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.