Novel Rapid Noncompetitive Immunodiagnostics for Mycotoxins:Dissertation

Mycotoxins are among the most important contaminants of agricultural products. These fungal secondary metabolites cause significant annual crop losses and different adverse effects for humans and animals consuming mycotoxin contaminated food or feed. Mycotoxins are a global problem and the significance of their measurement and control is increasing all the time. Novel solutions are needed to improve rapid high-throughput methods for screening large batches of food materials for mycotoxin contamination. In this work, recombinant antibodies were applied to develop rapid noncompetitive methods that hypothetically would improve both sensitivity and specificity of the mycotoxin assay. HT-2 toxin was selected as an example of a specific mycotoxin target. The first part of the work describes the development of a recombinant antibody pair for realizing a specific immune complex assay for HT-2 toxin. The first antibody recognizes the HT-2 toxin molecule, while the second antibody binds only to the immune complex formed by the first antibody and the target toxin. These two antibodies were then implemented in one-step homogeneous immune complex assay requiring only addition of the sample and measuring. The assay was demonstrated for HT-2 toxin determination in wheat samples. In the second part of the work, a simple noncompetitive ELISA assay was realized for HT-2 toxin detection in three important grain matrices wheat, barley and oats. These both developed assays were capable of measuring HT-2 toxin from naturally contaminated samples in a relevant concentration range. The last part of the work describes a proof-of-concept study for direct measurement of HT-2 toxin employing an ultra-sensitive graphene protected copper surface plasmon resonance method. The obtained detection limit in buffer was ~0.5 fg/mL, which is several orders of magnitude better than reported for conventional methods. The example of a direct label-free measurement of HT-2 toxin paves the way for future biosensing applications. The most evident contribution of this work is development of a first specific assay for HT-2 toxin without any cross-reactivity with other structurally related mycotoxins. This immune complex assay based on two recombinant antibodies, was demonstrated with two different assay platforms for HT-2 toxin. Similar technology can be adapted to develop specific assays also for other mycotoxins and for other small molecules, in general