<i>In vitro</i> affinity maturation of a single‐chain antibody against thyroxine based on computer‐aided design

Thyroxine (T4) plays a crucial role in regulating various physiological functions in the human body. As a key biomolecule for molecular recognition, single-chain variable fragments (scFvs) offer advantages, such as small molecular size, high specificity, and strong affinity, making them ideal alternatives to traditional antibodies for diagnostic applications. This study aimed to achieve in vitro affinity maturation of T4-specific scFvs through computational design, identify variants with enhanced affinity, and elucidate their interaction mechanisms with T4. A T4-specific scFv (T4-scFv) was constructed based on the previously reported anti-T4 Fab fragment (PDB ID: 5MHE) using homology modeling, followed by molecular dynamics optimization. Molecular docking and virtual mutagenesis of eight key residues (Ser91, Ser92, Pro94, Tyr180, Tyr181, Pro182, Pro186, and Ser226) were performed, yielding three affinity-enhanced variants. These variants were expressed in E. coli and evaluated experimentally using indirect competitive ELISA (IC-ELISA). The results showed that the IC₅₀ values decreased from 34.59 ng·mL^-1 (T4-scFv) to 18.30 ng·mL^-1 (scFv-M92), 15.26 ng·mL^-1 (scFv-MS), and 11.84 ng·mL^-1 (scFv-M226), demonstrating a 2.9-fold improvement in affinity for the best mutant. The optimized scFv-M226 also exhibited high specificity, with cross-reactivity to T3 and rT3 of less than 2.0%. A quantitative T4 detection method based on scFv-M226 was developed, which showed a wide linear range (1.0-200 ng·mL^-1) and a detection limit (IC₁₀) of 0.93 ng·mL^-1. These findings confirm the accuracy of the computationally guided affinity maturation strategy and highlight the potential of scFv-M226 for clinical detection and diagnostic monitoring of T4 in serum.