Markus Lacorn

AACCI Approved Methods Technical Committee Report: Collaborative Study on the Immunochemical Determination of Intact Gluten Using an R5 Sandwich ELISA

In 2008, the AACC International Protein Technical Committee (now the Protein and Enzymes Technical Committee) initiated a collaborative study of a method for determining gluten in selected foods using an R5 antibody sandwich ELISA system. The method has been approved as AACCI Approved Method 38-50.01. The new method has been validated for testing foods to determine that they conform to the newly defined Codex threshold of 20 mg of gluten/kg in total for gluten-free products.

AACCI Approved Methods Technical Committee Report: Collaborative Study on the Immunochemical Determination of Partially Hydrolyzed Gluten Using an R5 Competitive ELISA

In 2008, the AACC International Protein Technical Committee (now the Protein and Enzymes Technical Committee) initiated a collaborative study of a method for determining gluten in fermented products using an R5 competitive ELISA system. The method has been approved as AACCI Approved Method 38-55.01. The new method has been validated for testing fermented foods and beverages to determine whether they conform to the newly defined Codex threshold of 20 mg of gluten/kg in total for gluten-free products.

Determination of Residual Egg White Proteins in Red Wines during and after Fining

Methods are needed for detecting allergenic proteins in wine after fining processes, both for legal requirements and to estimate any risk for allergic individuals. One of the critical allergens in this case is the protein fraction from egg white. The efficacy of use of a commercial egg protein detection kit was evaluated using wines spiked with egg protein. For calibration, a whole egg powder reference material was used. There were no matrix effects and the detection limit was 0.27 mg whole egg powder/L. The intra- and interassay coefficients of variation were 7.3% and 11.9%, respectively. Recoveries of different egg preparations in spiked red wine were between 80 and 110%. A field study showed different concentrations of egg during fining, but no detectable or low values of ≤1 mg/L in the bottled wine.

Commercial ELISA Measurement of Allergens and Gluten: What We Can Learn from Case Studies

During the last decade, results from ELISA test kits have often been criticized as being flawed. Therefore, it may appear counterintuitive that ELISAs are used for most food allergen and gluten analytical needs. One reason, in addition to the nonavailability of comparable alternative methods, is the fact that the methods used underwent a long validation and learning period in the market. This publication presents several case studies on interference, cross-reactivity issues, calibrators, fragmented allergens and gluten, matrix influences, and misunderstood intended-use statements. Afterward, the relevant validation parameter LOD, LOQ, selectivity, and precision are discussed. Finally, a comprehensive list of practical recommendations for ELISA test kit users is presented.

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 devices 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.

How should we proceed with the standardization in case of allergen determination

Allergens cannot at present be measured with the trueness that attends the analysis of well-characterized chemical entities such as mycotoxins where the analytes are defined and identical in sample and calibrator. By nature, allergens are nearly always a mixture of different proteins with different mass fractions of each single protein. Allergens are furthermore in a commodity with different total protein contents in the presence of an unknown matrix. To circumvent these difficulties, reference materials for allergens would be useful but until now not realized due to biological variabilities of the materials, and the contradiction between medical issues and declaration of foods due to legislation. Examples for standardization efforts are given which are (1) peanut where a common reference material not intended for allergen determination is used and (2) gluten/gliadin where the extraction procedure, the antibody and a calibrator are standardized but proficiency tests are not fit-for-purpose due to inhomogeneity of the testing material. Furthermore, the analytical minimum requirements and the declaration of egg-white proteins, caseins and lysozyme in wine are reviewed. To show one negative example for standardization efforts, milk is discussed. The possibilities and limits of reference materials for the standardization of allergen determinations are discussed.

Measurement of Gluten in Food Products: Proficiency‐Testing Rounds as a Measure of Precision and Applicability

In 2008, Codex Alimentarius endorsed the R5 Enzyme‐Linked Immunosorbent Assay (ELISA) method as Method Type 1 for gluten measurement in gluten‐free foods. The most recognized R5 ELISA test kit is the RIDASCREEEN® Gliadin (R7001; manufacturer R‐ Biopharm). Beside collaborative tests that led to several international approved methods of this test kit, proficiency‐testing (PT) rounds are regularly performed in Europe by dif‐ ferent PT providers. Results from these rounds were analyzed regarding the number of participating labs with acceptable results for the RIDASCREEN® Gliadin. All PT rounds document the excellent consistency and comparability of results. The data show that the RIDASCREEN® Gliadin R5 ELISA is also applicable to cake mix, oat‐based foodstuff, infant soya formula, cookies, canned boiled sausage, gravy thickener, pasta, and potato dumpling. These rounds also included the analysis of blank matrices. It was found that more than 95% of all participating laboratories correctly detected these samples as nega‐ tive. Other gluten test kit manufacturers were analyzed as well, but due to the low num‐ ber of participants using these test kits results were often only analyzed in a qualitative manner questioning the comparability of these kits to the RIDASCREEN® Gliadin R5 ELISA.

Validation of the RIDASCREEN(®)FAST Milk Kit.

The RIDASCREEN(®)FAST Milk test is a sandwich ELISA for the rapid quantification of milk proteins in various foods. The specific antibodies target casein and β-lactoglobulin. Samples are extracted and can then be analyzed in less than 40 min. The calibration curve covers a range from 2.5 to 67.5 mg/kg milk protein. The assay was validated with cookies, infant formula, chocolate dessert, ice cream, and sausages. All negative samples were found well below the LOQ of 2.5 mg/kg. Recoveries of the spiked samples were mostly in the range of 80-120%. The LOD of the ELISA was found below 1 mg/kg. The analysis of 39 different substances of interest revealed that no cross-reactivity above the LOQ occurred. Ruggedness testing proved that variations in incubation temperature, reagent volume, incubation time, extraction temperature, and extraction time had no significant influence. The stability at 4-8°C of three independent lots was investigated and found to exceed 18 months. Very good lot-to-lot consistency and no significant loss of the analytical capacity over the shelf life were observed. Incurred cookies and chocolate dessert samples were prepared and analyzed by an independent laboratory; mean recoveries of 94.4 and 102.2% and mean SDs of 10.9 and 6.3%, respectively, were found. For the 0 mg/kg level for both materials, all samples tested returned values of <2.5 mg/kg. Therefore, the analytical performance claims of the manufacturer were confirmed.