Robustness testing of the xMAP Food Allergen Detection Assay: a multiplex assay for the simultaneous detection of food allergens.

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, its reliability must be assessed when conditions of the assay procedure are altered. This study was conducted to determine the effects on assay performance associated with changes in incubation temperature, amounts of the antibody bead cocktail, and concentrations of detection antibody and beta-mercaptoethanol in the reduced-denatured extraction buffer. The analysis of buffered-detergent extracts revealed lower responses at 22 degrees C than at 37 degrees C, but temperature had no effect on the analysis of reduced-denatured extracts. Changes in beta-mercaptoethanol and detection antibody concentrations had an effect on the detection of only milk in the reduced-denatured extracts. A slight change in the measured bead count was observed when one-fourth of the bead cocktail was used, and a large decrease in the bead count was noted when one-eighth of the recommended amount was used, but this number (> 25) was still sufficient to provide reliable results. Overall, the xMAP FADA was very robust to changes in the assay procedure, which may inadvertently occur.