ELISA technology for screening mycotoxin detection

What are mycotoxin and where can they be found

Mycotoxins can be describes as a family of poisonous nanoparticle secondary metabolites which can be generated from specific moulds. In general, they are able to grow on many different natural materials which can include foodstuff and crops for example: apple juice, dried fruit, coffee, nuts, cereals and many spices. Under suitable moisture and temperature conditions, in particular fungi are able to rapidly proliferate to generate a large number of mycotoxins. In general the growth of mould tends to occur during the time of harvest either just before, after or during storage. It is found on or within the food itself and is usually under humid, damp and warm conditions. Majority of mycotoxins are chemically stable and can survive most food processing procedures.

At present, there are more than 500 different mycotoxins which have been discovered and there is a gradual increase in this number has each year passes by. The most common mycotoxins that can are found to concern livestock and human health include: aflatoxins, patulin, fumonisins, ochratoxin A, nivalenol/deoxynivalenol and zearalenone. They can appear within the food chain due to the result of mould infection of crops. The exposure to various mycotoxins can occur both indirectly from animals that have been fed contaminated feed (for example; herd, hay, straw and fodder) or directly from eating infected food (for example, meat, dairy, poultry, cereal).

Types of mycotoxins

Listed below are some of the most common types of mycotoxins.

1. Fumonisins: these moulds are predominately discovered as contaminants in countries that have a temperate climate, Corn is an example of one of the most frequent contaminated product. There is also evidence to indicate that this mould may also be present in malt brewing and grains. Fumonisin B1 and B2 are typical examples.
2. Aflatoxins: belong to a family of mycotoxins which are produced by different strains of antimicrobial Aspergillus. There are many foodstuff where these mould can be found to grow, some of the most common are: cereals, oilseeds, corn, cotton seeds, peanuts, spices, unrefined vegetable oils, cocoa, coffee and dried fruits. Sixteen different types of aflatoxins have been discovered and the most common types are aflatoxin B1, B2, G1, G2, M1 and M2.
3. Ochratoxin: these are mycotoxins which are often produced from specific types of fungi, in particular aspergillus ochraceus or penicillium verrucosum. Naturally, they can be present in a number of different plants such as cocoa, beans, coffee, nuts and cereals. Ochatratoxin A, B and C are some of the common typical example of these mycotoxins.
4. Trichothecene: these are members of the sesquiterpene family of compounds and there are 150 chemically related mycotoxins which are present in this group. These mycotoxins are produced from Stachybotrys and they have been found in many different types of grains such as oats, wheat and maize. Satratoxin-H, T-2 mycotoxin and vomitoxin are common examples.
5. Zearalenone: are estrogenic metabolite which are formed from Gibberella and Fusarium species. It has a property of being heat stable and can be present throughout the world in many different cereal crops such as wheat, oats, rice, sorghum and maize. Typical example of toxic substances produced by Fusarium species include zearalenone, T-2 toxin, deoxynivalenol and diacetoxyscirpenol.

Screening mycotoxins using ELISA procedure

The enzyme linked immuno-sorbent assay (ELISA) is a laboratory based immunoassay procedure has been used over a decade within the UK and around the world in order to either screen or detect specific mycotoxins, it is a procedure that offers accuracy and precision.

It is often used to help in animal health welfare. It is fundamental in food safety analysis and some of the most common foods include: meat, honey, flour, wheat, shrimp, milk and pet food (dog food and cat food in particular). Mycotoxins are generally recognised as being safe but high levels can be dangerous. It does play a vital role in food processing, food security and human nutrition.

One of the main advantages of this laboratory immunoassay method is to provide a rapid means of analysis in order to eliminate negative samples and therefore reduce the overall analysis number. This technique is a popular laboratory based chemical experiment that relies on the ability of specific antibodies which are able to distinguish the three-dimensional structure of certain mycotoxins. This immunoassay is easy to use, highly specific, highly sensitive which makes it very accurate and precise laboratory test to carry out.

