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.

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.

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%).

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.

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.

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.

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.

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.

Application of Multiantigen Profiling To Detect Pecan.

A problem often encountered in the detection and identification of undeclared tree nut food allergens is the lack of analytical methods. This problem is accentuated by the current trend, whereby the primary methods used to detect food allergens are antibody-based enzyme-linked immunosorbent assays (ELISAs) and the development of analyte-specific antibodies takes months. The recently developed xMAP food allergen detection assay (xMAP FADA) has the ability to generate multiantigen profiles with tree nuts, thereby providing a potential solution to this problem. The xMAP FADA includes 22 antibodies targeting peanut, soy, and nine tree nuts. The high number of antibodies to a diverse group of tree nuts and legumes and the propensity of tree nuts to cross-react have enabled the development of multiantigen profiling, whereby an analyte reacts with the various antibodies to generate a profile. Recently, a question arose regarding the possible presence of pecan dust at a manufacturer of pecan products that also stored fresh produce. The lack of suitable pecan ELISAs created an analytical challenge that was resolved using multiantigen profiling with the xMAP FADA. Pecan was detected on swab samples by using multiantigen profiling and confirmed by DNA analysis. The use of multiantigen profiling provided an analytical capability beyond what was possible with an analyte-specific analytical method.

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.

Detection and Antigenic Profiling of Undeclared Peanut in Imported Garlic Using an xMAP Multiplex Immunoassay for Food Allergens.

A shipment of imported garlic powder was suspected of containing peanut. Samples (subs) collected from the shipment displayed considerable variability in peanut antigenicity when analyzed by enzyme-linked immunosorbent assay (ELISA). This raised questions regarding whether peanut was actually present, the amount present, and the basis for the variability in antigenic content. Analyses that used an xMAP multiplex assay for the detection of peanut and additional food allergens generated responses that were characteristic of peanut. Specifically, the relative intensities of two different peanut-specific antibodies coupled to beads (peanut-37 and -38) and the antigen profiles were identical to garlic controls spiked with peanut. In addition, the xMAP data did not indicate the presence of other allergens. Quantitative analyses indicated an approximately fivefold variation in peanut concentration among different subs. In contrast, within a sub, the apparent peanut concentration appeared constant. Particle size analyses of the garlic powder subs indicated a single distribution profile, with a peak at 380 µm. ELISA analysis of sieve-fractionated garlic powder from one of the subs indicated that slightly less than half of the detectable peanut was smaller than 212 µm, with the remainder almost evenly split between 212 and 300 µm and >300 µm. Modeling to predict possible oral exposure levels of peanut other than those directly measured requires additional research on the physicochemical properties of peanut and garlic, along with information on the production of the garlic powder.

Detection of Gluten during the Fermentation Process To Produce Soy Sauce.

Advances have been made to provide people with celiac disease (CD) access to a diverse diet through an increase in the availability of gluten-free food products and regulations designed to increase label reliability. Despite advances in our knowledge regarding CD and analytical methods to detect gluten, little is known about the effects of fermentation on gluten detection. The enzyme-linked immunosorbent assay (ELISA) and lateral flow devices routinely used by analytical laboratories and regulatory agencies to test for the presence of gluten in food were examined for their ability to detect gluten during the fermentation processes leading to the production of soy sauce, as well as in finished products. Similar results were observed irrespective of whether the soy sauce was produced using pilot-plant facilities or according to a homemade protocol. In both cases, gluten was not detected after moromi (brine-based) fermentation, which is the second stage of fermentation. The inability to detect gluten after moromi fermentation was irrespective of whether the assay used a sandwich configuration that required two epitopes or a competitive configuration that required only one epitope. Consistent with these results was the observation that ELISA, lateral flow devices, and Western immunoblot analyses were unable to detect gluten in commercial soy sauce, teriyaki sauce, and Worcestershire sauce. Although reports are lacking on problems associated with the consumption of fermented soy-containing sauces by consumers with CD, additional research is needed to determine whether all immunopathogenic elements in gluten are hydrolyzed during soy sauce production.

A Limited Survey of Dark Chocolate Bars Obtained in the United States for Undeclared Milk and Peanut Allergens.

