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

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 B-1, ochratoxin A, zearalenone, deoxynivalenol, T2-toxin. HT-2 toxin and fumonisin B-1. Mycotoxin specific monoclonal antibodies were coupled to different sets of microspheres and mycotoxins conjugated to the fluorescent protein Rphycoerythrin 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 lig kg(-1) for aflatoxin B-1, 2.5 mu g kg(-1) for ochratoxin A, 625 mu g kg(-1) for deoxynivalenol, 50 mu g kg(-1) for zearatenone, 1000 mu g kg(-1) for fumonisin B-1 and 25 mu g 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.