Validation of an ELISA test kit for the detection of total aflatoxins in grain and grain products by comparison with HPLC
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An ELISA Microtiter Plate, Total Aflatoxin Test called AgraQuant® was validated to measure total aflatoxins in a range from 4 to 40 ppb in corn, corn meal, corn gluten feed, corn gluten meal, corn germ meal, corn/soy blend, popcorn, sorghum, wheat, milled rice, soybeans, peanuts and cottonseed. The test is performed as a solid phase direct competitive ELISA using a horseradish peroxidase conjugate as the competing, measurable entity. For the test method, aflatoxins are extracted from ground samples with 70 % methanol and sample extracts plus conjugate are mixed and then added to the antibody-coated microwells. After 15 min incubation at room temperature, the plate is washed and enzyme substrate is added and allowed to incubate for an additional 5 min. Stop solution is then added and the intensity of the resulting yellow color is measured optically with a microplate reader at 450 nm. Results obtained from internal validation studies assessing accelerated stability indicate a minimum of 1 year shelf life for the kits; accuracy and precision are comparable to HPLC in the range of 0–320 ppb and limit of detection in corn is 2.5 ppb. Comparison of the method to HPLC, ability to detect individual aflatoxins and ruggedness of the test kits at 18–30°C determined this test to be rugged, sensitive, accurate, precise and effective comparable to HPLC for measuring total aflatoxins ranging from 4 to 40 ppb in the commodities evaluated.
KeywordsAgraQuant® ELISA HPLC test kit total aflatoxins validation
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- Richard, JL. 1998
Mycotoxins, toxicity and metabolism in animal–a systems approach overview.Miraglia, MEgmond, HPBrera, CGilbert, J eds. Mycotoxins and Phycotoxins–Developments in Chemistry, Toxicology and Food Safety.Alaken, Inc.FortCollins, CO363397Google Scholar
- Council for Agricultural Science and Technology (CAST). Mycotoxins: Risks in Plant, Animal and Human Systems. Task Force Report, no. 139, CAST, Ames, Iowa, 2003.Google Scholar
- Food and Agricultural Organisation. Worldwide Regulations for Mycotoxins 1995. A Compendium. FAO Food and Nutrition Paper 64, Rome, Italy, 1997.Google Scholar
- Trucksess, MW 2001
Rapid analysis (thin layer chromatographic and immunochemical methods) for mycotoxins in foods and feeds)Koe, WJSamson, RAEgmond, HPGilbert, JSabino, M eds. Mycotoxins and Phycotoxins in Perspective at the Turn of the Millennium.W.J. de KoeWageningen, The Netherlands2940Google Scholar
- Trucksess, MW, Koeltzow, DE 1995
Evaluation and application of immunochemical methods for mycotoxins in food.Nelson, JOKaru, AEWong, RB eds. Immunoanalysis of Agrochemical in Emerging Technologies.American Chemical SocietyWashington, DC326334Google Scholar
- Gilbert, J, Anklam, E 2002Validation of analytical methods for determining mycotoxins in foodstuffs.Trends Anal Chem21468486Google Scholar
- USDA/GIPSA. Design criteria and test performance specifications for quantitative aflatoxin test kits. U.S. Department of Agriculture, Federal Grain Inspection Service Quality Assurance and Research Division. 1998.Google Scholar
- Deshpande, S.S. Enzyme Immunoassays From Concept to Product Development, Chapman and Hall, International Thomson Publishing, New York: 996: 397 pp.Google Scholar