Identifying key priorities for research to protect the consumer with food hypersensitivity: A UK Food Standards Agency Priority Setting Exercise.

INTRODUCTION: Food hypersensitivity (FHS), including food allergy, coeliac disease and food intolerance, is a major public health issue. The Food Standards Agency (FSA), an independent UK Government department working to protect public health and consumers' wider interests in food, sought to identify research priorities in the area of FHS. METHODS: A priority setting exercise was undertaken, using a methodology adapted from the James Lind Alliance-the first such exercise with respect to food hypersensitivity. A UK-wide public consultation was held to identify unanswered research questions. After excluding diagnostics, desensitization treatment and other questions which were out of scope for FSA or where FSA was already commissioning research, 15 indicative questions were identified and prioritized by a range of stakeholders, representing food businesses, patient groups, health care and academia, local authorities and the FSA. RESULTS: 295 responses were received during the public consultation, which were categorized into 70 sub-questions and used to define 15 key evidence uncertainties ('indicative questions') for prioritization. Using the JLA prioritization framework, this resulted in 10 priority uncertainties in evidence, from which 16 research questions were developed. These could be summarized under the following 5 themes: communication of allergens both within the food supply chain and then to the end consumer (ensuring trust in allergen communication); the impact of socio-economic factors on consumers with FHS; drivers of severe reactions; mechanism(s) underlying loss of tolerance in FHS; and the risks posed by novel allergens/processing. DISCUSSION: In this first research prioritization exercise for food allergy and FHS, key priorities identified to protect the food-allergic public were strategies to help allergic consumers to make confident food choices, prevention of FHS and increasing understanding of socio-economic impacts. Diagnosis and treatment of FHS was not considered in this prioritization.

Microfluidic Separation Coupled to Mass Spectrometry for Quantification of Peanut Allergens in a Complex Food Matrix.

Peanut is an important food allergen, but it cannot currently be reliably detected and quantified in processed foods at low levels. A level of 3 mg protein/kg is increasingly being used as a reference dose above which precautionary allergen labeling is applied to food products. Two exemplar matrices (chocolate dessert and chocolate bar) were prepared and incurred with 0, 3, 10, or 50 mg/kg peanut protein using a commercially available lightly roasted peanut flour ingredient. After simple buffer extraction employing an acid-labile detergent, multiple reaction monitoring (MRM) experiments were used to assess matrix effects on the detection of a set of seven peptide targets derived from peanut allergens using either conventional or microfluidic chromatographic separation prior to mass spectrometry. Microfluidic separation provided greater sensitivity and increased ionization efficiency at low levels. Individual monitored transitions were detected in consistent ratios across the dilution series, independent of matrix. The peanut protein content of each sample was then determined using ELISA and the optimized MRM method. Although other peptide targets were detected with three transitions at the 50 mg/kg peanut protein level in both matrices, only Arah2(Q6PSU2)147-155 could be quantified reliably and only in the chocolate dessert at 10 mg/kg peanut protein. Recoveries were consistent with ELISA analysis returning around 30-50% of the incurred dose. MS coupled with microfluidic separation shows great promise as a complementary analytical tool for allergen detection and quantification in complex foods using a simple extraction methodology.

Gluten in Rice Flour and Baked Rice Products by G12 Sandwich ELISA: First Action 2014.03.

The Protein and Enzymes Technical Committee of American Association of Cereal Chemists initiated a collaborative study to confirm whether the G12 antibody-based sandwich ELISA test kit is able to detect gluten in the lower mg/kg (ppm) level. Twenty laboratories investigated 24 heat-treated and non-heat-treated blind-coded samples with incurred gluten levels up to 100 mg/kg. The method has been validated for testing foods to conform to the defined Codex thresholds for gluten in gluten-free products at less than 20 mg gluten/kg. The collaborative study showed that low levels of gluten could be detected by G12 Sandwich ELISA with reproducibility RSDR of 32% and repeatability RSDr of 16%. Incurred samples showed a recovery between 62 and 135%. It is recommended that the method be accepted by AOAC as Official First Action.

Validation of the AgraStrip® gluten G12 test kit.

Romer Labs , Inc. developed an immunochromatographic lateral flow assay for the qualitative detection of gluten in raw ingredients, processed foods, finished food products, and environmental surfaces, using the G12 antibody developed by Belén Morón. The G12 antibody targets a 33-mer peptide which is resistant to enzymatic digestion and heat denatiuration, as well as being the fragment of the gliadin protein.to which celiac disease sufferers react, making it a reliable analytical marker. This study was performed to validate the AgraStrip® GlutenG12 assay method under the guidance of the AOAC Peiformance Tested MethodsSM (PTM) program against AOAC Official Method of AnalysisSM 2012.01 in rice flour, bread, cookie, ice cream, and chocolate matrixes spiked with either purified gliadin or wheat gluten standard at 0, 3, 8, 15, and 25 ppm concentrations and tested at the 5, 10, and 20 ppm assay thresholds, as well as on, environmental surfaces. Stability, robustness, variation, and lot consistency studies were performed by spiking wheat gluten into a rice flour matrix at 0 and 15 ppm concentrations. The AgraStrip Gluten G12 assay was rigorously evaluated during this study and demonstrates its suitability as an AOAC PTM-certified gluten detection method.

