Food Analytical Methods

, Volume 11, Issue 5, pp 1444–1450 | Cite as

Development of an Indirect Competitive ELISA for Analysis of Alternariol in Bread and Bran Samples

  • Gurmit Singh
  • Ligia Velasquez
  • Beth Brady
  • Terry Koerner
  • Anne-Catherine Huet
  • Philippe Delahaut


Alternariol (AOH) is one of the major mycotoxins produced by various species of Alternaria fungi. Natural occurrences of AOH have been reported in various foods, including fruits; processed fruit products such as apple juice, tomato products; wheat and other grains; oilseeds and products thereof, such as sunflower seeds, oilseed rape meal, and flax seed/linseed; and pecans. In this study, AOH-specific polyclonal antibodies were generated and developed an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for monitoring AOH in bread and bran samples. The assay was very sensitive with a limit of detection (LOD) of 2.4 ± 0.6 ng/g and a half maximal inhibitory concentration (IC50) of 15.2 ± 2.6 ng/g in bread and a LOD of 8.4 ± 1.2 ng/g and IC50 of 52.8 ± 10.8 ng/g in bran extract. The assay was very specific to AOH and showed no cross-reactivity to alternariol monomethyl ether, altertoxin, altenuene, tentoxin, or tenuazonic acid. The effect of organic solvents on the assay was tested. The ELISA system tolerated methanol and acetonitrile as co-solvents at up to 5% content without significant loss of IC50 value. Recoveries in all cases were greater than 75%, and the results using this method were comparable to those obtained from mass spectrometry methods. We conclude that this method is suitable for rapid detection of AOH in bread and bran samples, without expensive analytical equipment or time-consuming sample preparation.


Mycotoxin Alternariol Polyclonal antibodies Indirect ELISA Food samples 



The authors thank Wendy Zhao, FRD Bureau of Chemical Safety, Health Canada, for providing the commercial samples which were analyzed by LC-MS/MS method in our division.

Compliance with Ethical Standards

Conflict of Interest

Gurmit Singh declares that he has no conflict of interest. Ligia Velasquez declares that she has no conflict of interest. Beth Brady declares that she has no conflict of interest. Terry Koerner declares that he has no conflict of interest. Anne-Catherine Huet declares that she has no conflict of interest. Philippe Delahaut declares that he has no conflict of interest.

Ethical Approval

This article does not contain any studies with human subjects. All animal experiments that described in the present study were performed in CER Groupe, Belgium, following all institutional and national guidelines for the care and use of laboratory animals.

