Surface Plasmon Resonance (SPR) Biosensor for the Detection of SARS-CoV-2 Using Autodisplyaed FV-antibodies on Outer Membrane of E. coli

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (NP) participates in viral genome packaging and abundantly produced when infected. In this work, SPR biosensor for the detection of SARS-CoV-2 in viral fluid using F-v-antibodies with the binding affinity to nucleocapsid protein (NP) of SARS-CoV-2. The F-V-antibodies with a specific binding activity to the SARS-CoV-2 NP were screened using the F-V-antibody library, which was expressed on the outer membrane of E. coli. F-V-antibodies comprised three complementarity-determining regions (CDRs) and four frame regions (FRs) of the heavy chain at the binding pocket of IgG. The FV-antibody library was prepared by performing site-directed mutagenesis and by using the autodisplay technology; FV-antibodies with specific binding activities to the nucleocapsid protein (NP) of SARS-CoV-2 were screened using NP-immobilized magnetic beads. First, E. coli isolates with the target F-V-antibody were screened, and the binding affinity (K-D) was estimated for the screened E. coli clones using FACS analysis. Then, the outer membrane (OM) of the screened E. coli clones with autodisplayed F-v-antibodies was obtained and layered on an SPR biosensor, and the binding curves of four different coronavirus (CoV) culture fluids, SARS-CoV-2, SARS-CoV, MERS-CoV, and CoV strain 229E, were compared. Finally, the F-V-antibodies of the screened E. coli clones were synthesized as peptides (11 amino acid residues), and the binding constants (K-D) to NP as well as the binding curves of the CoV strains in culture fluids were estimated. Using docking simulation, binding sites and interaction types between NP and each synthetic peptide were investigated. [Graphics]