In vitro affinity maturation of a single-chain antibody against thyroxine based on computer-aided design

Area of Science:

• Biomolecular Engineering
• Immunotechnology
• Computational Biology

Background:

• Thyroxine (T4) is vital for physiological regulation.
• Single-chain variable fragments (scFvs) offer advantages over traditional antibodies for diagnostics due to their size, specificity, and affinity.
• Developing high-affinity scFvs is crucial for accurate T4 detection.

Purpose of the Study:

• To perform in vitro affinity maturation of T4-specific scFvs using computational design.
• To identify T4-scFv variants with enhanced binding affinity.
• To elucidate the interaction mechanisms between optimized scFvs and T4.

Main Methods:

• Homology modeling and molecular dynamics were used to construct a T4-specific scFv (T4-scFv).
• Molecular docking and virtual mutagenesis identified key residues for affinity enhancement.
• Engineered variants were expressed in E. coli and evaluated using indirect competitive ELISA (IC-ELISA).

Main Results:

• Three affinity-enhanced variants (scFv-M92, scFv-MS, scFv-M226) were generated, with scFv-M226 showing a 2.9-fold improvement in affinity.
• scFv-M226 demonstrated high specificity, with <2.0% cross-reactivity to T3 and rT3.
• A quantitative T4 detection assay using scFv-M226 achieved a wide linear range (1.0-200 ng·mL⁻¹) and a low detection limit (0.93 ng·mL⁻¹).

Conclusions:

• The computationally guided affinity maturation strategy is effective for enhancing scFv binding.
• The optimized scFv-M226 exhibits high affinity and specificity for T4 detection.
• scFv-M226 holds significant potential for clinical T4 monitoring and diagnostics.