Development of a competitive lateral flow assay utilizing affinity-engineered receptor binding domain (ligand) and...

Novel infectious Betacoronaviruses are a threat to global public health; therefore, rapid broad-spectrum screening methods are required. However, traditional antibody-dependent lateral flow assay (LFA) is used to detect known viruses based on specific immune recognition. To ensure the robust detection of various Betacoronaviruses species with potential infection risk, we developed a competitive LFA system using the receptor binding domain (RBD) of SARS-CoV-2 spike protein as capture ligand and human angiotensin-converting enzyme 2 (hACE2) as receptor probe. Colloidal gold particles were used to label hACE2, and RBDs with binding activity were successfully expressed in Komagataella phaffii and purified. Considering the functional roles of the residues 498 and 501 of RBD in binding, several single and double mutations were introduced. Molecular docking and microscale thermophoresis were used to investigate the effect of these mutations; among them, Q498Y showed significantly enhanced binding affinity to hACE2. Subsequent specificity and sensitivity analyses demonstrated that the optimized assay employing the Q498Y RBD achieved a limit of detection of 1.66 μg/mL for the SARS-CoV-2 S1 protein. Additionally, cross-recognition of other Betacoronaviruses, such as civet-originated SARS-CoV and bat-originated RshSTT182/200 coronavirus, were effectively detected. This platform provides a proof-of-concept for the receptor-ligand-based detection of Betacoronaviruses with a contagious risk. It may be useful in point-of-care testing, customs quarantine processes, and in-field surveys.