More than 370-Fold Increase in Antibody Affinity to Estradiol-17β by Exploring Substitutions in the V_H-CDR3

Antibodies that specifically target biomarkers are essential in clinical diagnosis. Genetic engineering has assisted in designing novel antibodies that offer greater antigen-binding affinities, thus providing more sensitive immunoassays. We have succeeded in generating a single-chain Fv fragment (scFv) targeted estradiol-17β (E₂) with more than 370-fold improved affinity, based on a strategy focusing the complementarity-determining region 3 in the V_H domain (V_H-CDR3). Systematic exploration of amino acid substitutions therein, using a clonal array profiling, revealed a cluster of four substitutions, containing H99P and a serial substitution E100eN-I100fA-L100gQ that lead to a 90-fold increase in E₂-binding affinity. This substitution quartet in the V_H-CDR3, combined with the substitution cluster I29V/L36M/S77G in the V_L domain, resulted in a scFv fragment with a further increase in the affinity (K_a, 3.2 × 10¹⁰ M^-1). This enabled a highly sensitive enzyme-linked immunosorbent assay capable of detecting up to 0.78 pg/assay. The current study has, thus, focused on the significance of reevaluating the potential of mutagenesis targeting the V_H-CDR3, and encouraging the production and use of engineered antibodies that enable enhanced sensitivities as next-generation diagnostic tools.