The xMAP Food Allergen Detection Assay (xMAP FADA) can simultaneously detect 15 analytes (14 food allergens, plus gluten) in one analysis. The xMAP FADA typically employs two antibody bead sets per analyte, providing built-in confirmation that is not available with other antibody-based assays. Before an analytical method can be used, it is important to assess its reliability when conditions of the assay procedure are altered. This study reports the effects on assay performance associated with incubation temperature and varying amounts of bead cocktail, and also detection antibody and β-mercaptoethanol concentrations in the reduced-denatured extraction buffer. The analysis of buffered-detergent extracts displayed lower responses at 22°C compared to 37°C, while temperature had no effect on the analysis of reduced-denatured extracts. Changes in β-mercaptoethanol and detection antibody concentrations only displayed an effect on the detection of milk in the reduced-denatured extracts. A slight change in the measured bead count was observed when one-fourth the bead cocktail was used, and displayed a large decrease when one-eighth of the recommended amount was used, but this number (≥25) was still sufficient to provide reliable results. Overall, the xMAP FADA displayed an excellent robustness towards changes in the assay procedure, as may inadvertently occur.
ABSTRACT An estimated 0.1 to 0.2% of the North American population is allergic to sesame, and deaths due to anaphylactic shock have been reported. Detecting and quantifying sesame in various food samples is critical to safeguard the allergic population by ensuring accurate ingredient labeling. Because of the modular nature of the xMAP Food Allergen Detection Assay (FADA), it was possible through method extension to add sesame as a validated additional analyte. Because raw and toasted sesame are both commonly used and the two display significantly different antigenicity, three antibodies, one monoclonal and two polyclonal, were conjugated to bead sets to ensure reliable detection. The modified xMAP FADA successfully detected sesame incurred or spiked in baked muffins, spice mix, canola oil, and in both raw and toasted sesame oils with limit of quantitation values ≤ 1.3 ppm of sesame. Canola oil, sesame oil, toasted sesame oil, and olive oil inhibited sesame detection, as did the detection of sesame incurred in foods containing oil (e.g., hummus). Despite this inhibition, the xMAP FADA was still able to reliably detect sesame at levels throughout the dynamic range of the assay (22 to 750 ng of protein per mL) in all the foods examined. Further, the high signal-to-noise ratio of the lowest calibration standard and preliminary studies conjugating the antibodies at higher concentrations indicate an ability to increase the sensitivity of the assay should the need arise. HIGHLIGHTS Sesame was added to the validated xMAP Food Allergen Detection Assay. Both raw and toasted sesame were successfully detected when incurred in foods and oils. Assay sensitivity can be increased by conjugating antibodies at a higher concentration as needed.