Western blot analysis of fermented-hydrolyzed foods utilizing gluten-specific antibodies employed in a novel multiplex competitive ELISA.

Horseradish peroxidase (HRP) conjugated gluten-specific antibodies (G12, R5, 2D4, MIoBS, and Skerritt), from nine commercial gluten ELISA test kits, previously utilized in the development of a multiplex competitive ELISA for the detection of fermented-hydrolyzed gluten, were utilized in western blot analyses of 59 fermented-hydrolyzed foods from four food groups (beer, soy-based sauces, vinegar, and sourdough bread). The protein/peptide profiles generated by the nine gluten-specific antibodies varied in size distribution and intensity dependent on the type of food, with minor differences between related products. Cluster analysis of the estimated gluten concentration values (based on western blot band intensities relative to intact gluten standards at 2.5 µg/mL and 100 µg/mL) and that of the relative response of the nine gluten-specific antibodies to different gluten proteins/peptides, distinguished among the different categories of fermented-hydrolyzed foods; comparable to what was observed in the multiplex competitive ELISA. Further, unlike the competitive ELISA, the western blot analyses distinguished between the presence of antigenic proteinaceous materials and false positives due to the presence of binding inhibitors (as observed with four soy-based sauces and one vinegar). Limitations of western blot analysis often include lower sensitivity than the comparable competitive ELISA and problems quantitating gluten-derived peptides and proteins. As a result, western blot analysis provides an orthogonal approach that can be used to both confirm the multiplex competitive ELISA while also providing additional insight into the protein/peptide profile of fermented-hydrolyzed foods. Graphical abstract.

Cross-reactivity by botanicals used in dietary supplements and spices using the multiplex xMAP food allergen detection assay (xMAP FADA).

Food allergies affect some 15 million Americans. The only treatment for food allergies is a strict avoidance diet. To help ensure the reliability of food labels, analytical methods are employed; the most common being enzyme-linked immunosorbent assays (ELISAs). However, the commonly employed ELISAs are single analyte-specific and cannot distinguish between false positives due to cross-reactive homologous proteins; making the method of questionable utility for regulatory purposes when analyzing for unknown or multiple food allergens. Also, should the need arise to detect additional analytes, extensive research must be undertaken to develop new ELISAs. To address these and other limitations, a multiplex immunoassay, the xMAP® food allergen detection assay (xMAP FADA), was developed using 30 different antibodies against 14 different food allergens plus gluten. Besides incorporating two antibodies for the detection of most analytes, the xMAP FADA also relies on two different extraction protocols; providing multiple confirmatory end-points. Using the xMAP FADA, the cross-reactivities of 45 botanicals used in dietary supplements and spices commercially sold in the USA were assessed. Only a few displayed cross-reactivities with the antibodies in the xMAP FADA at levels exceeding 0.0001%. The utility of the xMAP FADA was exemplified by its ability to detect and distinguish between betel nut, saw palmetto, and acai which are in the same family as coconut. Other botanicals examined included allspice, amchur, anise seed, black pepper, caraway seed, cardamom, cayenne red pepper, sesame seed, poppy seed, white pepper, and wheat grass. The combination of direct antibody detection, multi-antibody profiling, high sensitivity, and a modular design made it possible for the xMAP FADA to distinguish between homologous antigens, provide multiple levels of built-in confirmatory analysis, and optimize the bead set cocktail to address specific needs.

A multiplex competitive ELISA for the detection and characterization of gluten in fermented-hydrolyzed foods.

