Isolation and characterization of high affinity VHH antibody fragments against alpha-cobratoxin
Camelid VHHs may provide better treatment for snake envenomation than conventional antivenom antibodies because of their smaller size (~16 kDa), better tissue permeability and lower immunogenicity. In this thesis, a phage-displayed VHH library with 4.2 x 109 functional clones was constructed from a Ilama hyperimmunized with crude Thai cobra (' Naja kaouthia') venom. After three rounds of panning against acobratoxin ([alpha]-Cbtx), a potent neurotoxin from the Thai cobra venom, 26 unique clones were found using monoclonal phage ELISA and confirmed by DNA sequencing. Surface plasmon resonance (SPR) analyses showed that the four selected anti-[alpha]-Cbtx VHH clones had dissociation constants (KD) in the low nanomolar range (0.4-25 nM)and that these four VHHs bound to the same or overlapping epitopes on [alpha]-Cbtx. An 'in vitro' muscle twitch assay showed that VHH C2 (KD = 0.4nM) effectively neutralized the paralytic effects of [alpha]-Cbtx at neuromuscular junctions.