Rapid cell-free antibody fragment production and binding analysis via fluorescence correlation.

This study presents a workflow for rapid production and functional characterization of antibody fragments using an E. coli-based cell-free protein synthesis (CFPS) system. We quantified binding interactions by fluorescence correlation spectroscopy (FCS), which enabled determination of dissociation constants (KD) between antibodies and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. As an initial validation, two conventionally expressed anti-RBD antibodies were analyzed to establish the reliability of the FCS-based binding measurements. To enable efficient cell-free production, strategies were developed to improve the solubility and yield of single-chain variable fragments (scFvs), including implementation of an established two-stage refolding workflow adapted for CFPS-derived proteins. This approach enabled recovery of functional scFvs from insoluble fractions. In addition, Fab fragments were successfully produced and characterized, with binding measurements confirming retention of antigen recognition. Together, these results demonstrate that CFPS can be combined with FCS to enable rapid, solution-phase evaluation of antibody fragment binding without reliance on conventional cell-based expression systems. This integrated platform provides a scalable approach for antibody screening and characterization, with potential applications in therapeutic antibody development and high-throughput discovery.