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.
In 2013 the U.S. Food and Drug Administration (FDA) defined the term “gluten-free” and identified a gap in the analytical methodology for detection and quantification of gluten in foods subjected to fermentation and hydrolysis. To ascertain the ability of current enzyme-linked immunosorbent assays (ELISAs) to detect and quantify gluten in fermented and hydrolyzed products, sorghum beer was spiked in the initial phases of production with 0, 20, and 200 μg/ml wheat gluten, and samples were collected throughout the beer production process. The samples were analyzed using five sandwich ELISAs and two competitive ELISAs and by sodium dodecyl sulfate–polyacrylamide gel electrophoresis with Western analysis employing four antibodies (MIoBS, R5, G12, and Skerritt). The sensitivity of the MIoBS ELISA (0.25 ppm) enabled the reliable detection of gluten throughout the manufacturing process, including fermentation, when the initial concentration of 20 μg/ml dropped to 2 μg/ml. The R5 antibody–based and G12 antibody–based sandwich ELISAs were unable to reliably detect gluten, initially at 20 μg/ml, after the onset of production. The Skerritt antibody–based sandwich ELISA overestimated the gluten concentration in all samples. The R5 antibody–based and G12 antibody–based competitive ELISAs were less sensitive than the sandwich ELISAs and did not provide accurate results for quantifying gluten concentration. The Western analyses were able to detect gluten at less than 5 μg/ml in the samples and confirmed the results of the ELISAs. Although further research is necessary before all problems associated with detection and quantification of hydrolyzed and fermented gluten are resolved, the analytical methods recommended by the FDA for regulatory samples can detect ≥20 μg/ml gluten that has undergone brewing and fermentation processes associated with the manufacture of beer.
In 2013, the U.S. Food and Drug Administration conducted a survey of green and white teas marketed in the northeastern United States for the presence of undeclared wheat. Based on the requirement for concurrence between the RIDASCREEN gliadin (R5) enzyme-linked immunosorbent assay (ELISA) and the Morinaga Institutes of Biological Science (MIoBS) wheat protein ELISA, none of the 20 products included in the survey tested positive for wheat, rye, barley, or gluten. However, eight of the teas generated responses indicative of the presence of gluten with the RIDASCREEN gliadin (R5), AgraQuant gluten G12, and Aller-Tek (Skerritt) sandwich ELISAs. Five of the eight teas generated responses indicative of >20 ppm of gluten using the RIDASCREEN and AgraQuant ELISA test kits, and all eight had ≥20 ppm based on the Aller-Tek ELISA. Extracts prepared using the RIDASCREEN validated protocol and the MIoBS validated sodium dodecyl sulfate plus β-mercaptoethanol (overnight) protocol were analyzed using both test kits. The extracts prepared using the RIDASCREEN protocol tested positive for gluten with both test kits. Western blot analyses of the two sets of extracts using the R5 and MIoBS antibodies to visualize the bands revealed the presence of antigenic proteins in both sets of extracts, although the profiles and band intensities were different and inconsistent with the ELISA results. These results raise questions regarding the screening procedures used to detect gluten and how the observation of a homologous antigenic element is defined.