Development of a Nanobody–Alkaline Phosphatase Fusion Protein for Detection of SARS-CoV-2 Spike Protein in a Fluorescence Enzyme Immunoassay

The continuous spread and evolution of severe acute respiratory symptom coronavirus 2 (SARS-CoV-2) necessitate the development of convenient and rapid detection methods. In this study, we developed a fluorescence enzyme immunoassay (FEIA) based on a nanobody (Nb)–alkaline phosphatase (ALP) fusion protein for detection of SARS-CoV-2 spike protein. The genetically modified anti-SARS-CoV-2 S-RBD Nb, Nb61, gene was fused with the ALP gene sequences via a flexible linker. Recombinant cloning was used to yield a recombinant prokaryotic expression plasmid, Nb61-ALP-His. The Nb61-ALP-His construct was transformed into E. coli BL21(DE3) and expressed in bacteria. Both Nb61 properties and ALP enzymatic activity were validated by colorimetric and fluorometric analysis. FEIA was optimized and established on the basis of the Nb61-ALP fusion protein. The detection limit of the FEIA was 3.18 ng/mL, with a linear range of 1.9–62.5 ng/mL. Comparison with a commercial kit indicated the reliability of the Nb61-ALP fusion-protein-based FEIA for monitoring the SARS-CoV-2 spike protein. This study highlights the potential of Nb-based enzyme immunoassays as a valuable tool for the rapid and accurate detection of SARS-CoV-2.