At present, the majority commercially available ELISA kits that can be used for detecting mycotoxins are working in the kinetic phase of antibody-antigen binding, this has the added advantage of reducing the incubation times into minutes rather than hours.

General steps involved in an ELISA test

• Extract mycotoxin from a ground sample with solvent.
• Mix sample extract with an enzyme coupled mycotoxin.
• Add this mix solution to an antibody coated microtiter wells.
• Mycotoxins in the sample extract or control standards are allowed to compete against the enzyme-conjugated mycotoxin for antibody binding sites on the microtiter wells that are not already occupied.
• A wash step is then carried out, followed by the addition of an enzyme substrate. This will result in producing a coloured solution. The intensity of the colour is inversely proportional to the amount of sample mycotoxin or standard that is present.
• Finally a solution is then added in order to stop the enzyme reaction.
• An ELISA reader is used to measure the intensity of the colour at an absorbance filter of 450nm.
• The reading obtained for the samples can be compared to the reading obtained for the standards used. A standard curve is drawn and an interpretative result for the sample readings is obtained.

ELISA kit methods are widely accepted as the favoured options for high throughput analysis since this procedure requires low sample volumes and the potential of less sample extract clean up when compared to other conventional methods such as HPLC and TLC.

Final Thoughts

Mycotoxins can be classified as nanoparticle secondary metabolism products from moulds and the subsequent uptake of mycotoxins through mouldy foodstuffs. These are responsible for causing mycotoxicosis, where even tiny concentrations are sufficient to cause a toxic effect, some of these effects can be fatal by damaging the immune system, gastro intestinal, lymphocyte, blood, intestinal tract, multiple myeloma, reproductive disorders, skin, stomach, liver, kidney damage and various cancer related diseases. International agency for research on cancer and World Health Organisation have carried out screening programs on herd animals in order to minimise serious toxicity and contaminants from entering the food chain.

For accurate prognosis it is vital to look out for key signs and chronic symptoms that can include: urination, induced coma, ingestion, complication in the eye and stress. ELISA kit procedures are the favoured choice for detecting mycotoxins, since they are simple, specific, sensitive, rapid and can be portable to be used outdoors in the field. They can be used as a screen or as a panel assay in order to provide accurate and reliable results with fewer observations of false positives. The exposure to mycotoxins has to be kept to a minimum in order to protect humans and animals. It can also have a great impact on food security and nutrition by reducing access to healthy food.

References

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2. Advances, challenges and opportunities for point-of-need screeningof mycotoxins in foods and feeds .Analyst. 2018 Feb 26;143 (5): 1015-1035. Review. Soares RRG. et al.
3. Some major mycotoxins and their mycotoxicoses–an overview. Int J Food Microbiol. 2007 Oct 20;119 (1-2): 3-10. Review. Richard JL.
4. Mycotoxins and animal health. Adv Vet Sci Comp Med. 1981; 25: 185-243. Pier AC.
5. Analysis of multiple mycotoxins in food. Methods Mol Biol. 2011; 747: 233-58. Hajslova J. et al.
6. Mycotoxin methodology. Food Addit Contam. 1995 May-Jun;12 (3): 395-403. Review. Scott PM.
7. Rapid test strips for analysis of mycotoxins in food and feed. Anal Bioanal Chem. 2009 Jan;393 (1): 67-71. Review. Krska R. and Molinelli A.
8. Risk screening of mycotoxins and their derivatives in dairy products using a stable isotope dilution assay and LC-MS/MS. J Sep Sci. 2021 Feb;44 (4): 782-792. Yang S. et al.

Related ELISA Kits

Fumonisin ELISA Kit
Aflatoxin Total ELISA Kit
Aflatoxin M1 ELISA Kit
Aflatoxin B1 ELISA Kit
T-2 Toxin ELISA Kit
Zearalenone ELISA Kit