Rapid and sensitive detection of SARS-CoV-2 based on a phage-displayed scFv antibody fusion with alkaline phosphatase and NanoLuc luciferase

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the subsequent pandemic have led to devastating public health and economic losses. The development of highly sensitive, rapid and inexpensive methods to detect and monitor coronaviruses is essential for family diagnosis, preventing infections, choosing treatments and programs and laying the technical groundwork for viral diagnosis. This study established one-step immunoassays for rapid and sensitive detection of SARS-CoV-2 by using a single-chain variable fragment (scFv) fused to alkaline phosphatase (AP) or NanoLuc (NLuc) luciferase. First, a high-affinity scFv antibody specific to the SARS-CoV-2 nucleocapsid (N) protein was screened from hybridoma cells-derived and phage-displayed library. Next, prokaryotic expression of the scFv-AP and scFv-NLuc fusion proteins were induced, leading to excellent antibody binding properties and enzyme catalytic activities. The scFv-AP fusion had a detection limit of 3 pmol per assay and was used to produce eye-readable biosensor readouts. Moreover, the scFv-NLuc protein was applied in a highly sensitive luminescence immunoassay, achieving a detection limit lower than 0.1 pmol per assay. Therefore, the scFv-AP and scFv-NLuc fusion proteins can be applied for the rapid and simple diagnosis of SARS-CoV-2 to safeguard human health and provide guidance for the detection of other pathogenic viruses. [Display omitted] •Generation of high-affinity scFv antibodies against the SARS-CoV-2 N protein via hybridoma and phage display technologies.•Fusion expression of the H6 scFv antibody and alkaline phosphatase (AP) or NLuc luciferase for one-step detection.•H6-AP fusion produces a signal that is visible to the naked eye, whereas H6-NLuc is more sensitive.•ScFv-enzyme fusions have enabled the development of immunoassays for highly sensitive and rapid detection of SARS-CoV-2.