Multiplex Detection of Food Allergens and Antigen Profiling of Cumin

Introduction: In late 2014, several cumin-containing products were recalled for the presence of undeclared peanuts and almonds detected using ELISA methods. The detection of multiple undeclared food allergens is difficult, especially when there is the possibility that homologous proteins may be present that can cross-react with the antibodies used in the ELISAs. Currently the only methods that can simultaneously detect multiple allergens entail the use of mass spectrometry, a time-consuming and expensive process.

Purpose: To develop an inexpensive, rapid, high-throughput, multiplex assay for the simultaneous detection and quantification of food allergens and gluten. The assay should also be able to distinguish between known food allergens and novel cross-reactive proteins.

Methods: Established antibodies, used in the detection of food allergens, were conjugated to paramagnetic, color-coded beads to generate two multi-analyte profiling (xMAP^®) assays. A 29-plex and a nine-plex were designed to detect non-denatured and denatured food allergen proteins, respectively. Included in each mixture were AssayCheX^TMProcess Control microspheres to confirm instrument and reagent optimal performance.

Results: The multiplex assays were able to detect all food allergens at concentrations less than 50 ppb (e.g., 50 ng/g or ng/ml). By comparing the intensities of the responses generated by cumin with the 30 antibodies included in the assays, it was shown that the cumin samples contain either a mixture of many undeclared allergens (i.e., peanut, almond, Brazil nut, cashew, coconut, hazelnut, macadamia, and pistachio) or a novel cross-reactive protein(s).

Significance: The development of a multiplex assay for food allergens makes it possible to analyze 48 samples in one day for the presence of 14 food allergens and gluten, a process that would otherwise take over one month using standard ELISA technology. Further, without the use of expensive equipment, it is now possible to identify the presence of novel cross-reactive proteins. This should enhance efforts to identify potentially allergenic foods.