ENZYME-LINKED-IMMUNOSORBENT-ASSAY FOR DETECTION OF ZEARALENONE IN CORN, WHEAT, AND PIG FEED - COLLABORATIVE STUDY

A direct competitive enzyme-linked immunosorbent assay (ELISA) screeni ng method for zearalenone in corn, wheat, and feed at 500 ng/g was eva luated by 23 collaborators (22 laboratories) in an international colla borative study. Eighteen samples of spiked or naturally contaminated c orn, wheat, and pig feed were prepared by the sponsoring laboratory an d sent for testing with complete test kits to participating collaborat ors in Canada, Italy, Sweden, The Netherlands, and the United States. Test samples were extracted with methanol-water solution (70 + 30) by shaking on a wrist-action shaker for 3 min. A portion of the extract w as mixed with an equal volume of zearalenone-enzyme conjugate, and the mixture was incubated with zearalenone-specific monoclonal antibodies coated onto microtiter wells. All test samples were assayed in duplic ate. One of 52 (2%) blanks was reported positive. Thirty-nine of the 5 2 (75%) samples that were spiked at 500 ng/g were reported as positive . Forty-nine of the 51 (96%) samples with concentrations at or above 1 000 ng/g were reported as positive. The overall incidence of false neg atives was 6.0% and the incidence of false positives was 22.7% by the ELISA method. Only one (3.4%) false negative was reported for samples containing greater than or equal to 800 ng/g. In the spectrophotometri c method, 8 collaborators determined approximate levels of zearalenone in test samples from standard curves constructed from spiked extracts (0-3000 ng/g of each commodity tested). This method gave and overall incidence of false negatives of 5.7% and false positives of 17.8%. Ave rage relative standard deviations, RSD(r) (repeatability) and RSD(R) ( reproducibility), were 11.6 and 25.1% for spiked samples and 11.7 and 33.1% for naturally contaminated samples, respectively. Standard curve s were constructed with each set of samples assayed. Comparison of abs orbance values from these standard curves indicate the performance of reagents and antibody used in the assay. The ELISA method has been ado pted first action by AOAC INTERNATIONAL as a screening method for zear alenone at greater than or equal to 800 ng/g in corn, wheat, and pig f eed.