Consumer-Led Investigation into Potential Issues That Arise When Testing Dairy Matrixes for Gluten With the NIMA Sensor.

BACKGROUND: Some consumers with celiac disease use personal, point-of-use gluten detection devices to test food. False-positive results may occur due to sampling, matrix effects, and sensor issues. OBJECTIVE: The purpose of the present study was to determine if the positive gluten results some users were obtaining when testing cream cheese and materials of similar consistency were false positives and, if so, what might be causing them to occur. METHODS: Cream cheese, soft cheese, and yogurt were tested for gluten using the Ridascreen Gliadin R7001 sandwich R5 ELISA and the Ridascreen Gliadin R7021 competitive R5 ELISA. Two test portions were taken, extracted, and tested from each homogenized material. Materials were also analyzed for gluten using a NIMA sensor, a personal, point-of-use gluten detection device. Multiple test portion weights were tested beginning at 0.13 to 0.17 g (the ideal weight of the test portion according to the NIMA sensor development team). RESULTS: Using the sandwich R5 ELISA and the competitive R5 ELISA, all materials tested below the lower LOD for gluten. Using a NIMA sensor, as the weight of the test portion tested increased, sensor results went from no gluten found, to gluten found, to no test result. CONCLUSION: The gluten found results using the NIMA sensor are likely false positives that appear to correspond with the weight and volume of the material tested, as well as the viscosity. There is also an apparent disconnect between the gluten found result reported by the sensor and an interpretation of the lateral flow device (LFD) strip result when assessed by eye which should also be taken into account. Ideally, NIMA sensor users should be advised on the weight amount of material to analyze and test portions should be weighed before being used with the NIMA sensor. However, this is not a practical solution when testing in many environments, including restaurants. HIGHLIGHTS: Slight variations in weight and volume of test materials can result in false positive results when testing dairy matrixes for gluten using the Nima sensor.

A chromatographic and immunoprofiling approach to optimising workflows for extraction of gluten proteins from flour.

Ingestion of gluten proteins from wheat, and related prolamin proteins from barley, rye, and oats, can cause adverse reactions in individuals with coeliac disease and IgE-mediated allergies. As there is currently no cure for these conditions, patients must practice avoidance of gluten-containing foods. In order to support patients in making safe food choices, foods making a "gluten-free" claim must contain no more than 20 mg/Kg of gluten. Mass spectrometry methods have the potential to provide an alternative method for confirmatory analysis of gluten that is complementary to analysis currently undertaken by immunoassay. As part of the development of such methodology the effectiveness of two different extraction procedures was investigated using wholemeal wheat flour before and after defatting with water-saturated butan-1-ol. A single step extraction with 50 % (v/v) propan-2-ol containing 2 M urea and reducing agent (buffer 1) was compared with a two-step extraction using 60 % (v/v) aqueous ethanol (buffer 2) followed by re-extraction of the pellet using buffer 1, using either wheel mixing under ambient conditions (19 °C) or sonication at 60 °C. The procedures were compared based on total protein extraction efficiency and the composition of the extracts determined using a combination of HPLC, SDS-PAGE and immunoblotting with a panel of four gluten-specific monoclonal antibodies. Defatting generally had a detrimental effect on extraction efficiency and sonication at 60 °C only improved extraction efficiency with buffer 2. Although the single-step and two-step procedures were equally effective at extracting protein from the samples, analysis of extracts showed that the two-step method gave a more complete extraction of gluten proteins. Future studies will compare the effectiveness of these procedures when applied in the sample workflows for mass spectrometry based methods for determination of gluten in food.

Characterization of G12 sandwich ELISA, a next-generation immunoassay for gluten toxicity.

In this work, a monoclonal antibody called G12, raised against the most immunotoxic peptide to celiac disease patients, was used to develop a sandwich ELISA. Preliminary results on cross-reactivities, recoveries, and extraction methods of the new assay are presented. The assay calibration was performed using material from the Prolamin Working Group. The antibody's specificity was determined by crossreactivity studies on different grains, nuts, oils, and starches. Recovery of the assay was determined by spiking experiments on common food matrixes, as well as on problematic matrixes. Furthermore, sample extraction methods using ethanol, cocktail solution, and a proprietary buffer have been compared.

Sensitization with 7S globulins from peanut, hazelnut, soy or pea induces IgE with different biological activities which are modified by soy tolerance.

