One-step immunoassay of SARS-CoV-2 using screened Fv-antibodies and switching peptides

Abstract

The Fv-antibodies were correponded to VH region of immunoglobulin G, which were composed of three complementarity determining regions (CDRs) for the specific binding of antigens. In this work, the Fv-antibodies against SARS-CoV-2 spike protein (SP) were screened from an autodisplayed Fv-antibody library which was expressed on E. coli outer membrane, and the receptor binding domain (RBD) of SP was used as a screening probe. The screened target clones were analyzed to have quantitative binding properties to the RBD, and the Fv-antibodies from the screened target clones were expressed as soluble proteins. The binding affinity (KD) of expressed Fv-antibodies to the RBD was estimated to be 70–85 nM using SPR biosensor. The specific binding properties of Fv-antibodies were analyzed for pseudo-virus particles with SARS-CoV-2 SP on the Lenti-virus envelope, such as wild type (Wuhan-1) and variants (Delta, Omicron BA.2, Omicron BA.4/5) using a SPR biosensor. The detection of real SARS-CoV-2 (Wild type, Wuhan-1) based on a SPR biosensor was also presented using the Fv-antibodies with the binding constant (KD) of cycle threshold value (Ct) = 33.8–32.9 (2.19–4.08 copies/μL) and LOD of 0.67–0.83 copies/μL (Ct = 35.5–35.2). Finally, one-step immunoassay based on switching peptide was demonstrated for the detection of the real SARS-CoV-2 (Wuhan-1) without any washing step. The binding constant (KD) was estimated to be Ct = 35.2–33.9 (0.83–2.04 copies/μL), and LOD was estimated to be 0.14–0.47 copies/μL (Ct = 37.8–36.0). Considering the LOD of the conventional RT-PCR (Ct = 35), the LOD of the one-step immunoassay based on the switching peptide was determined to be feasible for the medical diagnosis of COVID-19.