Analysis of Gluten in Dried Yeast and Yeast-Containing Products.

Yeast are commonly used in the preparation of foods and beverages such as beer and bread and may also be used on their own as a source of nutrients and flavoring. Because of the historical connection of yeast to products made from wheat and barley, consumers maintaining a gluten-free diet can have concerns about the safety of yeast ingredients. Analyzing the safety of yeast and yeast-containing products presents some difficulties, as the yeast organisms actively degrade any gluten in the product, raising questions on the appropriateness of detection by traditional antibody-based methods. This study examines a variety of yeast and yeast-containing products by competitive ELISA and liquid chromatography-mass spectrometry for the estimated level of gluten proteins. While samples such as yeast extracts and nutritional yeast contained gluten levels below the 20 mg/kg (or parts per million, ppm) threshold defined by Codex Alimentarius, one baking yeast and a nutritional yeast supplement sample contained higher levels of gluten. This study demonstrates that both competitive ELISA and liquid chromatography-mass spectrometry provide similar results in the detection of wheat and barley gluten in yeast-containing products.

The Use of Visual Examination for Determining the Presence of Gluten-Containing Grains in Gluten Free Oats and Other Grains, Seeds, Beans, Pulses, and Legumes.

Obtaining representative test samples for antibody-based testing is challenging when analyzing whole grains for gluten. When whole grains are ground into flour for testing, confocal microscopy studies have shown that gluten tends to exist as aggregates within the starch background, making single-sample testing inaccurate and complicating the ability to arrive at an accurate average from multiple samples. In addition, whole-grain products present a unique risk to gluten free consumers, in that any contamination is localized to specific servings rather than being distributed across the product lot. This makes parts-per-million values less relevant for whole-grain products. Intact grains, seeds, beans, pulses, and legumes offer an alternative opportunity for gluten detection, in that contaminating gluten-containing grains (GCGs) are visible and identifiable to the trained eye or properly calibrated optical sorting equipment. The purpose of the current study was to determine a Gluten Free Certification Organization threshold level for the maximum number of GCGs within a kilogram of nongluten grains sold as specially processed gluten free product and to determine the feasibility of this threshold by evaluating visual examination data from two major oat processors.

Stakeholders' Guidance Document for Consumer Analytical Devices with a Focus on Gluten and Food Allergens.

Until recently, analytical tests for food were performed primarily in laboratories, but technical developments now enable consumers to use devices to test their food at home or when dining out. Current consumer devices for food can determine nutritional values, freshness, and, most recently, the presence of food allergens and substances that cause food intolerances. The demand for such products is driven by an increase in the incidence of food allergies, as well as consumer desire for more information about what is in their food. The number and complexity of food matrixes creates an important need for properly validated testing devices with comprehensive user instructions (definitions of technical terms can be found in ISO 5725-1:1994 and the International Vocabulary of Metrology). This is especially important with food allergen determinations that can have life-threatening consequences. Stakeholders-including food regulators, food producers, and food testing kit and equipment manufacturers, as well as representatives from consumer advocacy groups-have worked to outline voluntary guidelines for consumer food allergen- and gluten-testing devices. These guidelines cover areas such as kit validation, user sampling instructions, kit performance, and interpretation of results. The recommendations are based on (1) current known technologies, (2) analytical expertise, and (3) standardized AOAC INTERNATIONAL allergen community guidance and best practices on the analysis of food allergens and gluten. The present guidance document is the first in a series of papers intended to provide general guidelines applicable to consumer devices for all food analytes. Future publications will give specific guidance and validation protocols for devices designed to detect individual allergens and gluten, as statistical analysis and review of any validation data, preferably from an independent third party, are necessary to establish a device's fitness-for-purpose. Following the recommendations of these guidance documents will help ensure that consumers are equipped with sufficient information to make an informed decision based on an analytical result from a consumer device. However, the present guidance document emphasizes that consumer devices should not be used in isolation to make a determination as to whether a food is safe to eat. As advances are made in science and technology, these recommendations will be reevaluated and revised as appropriate.

