Fluorescence polarization as a tool for the determination of deoxynivalenol in wheat

Abstract

The mould Fusarium graminearum is found worldwide as a pathogen of cereal grains, in particular of wheat and maize, and it produces a mycotoxin known as deoxynivalenol (DON or vomitoxin). Each year, the presence of this compound and related trichothecenes causes substantial losses to agricultural productivity. Rapid methods for the measurement of the toxin in grains are required to monitor and divert effectively contaminated grain from the food supply. A fluorescence polarization (FP) immunoassay using a previously described monoclonal antibody for DON was developed. The assay was based on the competition of unlabeled DON from a sample with a fluorescently tagged DON, DON-fluorescein (DON-FL), for a DON-specific monoclonal antibody in solution. The FP of the tagged DON was increased upon binding with the antibody. In the presence of free toxin, less of the DON-FL was bound and the polarization signal was decreased. The assays were very simple to perform, requiring only mixing of an aqueous extract of wheat with the DON-FL and antibody. The sensitivity of the assay was strongly dependent upon the time between mixing of the sample with the tracer and measurement of the fluorescence polarization, with midpoints for the competition curves ranging from 0.03 µgml-1 with a 15-s incubation to >1 µgml-1 with a 12-min incubation. Samples of wheat naturally contaminated with DON were evaluated by FP and by an HPLC-UV method, with a good correlation (r 2 = 0.97). Although the FP method tended to overestimate DON slightly in the wheat samples, by ˜20%, the assay was easy to use and very useful for the screening of wheat.