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This document provides supplemental guidance on specifications for the development and implementation of studies to validate the performance characteristics of quantitative ELISA methods for the determination of food allergens. It is intended as a companion document to other existing publications on method validation. The guidance is divided into two sections: information to be provided by the method developer on various characteristics of the method, and implementation of a multilaboratory validation study. Certain criteria included in the guidance are allergen-specific. Two food allergens, egg and milk, are used to demonstrate the criteria guidance. These recommendations will be the basis of the harmonized validation protocol for any food allergen ELISA method, whether proprietary or nonproprietary, that will be submitted to AOAC and/or regulatory authorities or other bodies for status recognition. Regulatory authorities may have their own particular requirements for data packages in addition to the guidance in this document. Future work planned for the implementation and validation of this guidance will include guidance specific to other priority allergens.

A competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect hazelnut by using polyclonal antibodies generated against a protein extract of roasted hazelnut. No cross-reactivity was observed in tests against 39 commodities, including many common allergens, tree nuts, and legumes. Hazelnut protein standard solutions at 0.45 ng/mL [inhibition concentration (IC80) of the competitive test] were clearly identified by the ELISA. An extraction and quantification method was developed and optimized for chocolate, cookies, breakfast cereals, and ice cream, major food commodities likely to be cross-contaminated with undeclared hazelnut during food processing. No sample cleanup was required when extracts were diluted 10-fold. Recovery results were generated with blank matrixes spiked at 4 levels from 1 to 10 microg/g hazelnut protein. With the developed extraction and sample handling procedure, hazelnut proteins were recovered at 64-83% from chocolate and at 78-97% from other matrixes. A confirmatory technique was developed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western transfer. The developed methods were applied to a small market survey of chocolate products and allowed the identification of undeclared hazelnut in these products.

An enzyme-linked immunosorbent assay (ELISA) was developed to determine the presence of egg proteins in foods. The polyclonal antibodies developed were specific to whole egg proteins and did not cross-react with any of the 38 nuts, legumes, or other common food ingredients tested. The concentrations of egg proteins that will inhibit 50% of antibody-antigen binding, IC50, were 3-7 ng/mL, and the linear range was 0.5-62.5 ng/mL. The detection limit was 0.2 ppm for various foods. Recoveries ranged from 67 to 96%. The intra- and inter-assay coefficients of variation in this procedure were 10-13% for ice cream spiked at 0.8 and 1.6 ppm. The ELISA has been applied to ice creams, noodles, pasta, and breads. Egg proteins were identified in all declared egg products, and no false positives were found.

Food allergy presents a problem for many parts of society, including sensitive subjects, schools, health authorities, and the food industry. Once food allergy is diagnosed, dietary avoidance is the principle method of management. Because trace levels of peanuts can elicit an adverse to fatal reaction, unintentional exposure to the offending allergens may have devastating consequences to sensitive individuals. However, determination of trace amounts of unintentional peanut contamination in our food supply is very difficult. Recently, we developed polyclonal antibodies specific to peanut proteins that do not cross-react with 22 legumes, tree nuts, or other common snack ingredients. An antiserum containing the polyclonal antibodies was used to develop a sensitive enzyme-linked immunosorbent assay (ELISA) for determination of peanut proteins in snack foods. This study reports the first successful ELISA test to detect trace amounts of peanut allergens in a variety of foods. The concentration of peanut protein that inhibited 50% of antibody-antigen binding (IC50) was 12 ng/mL, the linear range was 1-63 ng/mL, and the detection limit was 400 ng/g (ppb) for the various foods tested. Recoveries ranged from 68 to 90%, with coefficients of variation of 2-22%, depending on the commodity. Using this new procedure, allergy-related complaint samples from various food groups were analyzed, and undeclared peanut proteins were identified in some products.