The Use of Visual Examination for Determining the Presence of Gluten-Containing Grains in Gluten Free Oats and Other Grains, Seeds, Beans, Pulses, and Legumes.

Obtaining representative test samples for antibody-based testing is challenging when analyzing whole grains for gluten. When whole grains are ground into flour for testing, confocal microscopy studies have shown that gluten tends to exist as aggregates within the starch background, making single-sample testing inaccurate and complicating the ability to arrive at an accurate average from multiple samples. In addition, whole-grain products present a unique risk to gluten free consumers, in that any contamination is localized to specific servings rather than being distributed across the product lot. This makes parts-per-million values less relevant for whole-grain products. Intact grains, seeds, beans, pulses, and legumes offer an alternative opportunity for gluten detection, in that contaminating gluten-containing grains (GCGs) are visible and identifiable to the trained eye or properly calibrated optical sorting equipment. The purpose of the current study was to determine a Gluten Free Certification Organization threshold level for the maximum number of GCGs within a kilogram of nongluten grains sold as specially processed gluten free product and to determine the feasibility of this threshold by evaluating visual examination data from two major oat processors.

The Celiac Patient Antibody Response to Conventional and Gluten-Removed Beer.

Enzymatic digestion, or hydrolysis, has been proposed for treating gluten-containing foods and beverages to make them safe for persons with celiac disease (CD). There are no validated testing methods that allow the quantitation of all the hydrolyzed or fermented gluten peptides in foods and beverages that might be harmful to CD patients, making it difficult to assess the safety of hydrolyzed products. This study examines an ELISA-based method to determine whether serum antibody binding of residual peptides in a fermented barley-based product is greater among active-CD patients than a normal control group, using commercial beers as a test case. Sera from 31 active-CD patients and 29 nonceliac control subjects were used to assess the binding of proteins from barley, rice, traditional beer, gluten-free beer, and enzymatically treated (gluten-removed) traditional beer. In the ELISA, none of the subjects' sera bound to proteins in the gluten-free beer. Eleven active-CD patient serum samples demonstrated immunoglobulin A (IgA) or immunoglobulin G (IgG) binding to a barley extract, compared to only one nonceliac control subject. Of the seven active-CD patients who had an IgA binding response to barley, four also responded to traditional beer, and two of these responded to the gluten-removed beer. None of the nonceliac control subjects' sera bound to all three beer samples. Binding of protein fragments in hydrolyzed or fermented foods and beverages by serum from active-CD patients, but not nonceliac control subjects, may indicate the presence of residual peptides that are celiac-specific.

Evaluation of qualitative and quantitative immunoassays to detect barley contamination in gluten-free beer with confirmation using LC/MS/MS.

To meet the need for the detection and quantitation of barley gluten in beer, qualitative screening and quantitative immunoassays based on the monoclonal antigluten antibody 401/21 (Skerritt) were validated in a single laboratory. Sample replicates were tested at each stage of beer production using multiple yeast strains and methods of end-stage protein removal. Quantitation was performed using barley-specific standards based on barley flour extracts. Immunoassay results were confirmed using LC/MS/MS for barley-specific peptides. The LOD for the qualitative screening test was 5 mg/L barley gluten. Recovery for the barley-spiked worts ranged from 81 to 128% in the quantitative ELISA assay; the LOD was <1 mg/L, and the LOQ was 5 mg/L. Both screening and confirmation methods were found to be fit for the purposes of detection of low levels of barley gluten in beer.

EZ Gluten for the qualitative detection of gluten in foods, beverages, and environmental surfaces.

