Botulinum neurotoxin type A (BoNT A) is one of the most potent toxins to humans and has become an imminent threat in biological warfare. Administered as either a pre- or post-exposure prophylaxis, high affinity neutralizing antibodies can potentially provide protection against the disease botulism. Human single-chain variable fragment antibodies (scFvs) that recognize BoNT A have previously been identified, including one antibody that can almost completely neutralize BoNT A. While extremely promising, it would be beneficial to enhance the affinity of these antibodies towards BoNT A. This will be achieved by subjecting the scFVs to affinity maturation/molecular evolution methods in combination with phage-display. The most promising antibodies will then be converted to a more stable format for further characterization. The ability of the antibodies to protect against the cytotoxic effects of BoNT A will be determined with an in vitro cell-based assay. Additionally, binding affinity and specificity of these antibodies will be investigated using BIAcore. The association rate constant, dissociation rate constant, and equilibrium dissociation constant for antibody-BoNT A binding will be obtained for the evolved antibodies.