A novel competitive ELISA was developed utilizing the G12, R5, 2D4, MIoBS, and Skerritt antibody-HRP conjugates employed in nine commercial ELISA test kits that are routinely used for gluten detection. This novel multiplex competitive ELISA simultaneously measures gliadin-, deamidated gliadin-, and glutenin-specific epitopes. The assay was used to evaluate 20 wheat beers, 20 barley beers, 6 barley beers processed to reduce gluten, 15 soy sauces, 6 teriyaki sauces, 6 Worcestershire sauces, 6 vinegars, and 8 sourdough breads. For wheat beers, the apparent gluten concentration values obtained by the G12 and Skerritt antibodies were typically higher than those obtained using the R5 antibodies. The sourdough bread samples resulted in higher apparent gluten concentration values with the Skerritt antibody, while the values generated by the G12 and R5 antibodies were comparable. Although the soy-based sauces showed non-specific inhibition with the multiple R5 and G12 antibodies, their overall profile was distinguishable from the other categories of fermented foods. Cluster analysis of the apparent gluten concentration values obtained by the multiplex competitive ELISA, as well as the relative response of the nine gluten-specific antibodies used in the assay to different gluten proteins/peptides, distinguishes among the different categories of fermented-hydrolyzed foods by recognizing the differences in the protein/peptide profiles characteristic of each product. This novel gluten-based multiplex competitive ELISA provides insight into the extent of proteolysis resulting from various fermentation processes, which is essential for accurate gluten quantification in fermented-hydrolyzed foods. Graphical abstract A novel multiplex competitive ELISA for the detection and characterization of gluten in fermented-hydrolyzed foods.

Cross-reactivity profiles of legumes and tree nuts using the xMAP® multiplex food allergen detection assay.

The homology between proteins in legumes and tree nuts makes it common for individuals with food allergies to be allergic to multiple legumes and tree nuts. This propensity for allergenic and antigenic cross-reactivity means that commonly employed commercial immunodiagnostic assays (e.g., dipsticks) for the detection of food allergens may not always accurately detect, identify, and quantitate legumes and tree nuts unless additional orthogonal analytical methods or secondary measures of analysis are employed. The xMAP® Multiplex Food Allergen Detection Assay (FADA) was used to determine the cross-reactivity patterns and the utility of multi-antibody antigenic profiling to distinguish between legumes and tree nuts. Pure legumes and tree nuts extracted using buffered detergent displayed a high level of cross-reactivity that decreased upon dilution or by using a buffer (UD buffer) designed to increase the stringency of binding conditions and reduce the occurrence of false positives due to plant-derived lectins. Testing for unexpected food allergens or the screening for multiple food allergens often involves not knowing the identity of the allergen present, its concentration, or the degree of modification during processing. As such, the analytical response measured may represent multiple antigens of varying antigenicity (cross-reactivity). This problem of multiple potential analytes is usually unresolved and the focus becomes the primary analyte, the antigen the antibody was raised against, or quantitative interpretation of the content of the analytical sample problematic. The alternative solution offered here to this problem is the use of an antigenic profile as generated by the xMAP FADA using multiple antibodies (bead sets). By comparing the antigenic profile to standards, the allergen may be identified along with an estimate of the concentration present. Cluster analysis of the xMAP FADA data was also performed and agreed with the known phylogeny of the legumes and tree nuts being analyzed. Graphical abstract The use of cluster analysis to compare the multi-antigen profiles of food allergens.

Multiplex detection of food allergens and gluten.

To help safeguard the food supply and detect the presence of undeclared food allergens and gluten, most producers and regulatory agencies rely on commercial test kits. Most of these are ELISAs with a few being PCR-based. These methods are very sensitive and analyte specific, requiring different assays to detect each of the different food allergens. Mass spectrometry offers an alternative approach whereby multiple allergens may be detected simultaneously. However, mass spectrometry requires expensive equipment, highly trained analysts, and several years before a quantitative approach can be achieved. Using multianalyte profiling (xMAP®) technology, a commercial multiplex test kit based on the use of established antibodies was developed for the simultaneous detection of up to 14 different food allergens plus gluten. The assay simultaneously detects crustacean seafood, egg, gluten, milk, peanut, soy, and nine tree nuts (almond, Brazil nut, cashew, coconut, hazelnut, macadamia, pine nut, pistachio, and walnut). By simultaneously performing multiple tests (typically two) for each analyte, this magnetic bead-based assay offers built-in confirmatory analyses without the need for additional resources. Twenty-five of the assays were performed on buffer extracted samples, while five were conducted on samples extracted using reduced-denatured conditions. Thus, complete analysis for all 14 allergens and gluten requires only two wells of a 96-well microtiter plate. This makes it possible to include in a single analytical run up to 48 samples. All 30 bead sets in this multiplex assay detected 5 ng/mL of food allergen and gluten with responses greater than background. In addition, 26 of the bead sets displayed signal/noise ratios of five or greater. The bead-based design makes this 30-plex assay expandable to incorporate new antibodies and capture/detector methodologies by ascribing these new detectors to any of the unassigned bead sets that are commercially available.

