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

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 (K_D) 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™ 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.