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%).

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.

The Effect of Different Methods of Fermentation on the Detection of Milk Protein Residues in Retail Cheese by Enzyme-Linked Immunosorbent Assay (ELISA).

Milk and milk products are among the most important allergenic food ingredients, both in the United States and throughout the world; cheeses are among the most important of these milk products. Milk contains several major antigenic proteins, each with differing susceptibilities to proteolytic enzymes. The extent of proteolysis in cheese varies as a result of conditions during manufacture and ripening. Proteolysis has the potential to degrade antigenic and allergenic epitopes that are important for residue detection and elicitation of allergic reactions. Commercial enzyme-linked immunosorbent assays (ELISAs) are not currently validated for use in detecting residues in hydrolyzed or fermented food products. Eighteen retail cheeses produced using 5 different styles of fermentation were investigated for detectable milk protein residues with 4 commercial ELISA kits. Mozzarella, Swiss, Blue, Limburger, and Brie cheeses were assessed. The Neogen Veratox® Casein and Neogen Veratox® Total Milk kits were capable of detecting milk residues in most cheeses evaluated, including blue-veined cheeses that exhibit extensive proteolysis. The other 2 ELISA kits evaluated, r-Biopharm® Fast Casein and ELISA Systems™ Casein, can detect milk residues in cheeses other than blue-veined varieties. ELISA results cannot be quantitatively compared among kits. The quantitative reliability of ELISA results in detection of cheese residues is questionable, but some methods are sufficiently robust to use as a semi-quantitative indication of proper allergen control for the validation of cleaning programs in industry settings. PRACTICAL APPLICATION: Many commercially available enzyme-linked immunosorbent assays (ELISAs) are not validated for detection of allergenic residues in fermented or hydrolyzed products. This research seeks to determine if commercial milk ELISAs can detect milk residues in varieties of cheese that have undergone different styles of fermentation and different degrees of proteolysis. Only certain milk ELISA kits are capable of detecting residues in all varieties of cheese. However, commercial milk ELISA kits are capable of semiquantitative detection of cheese residues in foods, or in industry settings for the validation of allergen cleaning programs.

Effect of proteolysis during Cheddar cheese aging on the detection of milk protein residues by ELISA.

Cow milk is a common allergenic food, and cow milk-derived cheese retains an appreciable level of allergenicity. The specific and sensitive detection of milk protein residues in foods is needed to protect milk-allergic consumers from exposure to undeclared milk protein residues contained in foods made with milk or milk-derived ingredients or made on shared equipment or in shared facilities with milk or milk-derived ingredients. However, during cheese ripening, milk proteins are degraded by chymosin and milk-derived and bacterial proteases. Commercial allergen-detection methods are not validated for the detection of residues in fermented or hydrolyzed products. The objective of this research was to evaluate commercially available milk ELISA kits for their capability to detect milk protein residues in aged Cheddar cheese. Cheddar cheese was manufactured at a local dairy plant and was aged at 5°C for 24 mo, with samples removed at various time points throughout aging. Milk protein residues and protein profiles were measured using 4 commercial milk ELISA kits and sodium dodecyl sulfate-PAGE. The ELISA data revealed a 90% loss of milk protein residue signal between the youngest and oldest Cheddar cheese samples (0.5 and 24 mo, respectively). Sodium dodecyl sulfate-PAGE analysis showed protein degradation throughout aging, with the highest level of proteolysis observed at 24 mo. Results suggest that current commercial milk ELISA methods can detect milk protein residues in young Cheddar cheese, but the detection signal dramatically decreases during aging. The 4 evaluated ELISA kits were not capable of detecting trace levels of milk protein residues in aged cheese. Reliable detection of allergen residues in fermented food products is critical for upholding allergen-control programs, maintaining product safety, and protecting allergic consumers. Furthermore, this research suggests a novel use of ELISA kits to monitor protein degradation as an indication of cheese ripening.

Commercial Milk Enzyme-Linked Immunosorbent Assay (ELISA) Kit Reactivities to Purified Milk Proteins and Milk-Derived Ingredients.

Numerous commercial enzyme-linked immunosorbent assay (ELISA) kits exist to quantitatively detect bovine milk residues in foods. Milk contains many proteins that can serve as ELISA targets including caseins (α-, ß-, or κ-casein) and whey proteins (α-lactalbumin or ß-lactoglobulin). Nine commercially-available milk ELISA kits were selected to compare the specificity and sensitivity with 5 purified milk proteins and 3 milk-derived ingredients. All of the milk kits were capable of quantifying nonfat dry milk (NFDM), but did not necessarily detect all individual protein fractions. While milk-derived ingredients were detected by the kits, their quantitation may be inaccurate due to the use of different calibrators, reference materials, and antibodies in kit development. The establishment of a standard reference material for the calibration of milk ELISA kits is increasingly important. The appropriate selection and understanding of milk ELISA kits for food analysis is critical to accurate quantification of milk residues and informed risk management decisions.