Distinction of Signals Generated by Allergens from Cross-Reactivity in Botanicals Used in Dietary Supplements and Spices Using the xMAP Food Allergen Detection Assay.

The xMAP Food Allergen Detection Assay (xMAP FADA) is a powerful analytical method by virtue of its ability to simultaneously detect multiple antigenic elements with a repertoire of antibodies targeting 15 food allergens plus gluten. Further, by incorporating multiple levels of redundancy, it can also be used to distinguish between homologous cross-reactive analytes. The power of its analytical capabilities is especially critical when working with botanicals. In this research, 95 botanicals used in dietary supplements and spices were analyzed for cross-reactivity with common food allergens and gluten using the xMAP FADA. Complementary antibody ratios were calculated, and, with most samples, ratios generated by homologous cross-reactive epitopes were easily distinguished from true reactivity. In very few cases, sample ratios were comparable to the ratios generated by the calibration standards, indicating the probable detection of relatively minor quantities of target food allergen. With the xMAP FADA, distinguishing signal indicating target allergen detection from cross-reactivity in botanicals is possible using redundant antibodies and multiple confirmatory end points.

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

The xMAP food allergen detection assay (xMAP FADA) is an advanced multiplex immunoassay with multiple antibodies for each of 15 target food allergens and gluten, allowing for signal confirmation and antigenic profiling to occur in a single analysis. Botanicals used as spices are complex matrices for allergen analysis because they can exhibit inherent cross-reactivity with antibodies employed by the assays. Preliminary examinations of botanicals revealed chili peppers to have notably high levels of cross-reactivity with Brazil nut and hazelnut antibody bead sets in the xMAP FADA. This in-depth investigation of 29 pre-ground and whole chili peppers indicated Brazil nut and hazelnut cross-reactivity to be consistent among most members of genusCapsicum, although cross-reactive signals generated by chili peppers were distinguishable from signals indicative of target allergen detection. Using the requirements that complementary antibodies used in the assay generated positive responses and that the various secondary end points were characteristic of the target analytes, xMAP FADA reactivity to chilis of the genus Capsicum was categorized as cross-reactivity instead of confirmed detection of target allergenic foods.

Multiplex-Competitive ELISA for Detection and Characterization of Gluten during Yogurt Fermentation: Effects of Changes in Certain Fermentation Conditions on Gluten Protein Profiles and Method Reproducibility Assessment.

The protein/peptide profiles of gluten during yogurt fermentation were evaluated using an optimized multiplex-competitive ELISA by preparing yogurts incurred with gluten at different concentrations and by varying certain fermentation conditions. Analysis indicated that epitope-specific responses with antibody binding to glutenin epitopes decreased less during longer fermentation times or at higher starter culture concentrations relative to gliadins. Incomplete proteolysis was observed after 24 h of fermentation, which became more efficient as fermentation time was increased. Western blot confirmed the results of ELISA. Cluster analysis indicated that out of the investigated parameters, fermentation time is the only parameter that could affect the overall gluten protein/peptide profiles during yogurt fermentation. This parameter needs consideration in evaluating the suitability of calibrant(s) to be used with the multiplex-competitive ELISA or any other methods to ensure accurate quantitation of gluten in yogurts and potentially in other foods with similar fermentation chemistry. A small-scale multilaboratory evaluation indicated that the multiplex-competitive ELISA has good analytical reproducibility (average interlaboratory % CV of 28-41%).

Detection of hazelnuts and almonds using commercial ELISA test kits.

Three commercial sandwich enzyme-linked immunosorbent assay (ELISA) test kits for the detection of hazelnuts and almonds were evaluated. Limits of detection and dynamic ranges were determined for hazelnuts and almonds spiked into cooked oatmeal, dipping chocolate, and muffins (baked). The limit of detection values varied from 1 to 38 µg/g, depending on the food matrix and ELISA test kit. Percent recoveries based on the standards supplied with the test kits varied from 10% to 170%. It is impossible to ascertain whether the percent recoveries reflect the performance of the ELISAs or differences between the protein content of the nuts used to spike the samples and the test kit standards. Unfortunately, reference materials do not exist that can be used to compare the results from different test kits and standardize the test kit standards. Also, insufficient knowledge regarding the epitope specificity of the antibodies used in the ELISAs further hinders interpretation of the results generated by the different test kits.

