6-Plex microsphere immunoassay with imaging planar array detection for mycotoxins in barley

Abstract

Mycotoxins are produced by fungi as secondary metabolites. They often multi-contaminate food and feed commodities posing a health risk to humans and animals. A fast and easy to apply multiplex screening of these commodities could be useful to detect multi-contamination. For this, we developed a semi-quantitative 6-plex immunoassay using a suspension array of paramagnetic colour-coded microspheres combined with imaging planar array detection for the mycotoxins aflatoxin B₁, ochratoxin A, zearalenone, deoxynivalenol, T2-toxin, HT-2 toxin and fumonisin B₁. Mycotoxin specific monoclonal antibodies were coupled to different sets of microspheres and mycotoxins conjugated to the fluorescent protein R-phycoerythrin served as reporter molecules. Competition between free mycotoxins in the sample and mixed reporter molecules for antibody binding sites on mixed microspheres created a multiplex direct inhibition immunoassay. The reagents were selected for no or low cross-interactions between the assays and cross-reactions with metabolites and possible masked forms were determined. A within-laboratory validation was carried out using blank and spiked barley samples. Furthermore, the 6-plex was used to screen available barley, and malted barley, reference materials. The validation showed very high inter and intra-day precision for all samples with a maximum relative standard deviation value of 10%. The screening assay allows easy and rapid multiplex detection of the target mycotoxins in barley according to EU legislation. With a cut off factor of 50%, based on the EU maximum levels, we were able to screen at 2 μg kg⁻¹ for aflatoxin B₁, 2.5 μg kg⁻¹ for ochratoxin A, 625 μg kg⁻¹ for deoxynivalenol, 50 μg kg⁻¹ for zearalenone, 1000 μg kg⁻¹ for fumonisin B₁ and 25 μg kg⁻¹ for T-2 toxin. Thanks to the transportable planar array system, the developed 6-plex has potential for future on-site testing. Future implementation of this method as a pre-screening tool, prior to instrumental analysis, is highly attractive since costly LC-MS/MS analysis of samples below the maximum levels can be avoided.