Protein Microarrays: Simultaneous Detection of Mycotoxins in Foodstuffs and Animal Feed

The development of fast and sensitive methods to detect various biological toxins and food pathogens is of great importance. Traditional methods employ high pressure liquid chromatography and mass spectrometry, preceded by significant steps of sample preparation to eliminate interference from other components like sample matrix, or utilize immunological techniques like ELISA. Despite the high detection sensitivity of classical immunoassays, these methods do not allow to carry out simultaneous detection of several toxins. Microarrays open a new perspective. They allow parallel analyses of samples for many parameters using minute amount of sample material and reagents.

We have developed an antibody-based microarray technique for the multiplexed detection of mycotoxins in foodstuffs or animal feed. Mycotoxins such as zearalenone, ochratoxin A, deoxynivalenol and aflatoxin B1 are small toxic products formed as secondary metabolites by a few fungal species that colonise crops, contaminate them in the field or after harvest, and result in toxic effects on humans and animals. Our quantitative detection method is based on a multiplexed competitive immunoassay: toxin-specific monoclonal antibodies are immobilized on epoxy-coated slides, mycotoxin standards (labelled with different fluorophores) and unlabelled toxins in the sample of interest compete for binding sites of spotted antibodies and thereby enable a correct quantification. All antibodies and toxins were thoroughly tested and show no cross-reactivity. We could also reduce the steps of sample preparation to a minimum by using a simple extraction routine, which shows for the different crop matrices tested no significant influence on the results. The shelf-life of our spotted arrays is about 8 months, fluorescence signals are stable for at least one month, if stored in the dark. In general, the presented antibody microarray enables the rapid detection of mycotoxins in a sub-ppm range with excellent recovery rates.