title: Validation of an ELISA test kit for the detection of total aflatoxins in grain and grain products by comparison with HPLC.
abstract: 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 degrees 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.
authors.affiliation: Romer Labs Singapore Pte. Ltd., 3791 Jalan Bukit Merah 08-08, e-Centre@redhill, 159471, Singapore.
authors.forename: Zhongming
authors.initials: Z
authors.lastname: Zheng
authors.forename: Craig W
authors.initials: CW
authors.lastname: Humphrey
authors.forename: Robin S
authors.initials: RS
authors.lastname: King
authors.forename: John L
authors.initials: JL
authors.lastname: Richard
mesh_terms: D000348:Aflatoxins / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D002523:Edible Grain / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards*; D007887:Fabaceae / Q000737:chemistry; D005506:Food Contamination; D006801:Humans; D011933:Reagent Kits, Diagnostic / Q000592:standards*; D015203:Reproducibility of Results
chemical_list: D000348:Aflatoxins; D011933:Reagent Kits, Diagnostic