A Novel System for Discovering High-affinity Antibody Mutants That Enables Immunoassays with Higher Sensitivities —Development and Application of Clonal Array Profiling (CAP)—

Antibody engineering is a powerful method used to generate high-affinity antibodies that enables sensitive immunoassays. It is commonly performed with the following steps: First, antibody fragments (e.g., single-chain Fv fragments; scFvs) with various mutations are displayed on the surface of filamentous bacteriophages to generate a diverse scFv-phages library. Then, rare clones with improved affinities are selected from the library via “panning” against target antigens immobilized on solid phases. However, this process often fails because of biased proliferation of scFv-phage clones and competition with large excesses of clones with weaker affinities. To overcome these limitations, we developed a clonal array profiling (CAP) method in which scFv-phage members in a library are individually examined for their antigen-binding ability. The advantage of CAP over conventional panning was evident in a comparative study that explored a library of anti-cortisol scFvs. CAP isolated five scFv mutants with >30-fold enhanced affinity (Ka) compared with wild-type scFv, enabling >11-fold more sensitive immunoassays. In contrast, no clones showing >5-fold higher Ka were found via panning. Considering the unique features of the primary structures of the improved scFvs found via CAP, we constructed new anti-cortisol scFv libraries where amino acid substitutions or insertions were introduced into the heavy-chain framework region 1 (VH-FR1). As expected, we obtained 21 mutants with >15-fold enhanced affinities. This VH-FR1-targeting mutagenesis also succeeded in generating affinity-matured scFvs against psilocybin, a hallucinogenic compound found in some mushrooms, which could be applied for developing on-site identification systems for hallucinogenic mushrooms, e.g. as immunochromatography devices.