Mycotoxin multiplex microsphere immunoassays: Screening from ingredients to beer

Food and feed can be contaminated with microorganisms, parasites and chemical substances, which can have light or severe toxicological effects. Therefore the European Union (EU) has established food and feed safety regulations. Combined with organized control strategies, they help to ensure the safety of food and feed that enter the production chain. Mycotoxins are produced by fungi and can be major food and feed contaminants. Upon ingestion, mycotoxins can cause mycotoxicosis. The symptoms of mycotoxicosis can be both acute and chronic, and range from nausea to cancer, and even death. Grain commodities are the basis for food and feed, and these can be contaminated by what are considered the most important mycotoxins in terms of agriculture and animal production; zearalenone (ZEN), deoxynivalenol (DON), T-2 toxin (T-2), aflatoxins (AFs), ochratoxin A (OTA) and fumonisins (FBs). The main aim of the research in this thesis, was to develop a multiplex immunoassay method capable of detecting the main 6 mycotoxins, and their modified forms, in grain-based feed and food commodities. For the development of the mycotoxin multiplex immunoassays, we implemented the multi-Analyte profiling (xMAP) technology from Luminex. This xMAP technology combines unique color-encoded microspheres in a suspension array format, which can be analysed by dedicated analyser platforms consisting of dedicated flow cytometers and a planar array analyser. The developed multiplex should be able to screen for the main six mycotoxins at food and feed ML levels in a wide range of matrices. This thesis gives a comprehensible look into the main mycotoxins. Their history, toxicity, occurrence and EU recommendations and legislation are summarized. A special focus is on the modified mycotoxins. These are the free forms of mycotoxins that are modified by living organisms like plants, bacteria and fungi. Since antibodies are the key biorecognition molecules for the developed multiplex immunoassays presented in this thesis, they are shortly explained, followed by their implementation in the multiplex mycotoxin detection immunoassays.

For the multiplex screening of food and feed matrices a semi-quantitative multiplex competitive immunoassay was developed for the detection of ZEN, DON, T-2, AFs, OTA and FBs. In the intial indirect format, protein-mycotoxin conjugates were coupled to the unique xMAP microspheres, who were then incubated with free mycotoxins (in samples) and mycotoxin specific antibodies. The developed method was critically tested by applying it to contaminated feed reference samples and the results were critically compared to confirmatory instrumental analysis of the same reference samples. The second approach presented is a faster direct detection format, introducing fluorescent mycotoxin-protein conjugates. In the direct format, monoclonal antibodies (mAbs) were coupled to the microspheres and incubated with free mycotoxins and the fluorescent mycotoxin-conjugates. The new 6-plex direct multiplex was applied to barley samples, for a successful in-house validation as a screening method. The cut-off factors were set at 50% of the EU maximum levels (MLs) for food: 2 µg/kg for AFB1, 2.5 µg/kg for OTA, 625 µg/kg for DON, 50 µg/kg for ZEN, 1000 µg/kg for FB1 and 25 µg/kg for T-2. The validation showed very high inter and intra-day precision for all samples. The direct 6-plex was also used to screen available barley and malted barley reference materials. The developed direct 6-plex assay showed potential for future implementation as a semi-quantitative pre-screening method for regulated mycotoxins, prior to instrumental analysis (like LC-MS/MS). Simplifications were introduced that could make the method more suitable for future on-site application. These simplifications comprised; washing steps by a handheld magnetic plate instead of an automated washer, addition of pre-mixed reagents from dropper bottles, instead of laboratory pipettes, addition of samples by disposable fixed volume micropipettes, and a sample extraction time of just 1 minute, additionally omitting centrifugation. Preliminary in-house validation of the simplified multiplex screening assay, using an on-site extraction procedure, was successful at EU maximum levels using the portable MAGPIX planar array analyzer.

A major part of this thesis is dedicated to a large global survey for mycotoxin occurence in beer with the aforementioned direct 6-plex. Besides the high number of screened samples, 1000 in total, there was a really strong focus on beers from the emerging craft beer scene. Besides the application of the 6-plex, a confirmatory analysis method (LC-MS/MS) was developed and applied to a selection of samples based on the acquired screening results. The major mycotoxins detected were DON and its plant metabolite deoxynivalenol-3-β-D-glucopyranoside (D3G). The 6-plex immunoassay reported the sum of DON and D3G (DON+D3G) contaminations ranging from 10 to 475 µg/L in 406 beers, of which 73% were craft beers. The popular craft beer style imperial stout, had the highest percentage of samples suspected positive (83%) with 29% of all imperial stout beers having DON+D3G contaminations above 100 µg/L. LC-MS/MS analysis showed that industrial pale lagers from Italy and Spain, predominantly contained FBs (3 – 69 µg/L). Besides FBs, some African traditional beers also contained aflatoxins (0.1 - 1.2 µg/L). The presence of OTA, T-2, HT-2, ZEN, β-zearalenol, 3/15-acetyl-DON, nivalenol and the conjugated mycotoxin zearalenone 14-sulfate were confirmed in some beers. In 27 craft beers, DON+D3G concentrations occurred above (or at) the Tolerable Daily Intake (TDI) level, which may have a health impact upon consumption of these beers.

The increased relevance of modified mycotoxins, and their inclusion in the process of the development of the presented mycotoxin multiplexes in this thesis, showed the importance of having access to novel reference substances. Therefore Cunninghamella strains were introduced as bioconjugation models. Liquid media cultures of Cunninghamella were fortified with a range of mycotoxins. The chosen Cunninghamella strains were able to produce a range of ZEN metabolites, including zearalenone-14-β-D-glucopyranoside (Z14G), zearalenone-16-β-D-glucopyranoside (Z16G) and zearalenone-14-sulphate (Z14S).