Multi-laboratory validation of the xMAP—Food Allergen Detection Assay: A multiplex, antibody-based assay for the simultaneous detection of food allergens

Abstract

The increasing prevalence of individuals with multiple food allergies and the need to distinguish between foods containing homologous, cross-reactive proteins have made the use of single-analyte antibody-based methods (e.g., ELISAs) sometimes insufficient. These issues have resulted in the need to conduct multiple analyses and sometimes employ orthogonal methods like mass spectrometry or DNA-based methods for confirmatory purposes. The xMAP Food Allergen Detection Assay (xMAP FADA) was developed to solve this problem while also providing increased throughput and a modular design suitable for adapting to changes in analytical needs. The use of built-in redundancy provides the xMAP FADA with built-in confirmatory analytical capability by including complementary antibody bead sets and secondary analytical end points (e.g., ratio analysis and multi-antibody profiling). A measure of a method's utility is its performance when employed by analysts of varying expertise in multiple laboratory environments. To gauge this aspect, a multi-laboratory validation (MLV) was conducted with 11 participants of different levels of proficiency. The MLV entailed the analysis of incurred food samples in four problematic food matrices, meat sausage, orange juice, baked muffins, and dark chocolate. Except for a couple of instances, involving two confirmatory components in the analysis of baked muffins, the allergenic foods were detected by all participants at concentrations in the analytical samples comparable to � 10 μg/g in the original food sample. In addition, despite high levels of inter-lab variance in the absolute intensities of the responses, the intralaboratory reproducibility was sufficient to support analyses based on the calibration standards and direct comparison controls (DCCs) analyzed alongside the samples. In contrast, ratio analyses displayed inter-laboratory %CV (RSD R) values < 20%; presumably because the ratios are based on inherent properties of the antigenic elements. The

Detection of Allergenic Proteins in Foodstuffs: Advantages of the Innovative Multiplex Allergen Microarray-Based Immunoassay Compared to Conventional Methods

“…Therefore, this immunochemical method cannot detect allergenic proteins that are damaged or unfolded, for instance after the application of processing procedures. A disadvantage of this method is that generally only an individual allergen for each test can be analyzed, although in some cases the detection of more than one allergen can be possible [ 86 ].…”

Quantification of rice α‐globulin allergen using liquid chromatography–tandem mass spectrometry combined with cysteine‐specific modifier and extended stable isotope‐labeled peptide

“…In contrast to allergens in fish, milk and many other foods, there are scarce determination pipeline and thresholds to support the supervising and labeling of the allergens in rice and food products. Over the years, conventional immunoaffinity methods such as antibody‐based enzyme‐linked immunosorbent assay (ELISA) have been widely adopted as standard methods to determine food allergens in many countries (Garber et al, 2020; Hirano et al, 2016). However, the quantitation result of immunoassay can be vulnerable to cross‐reactivity and interference, and is very difficult to multiplex (Ahsan et al, 2016).…”

Cross-Reactivity of Chili Peppers (Capsicum sp.) with the xMAP Food Allergen Detection Assay

“…The xMAP food allergen detection assay (xMAP FADA) is a potential solution to challenges posed by single-analyte ELISAs. In addition to including at least two sandwich-based antibody assays for every target antigen, except for crustacean, it is currently capable of simultaneously detecting 15 food allergens plus gluten. , Furthermore, the assay has two complementary extraction protocols to address specific needs. The buffered detergent extraction protocol employs UD (105 mM sodium phosphate, 75 mM sodium chloride, 2.5% non-fat dry milk, and 0.05% Tween 20) or PBST (phosphate-buffered saline with Tween 20) buffer, while the reduced denatured extraction protocol uses a sodium dodecyl sulfate (SDS)- and β-mercaptoethanol-based buffer .…”