The EZ Gluten assay is a rapid immunochromatographic screening method for qualitative detection of gluten in raw and cooked foods and beverages and on environmental surfaces. This AOAC Performance Tested Method study evaluated the EZ Gluten assay as an effective method for the detection of gluten in four selected matrixes: rice flour, cooked dough, beer, and dog food. In addition, the method was evaluated for its effectiveness in detecting gluten contamination of > or =1 microg/2 in.2 (25 cm2) stainless steel surface area. The EZ Gluten demonstrated 100% specificity [probability of detection (POD) 0.00, confidence interval (CI) 0.00-0.01] and 99% sensitivity (POD 0.99, CI 0.97-0.995) at the 10 ppm level for all four matrixes, and 100% specificity (POD 0.00, CI 0.00-0.11) and sensitivity (POD 1.00, CI 0.886-1.00) at the 1 microg level on the stainless steel surface. Independent laboratory testing confirmed the internal validation results in one matrix and on the stainless steel surface. Lot-to-lot, stability, and robustness studies provided evidence that the EZ Gluten is a rugged, consistent method for the detection of gluten at levels as low as 10 ppm.

The ARID family transcription factor bright is required for both hematopoietic stem cell and B lineage development.

Bright/Arid3a has been characterized both as an activator of immunoglobulin heavy-chain transcription and as a proto-oncogene. Although Bright expression is highly B lineage stage restricted in adult mice, its expression in the earliest identifiable hematopoietic stem cell (HSC) population suggests that Bright might have additional functions. We showed that >99% of Bright(-/-) embryos die at midgestation from failed hematopoiesis. Bright(-/-) embryonic day 12.5 (E12.5) fetal livers showed an increase in the expression of immature markers. Colony-forming assays indicated that the hematopoietic potential of Bright(-/-) mice is markedly reduced. Rare survivors of lethality, which were not compensated by the closely related paralogue Bright-derived protein (Bdp)/Arid3b, suffered HSC deficits in their bone marrow as well as B lineage-intrinsic developmental and functional deficiencies in their peripheries. These include a reduction in a natural antibody, B-1 responses to phosphocholine, and selective T-dependent impairment of IgG1 class switching. Our results place Bright/Arid3a on a select list of transcriptional regulators required to program both HSC and lineage-specific differentiation.

Recognition of gliadin and glutenin fractions in four commercial gluten assays.

Gluten sensitivity affects nearly 1% of the population in the United States and Europe. To help these consumers avoid the health issues that result from gluten consumption, the U.S. Food and Drug Administration is attempting to establish a definition and testing protocol for gluten-free foods. Establishing this protocol depends on accurate tests that can detect and quantitate gluten. There are multiple immunoassays available for the quantitation of gluten, and most are based on one of two antibodies. These antibodies, known as the Skerritt and R5 antibodies, were examined through the use of four commercial test kits for their ability to detect the two main components of gluten, known as gliadin and glutenin, in wheat. Commercial tests based on the Skerritt and R5 antibodies demonstrated differing affinities for gliadin and glutenin, with the Skerritt-based tests recognizing glutenins more strongly, and the R5 tests recognizing gliadins more strongly. Analysis of 40 processed food samples of unknown gluten content revealed differences in gluten detection and quantitation between the Skerritt-based and R5-based assays. These discrepancies in test results may be the result of the antibody affinity differences between the Skerritt- and R5-based tests, the solubility differences between gliadins and glutenins, or a combination of these and other factors.

Foxp1 is an essential transcriptional regulator of B cell development.

Forkhead transcription factors are key participants in development and immune regulation. Here we demonstrate that absence of the gene encoding the forkhead transcription factor Foxp1 resulted in a profound defect in early B cell development. Foxp1 deficiency was associated with decreased expression of all B lineage genes in B220+ fetal liver cells as well as with a block in the transition from pro-B cell to pre-B cell involving diminished expression of recombination-activating genes 1 and 2. Foxp1 bound to the Erag enhancer and was involved in controlling variable-(diversity)-joining recombination of the gene encoding immunoglobulin heavy chain in a B cell lineage-specific way. Our results identify Foxp1 as an essential participant in the transcriptional regulatory network of B lymphopoiesis.