Undeclared allergens in chocolate products have been responsible for numerous allergen-related recalls in the United States. A survey was conducted to determine the prevalence of undeclared milk and peanut in 88 and 78 dark chocolate bars, respectively. Concentrations of milk (as nonfat dry milk) or peanut in three samples of each chocolate product were determined with two milk- or peanut-specific enzyme-linked immunosorbent assay kits. In 75% of the chocolate bar products with a milk advisory statement, milk concentrations were above the limit of quantitation (2.5 µg/g [ppm]), with the majority having concentrations >1,000 ppm. An additional 67% of chocolate bars with a "traces of milk" statement contained 3 to 6,700 ppm of milk. Fifteen percent of chocolates labeled dairy free or lactose free and 25% labeled vegan were positive for milk, all with concentrations >1,000 ppm. Even for chocolates with no reference to milk on the label, 33% of these products contained 60 to 3,400 ppm of milk. The survey of chocolate products for peanuts revealed that 8% of products with an advisory statement contained peanut, with the highest concentration of 550 ppm. All nine chocolates bearing the peanut-free or allergen-free statement were negative for peanut, but 17% of chocolates with no label statement for peanut were positive for peanut at concentrations of 9 to 170 ppm. Evaluation of multiple lots of four chocolate products revealed that milk was consistently present or absent for the products investigated, but mixed results were obtained when multiple lots were tested for peanut. This study indicates that a large proportion of dark chocolate bars contain undeclared milk. The type of advisory statement or the absence of a milk advisory statement on products did not predict the amount or absence of milk protein. In contrast, a lower proportion of chocolates containing undeclared peanut was found. Consumers with food allergies should be cautious when purchasing dark chocolate products, particularly those that have an advisory label statement.

Presence of Undeclared Food Allergens in Cumin: The Need for Multiplex Methods.

Beginning in the autumn of 2014, millions of dollars of food and over 675 products were recalled in the United States due to the presence of undeclared peanut, attributed to cumin used in the manufacture of the products. Initial analyses also indicated the presence of almond. Subsequent research showed that the presence of peanut and almond did not fully explain the analytical results for the cumin samples. Using a combination of mass spectrometry, DNA-based methods (i.e., PCR and Sanger DNA Sequencing), microscopy, and antibody-based technologies (i.e., ELISA, Western blot analysis, and a novel xMAP multiplex assay) the presence of peanut was confirmed. Screening for secondary sources of adulteration (e.g., tree nuts, mahleb, peach, and cherry) supported the assessment that the cumin contained multiple contaminants. These results demonstrate the limitations of single analyte-specific assays and the need for orthogonal multiplex methods to detect food allergens irrespective of varietal or other differences.

Effects of a Proline Endopeptidase on the Detection and Quantitation of Gluten by Antibody-Based Methods during the Fermentation of a Model Sorghum Beer.

The effectiveness of a proline endopeptidase (PEP) in hydrolyzing gluten and its putative immunopathogenic sequences was examined using antibody-based methods and mass spectrometry (MS). Based on the results of the antibody-based methods, fermentation of wheat gluten containing sorghum beer resulted in a reduction in the detectable gluten concentration. The addition of PEP further reduced the gluten concentration. Only one sandwich ELISA was able to detect the apparent low levels of gluten present in the beers. A competitive ELISA using a pepsin-trypsin hydrolysate calibrant was unreliable because the peptide profiles of the beers were inconsistent with that of the hydrolysate calibrant. Analysis by MS indicated that PEP enhanced the loss of a fragment of an immunopathogenic 33-mer peptide in the beer. However, Western blot results indicated partial resistance of the high molecular weight (HMW) glutenins to the action of PEP, questioning the ability of PEP in digesting all immunopathogenic sequences present in gluten.

Application of Microwave Irradiation and Heat to Improve Gliadin Detection and Ricin ELISA Throughput with Food Samples.

The utility of microwave irradiation to accelerate the onset of equilibrium and improve ELISA performance was examined using ELISAs for the detection of the plant toxin ricin and gliadin. The ricin ELISA normally requires several one hour incubations at 37 °C, a total assay time of approximately five hours, and employs a complex buffer containing PBS, Tween-20®, and non-fat milk. Different energy levels and pulse designs were compared to the use of abbreviated incubation times at 37 °C for the detection of ricin in food. The use of microwave irradiation had no significant advantage over the application of heat using an oven incubator and performed worse with some foods. In contrast, a gliadin ELISA that relied on 30 min incubation steps at room temperature and a salt-based buffer performed better upon irradiation but also displayed improvement upon incubating the microtiter plate at 37 °C. Whether microwave irradiation was advantageous compared to incubation in an oven was inconclusive. However, by abbreviating the incubation time of the ricin ELISA, it was possible to cut the assay time to less than 2 hours and still display LOD values < 10 ppb and recoveries of 78%-98%.