Based on a three-dimensional molecular model of the variable region of a monoclonal antibody (Ab) TT1, in which the complementarity determining regions (CDRs) associate to form a cavity large enough to accommodate a single molecule of tri-iodothyronine (T3) or thyroxine (T4), we designed TT1 mutants with one amino acid substitution as candidates which have their binding affinity for T4 reduced but retain the same affinity for T3. Each candidate was subsequently tested by site-directed mutagenesis, transient expression in COS cells, and surface plasmon resonance (SPR) analysis for its binding ability for T3- or T4-conjugates with alkaline phosphatase (AP). Of the candidates, the Ab with serine in place of glycine at position 92 of the light chain (L;G92S) and the Ab with alanine in place of leucine at position 47 of the heavy chain (H;L47A) had the association constant (KA = kass / kdiss) for binding to T4-AP decreased by 5-fold, but retained the same KA for T3-AP.