Enzyme-linked immunosorbent assay detection of melamine in infant formula and wheat food products.

The adulteration of food products with melamine to inflate the nitrogen content necessitates the establishment of analytical methods that can distinguish between proteinaceous ingredients and such adulterants. The specificity and ability to detect melamine by two commercial enzyme-linked immunosorbent assay (ELISA) kits were evaluated along with three protocols for sample preparation. Both ELISAs displayed cross-reactivity with ammeline, but neither was able to detect ammelide or cyanuric acid, indicating either a requirement for the 4,6-diamino-1,3,5-triazine structure or inability to bind 1,3,5-triazine-4,6-diones. The limits of detection for melamine in powder infant formula ranged from 0.2 to 3 microg/g depending on the ELISA kit and the method used to prepare the sample. The limits of detection for melamine in liquid infant formula and wheat products were <1 microg/ml and <2.5 microg/g, respectively. The ELISA kits provide an effective alternative for the analysis of samples suspected of containing melamine without relying on extensive sample preparation or expensive instrumentation.

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

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 intra-laboratory 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 (RSDR) values < 20%; presumably because the ratios are based on inherent properties of the antigenic elements. The excellent performance of the xMAP FADA when performed by analysts of varying proficiency indicates a reliability sufficient to meet analytical needs.

Robustness Testing of the xMAP Food Allergen Detection Assay: A Multiplex Assay for the Simultaneous Detection of Food Allergens.

ABSTRACT: 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 ß-mercaptoethanol in the reduced-denatured extraction buffer. The analysis of buffered-detergent extracts revealed lower responses at 22°C than at 37°C, but temperature had no effect on the analysis of reduced-denatured extracts. Changes in ß-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.

Extension of xMAP Food Allergen Detection Assay To Include Sesame.

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.

Application of deadenylase electrochemiluminescence assay for ricin to foods in a plate format.

A recently developed bead-based deadenylase electrochemiluminescence assay for ricin is simple and sensitive in its ability to detect ricin, based on the catalytic activity of the toxin subunit, ricin A chain. The assay was modified to work in a 96-well plate format and evaluated by using juice samples. The plate-based assay, unlike the bead-based assay, includes wash steps that enable the removal of food particles. These steps minimize matrix effects and improve the signal-to-noise ratios and limits of detection (LOD). The LOD values for ricin in apple juice, vegetable juice, and citrate buffer by using the bead-based assay were 0.4, 1, and 0.1 microg/ml, respectively. In contrast, the LOD values for ricin by using the plate-based assay were 0.04, 0.1, and 0.04 microg/ml in apple juice, vegetable juice, and citrate buffer, respectively. The plate-based assay displayed three- to 10-fold lower LOD values than did the bead-based assay. Signal-to-noise ratios for the plate-based assay were comparable to those for the bead-based assay for ricin in citrate buffer, but 2- to 4.5-fold higher when the plate-based assay was used for analysis of juice samples.

Detection and Quantitation of Gluten in Fermented-Hydrolyzed Foods by Antibody-Based Methods: Challenges, Progress, and a Potential Path Forward.

Celiac disease (CD) affects ~1 in 141 individuals in the United States, requiring adherence to a strict gluten-free diet. The Codex Standard and the European Commission states that gluten level of gluten-free foods must not exceed 20 ppm. The FDA requires food bearing the labeling claim "gluten-free" to contain <20 ppm gluten. Accurate quantitation of gluten in fermented-hydrolyzed foods by antibody-based methods is a challenge due to the lack of appropriate reference materials and variable proteolysis. The recent uses of proteases (e.g., proline endopeptidases or PEP) to hydrolyze immunopathogenic sequences of gluten proteins further complicates the quantitation of immunopathogenic gluten. The commercially available antibody-based methods routinely used to detect and quantitate gluten are not able to distinguish between different hydrolytic patterns arising from differences in fermentation processes. This is a severe limitation that makes accurate quantitation and, ultimately, a detailed evaluation of any potential health risk associated with consuming the food difficult. Utilizing gluten-specific antibodies, a recently developed multiplex-competitive ELISA along with western blot analysis provides a potential path forward in this direction. These complimentary antibody-based technologies provide insight into the extent of proteolysis resulting from various fermentation processes and have the potential to aid in the selection of appropriate hydrolytic calibration standards, leading to accurate gluten quantitation in fermented-hydrolyzed foods.

