Anti-RBD Single-Chain Variable Fragments Library (scFv Library) Generation toward the Development of a Diagnostic...

The continued emergence of SARS-CoV-2 variants worldwide underscores the need for effective measures to combat the current and future outbreaks. The receptor-binding domain (RBD) of the spike glycoprotein plays a critical role in viral entry and represents a highly immunogenic target. In this study, we generated a mouse-derived single-chain variable fragment (scFv) phage display library against the RBD of the SARS-CoV-2 spike protein. Peptide ELISA-based epitope mapping demonstrated that selected monoclonal scFv phages exhibited broad epitope recognition and reactivity across multiple recently reported variants. In a SARS-CoV-2 pseudovirus neutralization assay, scFv C4 showed potent neutralizing activity against the wild-type, Gamma, and Delta variants, with IC50 values below 4 μg/mL. These findings highlight the potential utility of these scFvs as candidate reagents for the development of improved diagnostic platforms and as scaffolds for next-generation monoclonal antibody-based therapeutics.