BACKGROUND: It is not known why some foods sensitizing via the gastrointestinal tract are prevalent allergenic foods and others are not. Eating habits, processing, and the food matrix have been suggested to influence the allergenicity of a given food. Factors related to protein structure, such as stability to digestion, have also been suggested. 7S globulins from peanut, hazelnut, soy, and pea were studied to determine whether related proteins would induce a similar sensitization when removed from their 'normal' matrix. METHODS: Brown Norway rats (soy tolerant or nontolerant) were immunized i.p. 3 times with 100 µg purified peanut, hazelnut, soy, or pea 7S without adjuvant. Sera were analyzed for specific antibodies by different ELISAs (IgG1, IgG2a, and IgE), inhibition ELISA, and rat basophilic leukemia cell assay. RESULTS: The 4 related 7S globulins induced a response with an almost identical level of specific antibodies, but peanut 7S induced IgE of higher avidity than hazelnut and pea 7S which, again, had a higher avidity than IgE induced by soy 7S. Soy tolerance reduced the functionality of IgE without influencing antibody titers. CONCLUSIONS: Although the 4 7S globulins are structurally related allergens, they induce antibodies with different antigen-binding characteristics. Peanut 7S induces IgE of a higher avidity than hazelnut and pea 7S which, again, has a higher avidity than IgE induced by soy 7S. We also show that soy tolerance influences the function of antibodies to peanut 7S. These findings may help explain how antibodies of different clinical significances can develop in different individuals sensitized to the same allergen.

An ivermectin-sensitive glutamate-gated chloride channel from the parasitic nematode Haemonchus contortus.

Nematode glutamate-gated chloride channels are targets of the macrocyclic lactones, the most important group of anthelmintics available. In Xenopus laevis oocytes, channels formed by the GluClalpha3B subunit from the parasite Haemonchus contortus were more sensitive to l-glutamate (EC(50) = 27.6 +/- 2.7 microM) than those formed by the homologous subunit from Caenorhabditis elegans (EC(50) = 2.2 +/- 0.12 mM). Ibotenate was a partial agonist (EC(50) = 87.7 +/- 3.5 microM). The H. contortus channels responded to low concentrations of ivermectin (estimated EC(50) = approximately 0.1 +/- 1.0 nM), opening slowly and irreversibly in a highly cooperative manner: the rate of channel opening was concentration-dependent. Responses to glutamate and ivermectin were inhibited by picrotoxinin and fipronil. Mutating an N-terminal domain amino acid, leucine 256, to phenylalanine increased the EC(50) for l-glutamate to 92.2 +/- 3.5 microM, and reduced the Hill number from 1.89 +/- 0.35 to 1.09 +/- 0.16. It increased the K(d) for radiolabeled ivermectin binding from 0.35 +/- 0.1 to 2.26 +/- 0.78 nM. Two other mutations (E114G and V235A) had no effect on l-glutamate activation or ivermectin binding: one (T300S) produced no detectable channel activity, but ivermectin binding was similar to wild-type. The substitution of any aromatic amino acid for Leu256 had similar effects in the radioligand binding assay. Molecular modeling studies suggested that the GluCl subunits have a fold similar to that of other Cys-loop ligand-gated ion channels and that amino acid 256 was unlikely to play a direct role in ligand binding but may be involved in mediating the allosteric properties of the receptor.

Assessing Almond and Peanut Allergens Using Commercially Available Immunoanalytical Kits and LC-MS/MS: A Case Study.

With an ever-increasing allergic population and an emerging market for allergen-free foods, accurate detection of allergens in foods has never been more important. Although ELISA-based methods are the most widely used for detection of allergens in food, there is a need for the development of orthogonal approaches. A commercial ELISA detected a relatively high concentration of peanut and almond in an allergen-free product. However, another commercial ELISA declared a low peanut concentration and was negative for almond. Further testing using a commercial almond lateral-flow device confirmed the results from the second ELISA kit and demonstrated that the positive detection of almond was due to cross-reactivity. An MS method was used for final confirmation that the reported results were negative for both almond and peanut.

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.

Ion flow in a zeolitic imidazolate framework results in ionic diode phenomena.

Ionic transport (for applications in nanofluidics or membranes) and "ionic diode" phenomena in a zeolitic imidazolate framework (ZIF-8) are investigated by directly growing the framework from aqueous Zn(2+) and 2-methylimidazole as an "asymmetric plug" into a 20 µm diameter pore in a ca. 6 µm thin poly-ethylene-terephthalate (PET) film.

A multi-laboratory evaluation of a clinically-validated incurred quality control material for analysis of allergens in food.

A dessert matrix previously used for diagnosis of food allergies was incurred with pasteurised egg white or skimmed milk powder at 3, 6, 15 and 30 mg allergen protein per kg of dessert matrix and evaluated as a quality control material for allergen analysis in a multi-laboratory trial. Analysis was performed by immunoassay using five kits each for egg and milk (based on casein) and six 'other' milk kits (five based on ß-lactoglobulin and one total milk). All kits detected allergen protein at the 3 mg kg(-1) level. Based on ISO criteria only one egg kit accurately determined egg protein at 3 mg kg(-1) (p=0.62) and one milk (casein) kit accurately determined milk at 6 (p=0.54) and 15 mg kg(-1) (p=0.83), against the target value. The milk "other" kits performed least well of all the kits assessed, giving the least precise analyses. The incurred dessert material had the characteristics required for a quality control material for allergen analysis.