immunoassay based on Fv-antibodies could be used for the linear detection of SARS-CoV-2 NP, and the limit of detection (LOD) was estimated to be 9.6 nM (438 ng/mL) for Anti-NTD and 14.1 nM (639 ng/mL) for Anti-CTD. For the demonstration of one-step immunoassay for SARS-CoV-2, NATtrol (TM) SARS-CoV-2 real sample was used, and the LOD was estimated to be 29.7 copies/mL (Ct = 39.5) using Anti-NTD and 117.8 copies/mL (Ct = 38.0) using Anti-CTD. The measured LOD for the detection of SARS-CoV-2 using a one-step immunoassay based on the switching-peptide was considered feasible for the medical diagnosis of COVID-19. Finally, the interaction between the screened Fv-antibodies and SARS-CoV-2 NP was investigated using docking simulation.