Mycotoxins are a group of naturally occurring toxins produced by certain fungi, commonly known as moulds, which are harmful to humans, domestic animals and livestock. They are found in a wide range of foods and feeds. Mycotoxins can enter the food or feed chain through contaminated crops. Contamination may also occur postharvest during storage, transport, and processing stages of the food or feed supply chain. Mycotoxin occurrence is widespread throughout the world, mycotoxin producing fungi favour different climate conditions. Mycotoxins represent a major foodborne risk susceptible to climate change. Dueto health and welfare concerns, regulations and recommendations have been established in many countries for some mycotoxins.
The Clavicepsgenus of fungi produce ergot alkaloids; when these fungi infect the seed heads of plants during the flowering period, a wintering body (known as ergot, ergot body or sclerotium)is produced,which replaces the developing seed or grain. The sclerotia are harvested together with the cereals or grass, and if not removed before processing, can lead to contamination of cereal-based food and feed products, which can pose a risk to animal and human health.Alternaria, Aspergillus, Fusarium and Penicillum, genus of fungi also produce harmful mycotoxins.Mycotoxins of interest for food safety and regulatory purposes are aflatoxins (B1, B2, G1, G2 and M1), citrinin, deoxynivalenol, ergot alkaloids, fumonisins (FB1, FB2 and FB3), HT-2 toxin, patulin, ochratoxin A, T-2 toxin and zearalenone.
The availability of analytical methods enabling the detection of these compounds is therefore relevant for food and feed safety. In this article two immunoassay-based analytical tools are reported:anEnzyme-Linked Immunorbent Assay (ELISA) for the detection of a sum of 12 ergot alkaloids and abiochip-based immunoassay platform for the simultaneous detection of multiple mycotoxins from a single sample applying generic extraction protocol. Both applications facilitate the screening step of samples in the testing process.
Application of an ELISA to the detection of a sum of 12 main ergot alkaloids
The main ergot alkaloids produced by the Claviceps species which are contained in the sclerotia are ergometrine, ergotamine, ergosine, ergocristine, ergocryptine, ergocornine and their corresponding –inine epimers. For the detection of these compounds by ELISA, a competitive immunoassay format is used. The capture antibodies are immobilised and stabilised on 96 well microtitre plates. In the competitive reaction, the free analyte contained in the standard/sample competes for binding sites of the capture antibody with horseradish peroxidase labelled conjugate. Following incubation and washing steps, the enzyme substrate is added. The optical density is measured at 450nm once the colour reaction is stopped. The colour intensity is inversely proportional to the concentration of the ergot alkaloid present in the sample.
The ELISA reported in this article (validated based on Commission Regulation (EC) No 519/2014 as a semi-quantitative screening method), detects the sum of 12 main ergot alkaloids to test cereal-based feed, wheat, spelt, oats, barley and rye cereals and their milling products according to recently released (24th August 2021) Commission Regulation (EU) 2021/1399 amending Regulation (EC) No1881/2006. The ELISA is successfully assessed by fit-for-purpose approach according to Commission Regulation (EC) No 401/2006 as approved method of analysis. And is successfully assessed by fit-for-purpose approach according to Commission Regulation (EC) No 519/2014 for confirmatory method as an approved method for official control.The assay is standardized to the sum of 12 ergot alkaloids and the limits of detection (LODs) are 50 ppb for all matrices the ELISA is validated for.The single step sample preparation is simple and involves solvent solid/liquid extraction followed by dilution of collected supernatant. The ergot alkaloids detected with this platform are summarized in table 1.
Table 1. Ergot alkaloids detected by ELISA
Application of Biochip Array Technology to the simultaneous detection of multiple mycotoxins
Biochip Array Technology (BAT) provides a platform for the simultaneous determination of multiple mycotoxins from a single sample using miniaturized immunoassays with implications in the reduction of sample/reagent consumption and an increase in the output of test results.The core of the system is the biochip (9mm x 9mm) where the capture antibodies are immobilized and stabilised defining arrays of discrete test regions. Applying the same principle that a competitive ELISA, simultaneous chemiluminescent competitive immunoassays applied to the benchtop analyser Evidence Investigator, take place on the biochip surface.The chemiluminescent reactions produced at the different discrete test regions are simultaneously detected and recorded by a cooled charge coupled device (CCD) camera. The multiple data generated are reported and archived in a dedicated software for retrospective access.
BAT is employed for screening purposes and additionally enables the semi-quantitative detection of predominant and modified mycotoxins from cereals, cereals milling products as well as cereal based feed implementing simple, single step, generic solid/liquid extraction. As this platform enables simultaneous detection of multiple mycotoxins from a single sample, the screening capacity is significantly increased, saving time and reducing laboratory workload, usage of consumables and solvents. Myco arrays have been developed to accommodate 5, 6, 7, or 9 immunoassays. Because of the flexibility of this technology the arrays can be customised to accommodate the mycotoxins to be screened according to various jurisdictions, prevalence in a geographical region or other requirements. The mycotoxins detected with the biochip arrays are shown in table 2.
Moreover, cereals, directly used for consumption or as an ingredient of various feed and food commodities, may be naturally contaminated by one or several fungi simultaneously. Each fungal strain can produce either single or multiple mycotoxins. Because of the multi-analytical approach, BAT can detect co-occurrence of mycotoxins from a single food/feed sample. All validated matrices on Myco arrays followed Commission Regulation (EC) No519/2014 criteria. Additionally, there are currently over 50 matrices evaluated and this list is persistently expanding.
Optimal analytical performance of Myco 7 Array, which includes immunoassays for aflatoxin B1, aflatoxin G1, deoxynivalenol, fumonisin B1, ochratoxin A, T-2 toxin and zearalenone has been reported in the screening of cereals and their milling products as well as cereal based feed (Plotan et al. 2016, Freitas et al. 2019).
Table 2.Mycotoxins detected with biochip arrays
Mycotoxins are practically unavoidable contaminants of food and feed and may elicit adverse effects, which poses a major health concern. Mycotoxinsrepresent a major foodborne risk susceptible to climate change and their occurrence is widespread throughout the world. Regulations and recommendations have been established in many countries for some mycotoxins. Methodologies enabling their detection are then relevant for feed and food safety. The ELISA and BAT reported in this article, are immunoassay-based platforms that represent valuable analytical tools for the screening of mycotoxins in food/feed samplesfollowing simple, single step liquid extraction. The ELISA by detecting 12 main ergot alkaloids and multiplex BAT by detecting predominant and modified mycotoxins from a single sample.The use of these screening tools facilitate the testing process applied to assure safety of food and feed commodities.
Plotan M. et al. The use of biochip array technology for rapid multimycotoxin screening. Journal of AOAC International. 2016, 99: 878–889.
Freitas A., et al. Validation of a biochip chemiluminescent immunoassay for multi-mycotoxins screening in maize (Zea mays L.). Food Analytical Methods, 2019, 12: 2675–2684.