Abstract

Conventional antibody screening workflows, which rely on multi-step prokaryotic expression systems (e.g., molecular cloning, transformation, and protein purification), typically span weeks, limiting their efficiency. Here, we present a cell-free platform that integrates cell-free protein synthesis (CFPS) with bio-layer interferometry (BLI) to enable one-step affinity evaluation of nanobodies (Nbs) within hours. To illustrate our approach, we characterized an anti-aflatoxin B 1 -specific Nb (G8) and its mutants. By fusing Nbs with green fluorescent protein (GFP), we developed a GFP-based fluorescent calibration curve that enables rapid quantification of Nb concentration, thereby facilitating kinetic analysis using BLI. Comparative studies revealed high consistency between CFPS and traditional methods, with a Pearson correlation coefficient of 0.968, while reducing processing time by over 95%. The cell-free nature of our workflow allows for parallelized Nb mutagenesis screening and circumvents viability constraints, thus establishing a universal strategy for high-throughput antibody discovery and optimization.