Simultaneous Detection of Four Major Food Allergens Using a Multiplex Array.

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. ‘Generic’ immunoassays for ‘total protein’ do not measure specific allergens. Our aim was to validate a multiplex immunoassay capable of simultaneously measuring four major food allergens, Ara h 3, Ara h 6, Bos d 5 and shrimp tropomyosin.

METHODS: The multiplex array was developed on the Luminex xMAP system. Microspheres coupled to specific monoclonal antibodies were used for allergen capture and biotinylated specific mono- or polyclonal antibodies for detection. Reference standards formulated from purified natural allergens were used for calibration purposes. A full method validation was performed to determine parameters of linearity, range, limits of quantification and detection, accuracy and precision of the 4-plex food immunoassay.

RESULTS: The standard curves allow for quantification over a large dynamic range; 50-0.02ng/ml for Ara h 6, tropomyosin and Bos d 5, and 125-0.06ng/ml for Ara h 3. The lower limits of detection (LLOD) were as low as 0.02ng/ml and 0.06ng/ml. Intra- and inter- assay accuracy and precision results 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 four major food allergens. The multiplex array provides a sensitive and efficient tool for measuring specific allergens with potential application in food immunotherapy formulations and in the food industry.