Retrieving Dissociation-Resistant Antibody Mutants: An Efficient Strategy for Developing Immunoassays with Improved...

Previously, we generated high-affinity antibody mutants that enabled sensitive immunoassays by exploring diverse libraries of single-chain Fv fragments (scFvs) displayed on bacteriophage. To isolate rarely-occurring desirable clones, "panning" has commonly been performed but is often unsuccessful. Therefore, we previously developed a clonal array profiling (CAP) method, wherein scFv-displaying phage (scFv-Ph) clones in a library were examined individually regarding their ability to target antigens immobilized on microwells. Clones that showed strong reactivity were recovered via dissociation using an acidic treatment. The CAP successfully discovered cortisol-specific scFvs showing 17-31-fold improved K_a from libraries generated via site-directed insertions in a prototype anti-cortisol scFv (wt-scFv; K_a, 3.6 × 10⁸ M^-1), but their K_a did not exceed 1.1 × 10¹⁰ M^-1. In this study, to break this possible affinity ceiling, we devised a new system employing a dissociation-independent recovery. scFv-Phs were individually reacted to target antigen (cortisol) immobilized on microwells via a linker containing a disulfide bond. Following acidic and basic treatments to eliminate scFv-Phs with "ordinary affinities," dissociation-resistant scFv-Phs remaining on the microwells were retrieved via reductive cleavage of the disulfide bonds. This system allowed for a straightforward and efficient discovery of scFv mutants with 33-56-fold increased K_a (1.2-2.0 × 10¹⁰ M^-1), exceeding the previous affinity ceiling. These scFvs enabled an enzyme-linked immunosorbent assay for cortisol with 18-51-fold higher sensitivity than the assay performed using wt-scFv.