Single-Laboratory Validation of the Multiplex xMAP Food Allergen Detection Assay with Incurred Food Samples.

An xMAP Food Allergen Detection Assay (xMAP FADA) was developed to meet analytical needs when responding to complaints by individuals with multiple food allergies and to address potential ambiguities associated with cross-reactive proteins. A single-laboratory validation (SLV) was conducted to examine the reliability of the xMAP FADA to detect 15 analytes individually or as part of a mixture at more than six concentrations in four foods. The xMAP FADA reliably detected the analytes despite the incurred dark chocolate and incurred baked muffins displaying recoveries of 10-20% and <60%, respectively. The high reliability for recoveries less than 60% in part reflects the statistical strength of the design of the xMAP FADA. Only crustacean, egg, and milk incurred in dark chocolate were not reliably detected using the PBST-buffered-detergent protocol. Following the reduced-denatured protocol, no problems were encountered in the detection of milk, although egg did not display a dynamic response in dark chocolate. The ruggedness of the xMAP FADA was ascertained by the ability of novice analysts to detect food allergens in baked rice cookies. Despite one analyst losing >80% of the beads and the count for one bead set dropping to seven, the assay displayed only a decrease in precision (increased standard deviations) and a change in the ratios between complementary antibody pairs.

Rapid detection of ricin in cosmetics and elimination of artifacts associated with wheat lectin.

Ricin can be detected in cosmetics at 0.005 microg/mL in the analytical sample using lateral flow devices (LFDs). Wheat germ, an ingredient used in skin care products is also a potential source of wheat lectin. False positives were observed when wheat lectin was added to LFDs from two manufacturers, irrespective of whether the LFD was specific for ricin, Staphylococcus enterotoxin B (SEB), or botulinum toxin. In contrast, pea and peanut lectins did not cause false positives. Substitution of the buffer supplied with the LFDs with a buffer containing 2.5% non-fat milk powder eliminated the occurrence of false positives. This substitution increased the LOD to 0.01 microg/mL ricin, which is an acceptable level for screening cosmetics for contamination by ricin.

Identification of the RNA N-glycosidase activity of ricin in castor bean extracts by an electrochemiluminescence-based assay.

A simple electrochemiluminescence-based assay for RNA N-glycosidase activity has been modified to permit its use with authentic extracts of Ricinus communis (castor beans) and Abrus precatorius (jequirity seeds)--the natural sources of ricin and abrin. Modifications include the addition of an RNase inactivator to the reaction mixture, elimination of a signal-enhancing monoclonal antibody, and optimization of the incubation temperature. Concurrent testing with two substrates provides a diagnostic tool enabling castor bean toxins to be differentiated from a larger selection of N-glycosidase toxins than was previously examined.

Toxicity and detection of ricin and abrin in beverages.

The oral and intraperitoneal (i.p.) toxicities to female BALB/c mice of ricin and abrin in phosphate-buffered saline (PBS), spring water, apple juice, and half-and-half (only oral) were examined after brief (2 h) and prolonged (11 to 13 days) storage. The ricin and abrin samples prepared in PBS had oral toxicities consistent with those previous studies, indicating oral and i.p. 50% lethal doses of > 1 mg/kg of body weight and between 2 and 20 microg/kg of body weight, respectively. The toxicities of ricin and abrin in PBS were greater than those in apple juice and water. The oral toxicity of ricin and abrin in half-and-half appeared comparable to or less than that observed for the toxins in water. Spiked samples stored for a maximum of 11 days (13 for the abrin samples) at 4 degrees C induced similar numbers of fatalities as did samples stored for only 2 h. Enzyme-linked immunosorbent assays of the samples administered by i.p. injection indicated a decrease in detectable toxin at 0.5 microg/ml.