Informed Consent

Not applicable


  1. Ackermann Y, Curtui V, Dietrich R, Gross M, Latif H, Martlbauer E, Usleber E (2011) Widespread occurrence of low levels of alternariol in apple and tomato products, as determined by comparative immunochemical assessment using monoclonal and polyclonal antibodies. J Agric Food Chem 59(12):6360–6368. CrossRefGoogle Scholar
  2. Bauer JI, Gross M, Gottschalk C, Usleber E (2016) Investigations on the occurrence of mycotoxins in beer. Food Control 63:135–139. CrossRefGoogle Scholar
  3. Brugger EM, Wagner J, Schumacher DM, Koch K, Podlech J, Metzler M, Lehmann L (2006) Mutagenicity of the mycotoxin alternariol in cultured mammalian cells. Toxicol Lett 164(3):221–230. CrossRefGoogle Scholar
  4. Burkin AA, Kononenko GP (2011) Enzyme immunoassay of alternariol for the assessment of risk of agricultural products contamination. Appl Biochem Microbiol 47(1):72–76. CrossRefGoogle Scholar
  5. EFSA (2011) Scientific opinion on the risks for animal and public health related to the presence of Alternaria toxins in feed and food. EFSA J 9:2407CrossRefGoogle Scholar
  6. Fleck SC, Burkhardt B, Pfeiffer E, Metzler M (2012) Alternaria toxins: altertoxin II is a much stronger mutagen and DNA strand breaking mycotoxin than alternariol and its methyl ether in cultured mammalian cells. Toxicol Lett 214(1):27–32. CrossRefGoogle Scholar
  7. Hickert S, Gerding J, Ncube E, Hübner F, Flett B, Cramer B, Humpf HU (2015) A new approach using micro HPLC-MS/MS for multi-mycotoxin analysis in maize samples. Mycotoxin Research 31(2):109–115. CrossRefGoogle Scholar
  8. Hickert S, Bergmann M, Ersen S, Cramer B, Humpf HU (2016) Survey of Alternaria toxin contamination in food from the German market, using a rapid HPLC-MS/MS approach. Mycotoxin Research 32(1):7–18. CrossRefGoogle Scholar
  9. Juan C, Oueslati S, Mañes J (2016) Evaluation of Alternaria mycotoxins in strawberries: quantification and storage condition Food Additives and Contaminants - Part A Chemistry, Analysis, Control, Exposure and Risk Assessment 33:861–868, 5, DOI:
  10. Lehmann L, Wagner J, Metzler M (2006) Estrogenic and clastogenic potential of the mycotoxin alternariol in cultured mammalian cells. Food Chem Toxicol 44(3):398–408. CrossRefGoogle Scholar
  11. Liu GT et al. (1991) Relationships between Alternaria alternata and oesophageal cancer. IARC Sci Publ:258–262Google Scholar
  12. Liu GT, Qian YZ, Zhang P, Dong WH, Qi YM, Guo HT (1992) Etiological role of Alternaria alternata in human esophageal cancer. Chin Med J 105(5):394–400Google Scholar
  13. Logrieco A, Moretti A, Solfrizzo M (2009) Alternaria toxins and plant diseases: an overview of origin, occurrence and risks. World Mycotoxin J 2(2):129–140. CrossRefGoogle Scholar
  14. López P, Venema D, de Rick T, de Kok A, Scholten JM, Mol HGJ, de Nijs M (2016) Occurrence of Alternaria toxins in food products in The Netherlands. Food Control 60:196–204. CrossRefGoogle Scholar
  15. Noser J, Schneider P, Rother M, Schmutz H (2011) Determination of six Alternaria toxins with UPLC-MS/MS and their occurrence in tomatoes and tomato products from the Swiss market. Mycotoxin Research 27(4):265–271. CrossRefGoogle Scholar
  16. Ostry V (2008) Alternaria mycotoxins: an overview of chemical characterization, producers, toxicity, analysis and occurrence in foodstuffs. World Mycotoxin J 1(2):175–188. CrossRefGoogle Scholar
  17. Prelle A, Spadaro D, Garibaldi A, Gullino ML (2013) A new method for detection of five Alternaria toxins in food matrices based on LC-APCI-MS. Food Chem 140(1-2):161–167. CrossRefGoogle Scholar
  18. Schrader TJ, Cherry W, Soper K, Langlois I (2006) Further examination of the effects of nitrosylation on Alternaria alternata mycotoxin mutagenicity in vitro. Mutation Research - Genetic Toxicology and Environmental Mutagenesis 606(1-2):61–71. CrossRefGoogle Scholar
  19. Scott PM (2001) Analysis of agricultural commodities and foods for Alternaria mycotoxins. J AOAC Int 84(6):1809–1817Google Scholar
  20. Scott PM, Kanhere SR (2001) Stability of Alternaria toxins in fruit juices and wine. Mycotoxin Research 17(1):9–14. CrossRefGoogle Scholar
  21. Scott PM, Weber D, Kanhere SR (1997) Gas chromatography-mass spectrometry of Alternaria mycotoxin. J Chromatogr A 765(2):255–263. CrossRefGoogle Scholar
  22. Scott PM, Lawrence GA, Lau BPY (2006) Analysis of wines, grape juices and cranberry juices for Alternaria toxins. Mycotoxin Research 22(2):142–147. CrossRefGoogle Scholar
  23. Scott PM, Zhao W, Feng S, Lau BPY (2012) Alternaria toxins alternariol and alternariol monomethyl ether in grain foods in Canada. Mycotoxin Research:1–6Google Scholar
  24. Singh G, Brady B, Koerner T, Becalski A, Zhao T, Feng S, Godefroy SB, Huet AC, Delahaut P (2014) Development of a highly sensitive competitive indirect enzyme-linked immunosorbent assay for detection of acrylamide in foods and water. Food Anal Methods 7(6):1298–1304. CrossRefGoogle Scholar
  25. Solfrizzo M (2017) Recent advances on Alternaria mycotoxins Current Opinion in Food Science 17:57–61Google Scholar
  26. Zhao K, Shao B, Yang D, Li F, Zhu J (2015) Natural occurrence of Alternaria toxins in wheat-based products and their dietary exposure in China. PLoS One 10Google Scholar
  27. Zwickel T, Klaffke H, Richards K, Rychlik M (2016) Development of a high performance liquid chromatography tandem mass spectrometry based analysis for the simultaneous quantification of various Alternaria toxins in wine, vegetable juices and fruit juices. J Chromatogr A 1455:74–85. CrossRefGoogle Scholar

Copyright information

© Crown 2017

Authors and Affiliations

  • Gurmit Singh
    • 1
  • Ligia Velasquez
    • 1
  • Beth Brady
    • 1
  • Terry Koerner
    • 1
  • Anne-Catherine Huet
    • 2
  • Philippe Delahaut
    • 2
  1. 1.Food Research Division, Bureau of Chemical Safety, Food Directorate-HPFB, Health CanadaOttawaCanada
  2. 2.CER Groupe, Health DepartmentMarloieBelgium

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