The results show marked differences in specific peanut allergen profiles in peanut butter and flour and peanut preparations for clinical use. Roasting can increase Ara h 1 levels in peanut butter. Variability in allergen levels could affect the outcome of clinical trials of peanut OIT, especially with respect to Ara h 1. Specific allergen measurements will improve standardization and provide accurate dosing of peanut preparations that are being used for OIT.

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RATIONALE: Quantification of food allergens is increasingly important for dose assessments of food preparations used in oral immunotherapy (OIT), food allergy prevention, and monitoring safety in the food industry. Our aim was to develop and validate a multiplex immunoassay capable of simultaneously measuring eleven major food allergens from peanut, cow's milk, shellfish, egg, cashew, soy and hazelnut. METHODS: The multiplex array was developed on the Luminex xMAP system. Microspheres coupled to specific monoclonal antibodies were used for allergen capture. Biotinylated specific monoclonal or polyclonal antibodies were used for detection. Reference standards were formulated from natural or recombinant allergens, with purity established by mass spectrometry. A full method validation was performed to determine parameters of linearity, range, limits of quantification and detection, accuracy and precision of the multiplex food immunoassay. RESULTS: Full method validations were completed for the eleven major food allergens. The standard curves for all analytes allow for quantification over a broad dynamic range. The limits of detection (LLOD) were as low as 0.01ng/ml. Intra-and inter-assay accuracy and precision for three samples assayed in triplicate on four occasions passed acceptance criteria within the range of 70-130% recovery and a coefficient of variation of < _15%. CONCLUSIONS: A quantitative, accurate and precise multiplex immunoassay was validated for the simultaneous detection of eleven major food allergens. The multiplex array provides a sensitive and efficient tool for measuring specific food allergens, as opposed to generic food source proteins, with potential applications for risk assessment in the food industry and standardization of OIT products.

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