Selection, characterization, and application of a mycotoxin-specific single domain antibody fragment

15-Acetyl-deoxynivalenol (15-AcDON) is a low molecular weight (M.W. = 338 Da) sesquiterpenoid trichothecene mycotoxin associated with Fusarium Head Blight (FHB), a global disease of small grain cereals. Estimated economic losses from the most recent Canadian and US FHB epidemic during the mid 1990s range from ;A single-domain variable heavy chain (VHH) recombinant antibody (rAb) fragment specific to 15-AcDON was isolated from a hyper-immunized phagemid library. The dominant clone (NAT-267) was expressed and purified as a V HH monomer with a competitive binding affinity of 1.24 muM. The gene encoding the NAT-267 camelid single-domain antibody fragment (VHH) was expressed within the methylotropic yeast Pichia pastoris. Mycotoxin-mediated cytotoxicity was assessed by continuous measurement of cellular growth over time. At equivalent doses, 15-AcDON was significantly more toxic to wild-type P. pastoris than was DON, which, in turn, was more toxic than 3-AcDON. Intracellular expression of this toxin-specific VHH 'intrabody' within P. pastoris conveyed significant (p = 0.01) resistance to 15-AcDON cytotoxicity at doses ranging from 20 to 100 mug-mL-1, making this the first report of VHH-based sequestration of a haptenic mycotoxin. Furthermore, a biochemical transformation of DON to 15-AcDON was documented, which explained significant attenuation of the efficacy of DON at 100 and 200 mug-mL-1. This "proof of concept" model suggests that in planta VHH expression may lead to enhanced tolerance to mycotoxins and thereby serve as a novel tool to help limit Fusarium infection of commercial agricultural crops such as wheat and barley that are engineered to express this mycotoxin-specific VHH.