Phage display and selection of a site-directed randomized single-chain antibody Fv fragment for its affinity...

The affinity of an antibody Fv fragment was improved by semirational design involving site-directed randomization and phage display. On the basis of the predicted model of an anti-2-phenyloxazol-5-one (phOx) antibody Fv fragment, into which the ligand was inserted with the help of nuclear Overhauser enhancement (NOE) data, residues close to the hapten were identified. Seven of these residues in the third hypervariable regions of light and heavy chains were randomized in polymerase chain reactions (PCR) using degenerate oligonucleotides. Resulting clones were expressed as single-chain Fv (scFV) fragments on the surface of filamentous phage and selected for binding to phOx-conjugated bovine serum albumin. Selected Fv fragments were analyzed for hapten affinity by fluorescence quenching, and several mutants with improved affinities were identified. Phage selection on the basis of binding was very successful when phage scFv mutants differed in affinity by at least a factor of 6. Smaller differences did not result in predominant selection of the best binder. Combination of the two point mutations most crucial for improved hapten binding decreased the dissociation constant of the Fv for phOx 11-14-fold. Hapten binding of the improved Fv was analyzed in NOE experiments.