Attainment of 15-Fold Higher Affinity of a Fusarium-specific Single-Chain Antibody by Directed Molecular Evolution Coupled to Phage Display

Fusarium head blight (FHB) caused by Fusarium graminearum infection is a devastating disease of wheat, maize, and other cereals. A previously isolated chicken single-chain Fv antibody (scFv), CWP2, that conferred durable resistance in planta was subjected to directed evolution by error-prone PCR and DNA shuffling, generating a mutated library. Panning of the mutated library against cell wall–bound proteins (CWPs) from F. graminearum by phage display enriched phage clones that were used for a further round of DNA shuffling to construct a combinatorial library comprising 3 × 106 variants. Screening of this library by phage display for variants reactive against the CWPs led to the identification of a number of clones. Comparative enzyme-linked immunosorbent assay analyses revealed eight clones exhibiting a higher reactivity than the parent, CWP2, and containing four different single-chain antibody sequences. Surface plasmon resonance measurements confirmed that three mutated scFvs, CWPa, CWPb, and CWPd, displayed 15-fold, 11-fold, and 7-fold higher affinities, respectively, compared with CWP2. Three-dimension modeling of CWPa illustrates a conformational change bringing all six complementary domain regions on the antibody surface in one direction. These results provide promising unique resistance molecules for effective control of FHB and its associated mycotoxins in food/feed chains.