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Background: In September 2017, the AOAC INTERNATIONAL Stakeholder Panel for Alternative Methods adopted Standard Method Performance Requirement (SMPR®) 2017.021, “Quantitation of Wheat, Rye, and Barley Gluten in Oats,” as guidance for the validation of methods for measuring gluten in oat products. The SMPR requires prospective methods to demonstrate adequate recovery (50–200%) based on the analysis of a set of reference samples. Objective: This document provides specific methods and data on the preparation of such validation materials and their analysis by an R5 ELISA kit to demonstrate the SMPR recovery estimation procedure. Methods: Seven reference samples were made by spiking wheat, rye, and barley into gluten-free oat flour at two levels, 10 and 20 mg/kg. The levels of gluten were determined by a wet chemical method based on the Codex Alimentarius definition of gluten. Results: The recoveries for wheat, rye, and barley were 122, 425, and 349%, respectively, for the R5 ELISA kit. The wet chemical method for estimating gluten in a sample of pure grain demonstrated repeatability relative SDs ranging from 1.40 to 2.75%. Conclusions: The reference materials are suitable to estimate ELISA kit responses to wheat, rye, and barley and calculate recoveries. Highlights: A series of oat flours spiked with wheat, rye, and barley flours were developed to be used as reference materials. A wet chemical method was established to estimate gluten contents based on the Codex definition. The reference materials are available for purchase to support further method development and validation.

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.

Background: ELISA is a widely used method for aflatoxin M1 (AFM1) quantification in dairy products. This study was conducted to compare ELISA kits from different manufacturers for AFM1 quantification inmilk. Methods: High sensitivity ELISA kits (for up to 250 ng AFM1/L), including AgraQuant (Romer Labs), Bioshield M1ES (Prognosis Biotech), Helica 96 (Helica Biosystems), Veratox (Neogen, Inc.), and the medium sensitivity kit Immunolab AM1E01 (Immunolab GmbH; for 10–1000ng AFM1/L) were tested against a certified reference AFM1 whole milk having 44 ng AFM1/L and its 10-fold dilution, and a50 ng AFM1/L standard. In another experiment, Prognosis Bioshield M1UF (70–1000 ng AFM1/L), Romer’s AgraquantPlus (10–2000 ng AFM1/L), and Immunulab’s AM1E01 kits were compared totest a 500 ng AFM1/L solution. Results: In both of the experiments, the quantification ofthe tested AFM1 levels did not differ (P ≥ 0.310) between the kitsfrom various manufacturers. In the case of 4.4 ng AFM1/L, recovery of the toxin wasclose to the assigned value under the kit by Neogen, Inc. Incase the of 44 ng level, better recoveries were seen under the kits by Immunolab and Prognosis Biotech. In both the cases, low relative standard deviation (RSD) values were obtained only for the kit by Prognosis Biotech. In the case of the 50 and 500 ng AFM1/L solutions, the kits by Prognosis Biotech and Romer Labs provided recoveries close tothe assigned values as well as low RSD. Conclusions: These data indicate that all thestudied ELISA kit brands had comparable efficacy forAFM1 quantification. Recovery of the toxin and RSD may, however, differ between kits fromvarious manufacturers.

Background: It is important to analyze the presence of wheat/gluten in food to avoid wheat allergy or celiac disease. Objective: The Wheat/Gluten ELISA kit was developed to measure total wheat protein or gluten content in wheat, barley, and rye cereals as raw materials, and processed foods. Validation as to whether this kit is suitable for quantifying total wheat protein/gluten was carried out. Methods: The Wheat/Gluten ELISA kit was designed as a sandwich ELISA based on antigliadin polyclonal antibody. Selectivity, interference study, matrix study including incurred food, robustness, stability, and lot-to-lot consistency studies were conducted for the Wheat/Gluten ELISA kit. Incurred matrix studies were also conducted in an independent laboratory. Results: The analysis of 38 different substances revealed no cross-reactivity above the LOQ except for oats. Recoveries of the spiked samples were mostly in the range of 75–140%, including an independent laboratory result. The LOD of the ELISA was found to be 0.02–0.16 mg/kg. Robustness testing proved that extraction time and incubation time of first reaction and enzyme reaction had no significant influence on quantified value. The stability at 2–8°C was found to exceed 12 months. Good lot-to-lot consistency was observed. Conclusions: The Wheat/Gluten ELISA kit showed good analytical performance in the quantitative analysis of total wheat protein/gluten in the identified food products using the AOAC Performance Tested Method(s)SM program. Highlights: The Wheat/Gluten ELISA kit was validated and showed good analytical performance in the quantitative analysis of total wheat protein/gluten in food.

This study investigated the ability of two methods to detect adulteration of goat cheeses via the addition of cow's milk, with a negligible effect on the raw materials. Cheeses were produced from a mixture of goat's and cow's milk and were then analyzed by Fourier transform near-IR (FT-NIR) spectroscopy and competitive ELISA. The cheese spectra were scanned in the spectroscope in reflectance mode on an integrating sphere at 80 scans and a resolution of 4 cm−1. The spectra were evaluated via discriminant analysis, and a calibration was created via a partial least-squares algorithm to quantify the cow's milk admixture. A correlation coefficient of R = 0.999 was reached with a standard error of calibration of 0.0407. The results were statistically processed to a median value via a t-test. Adulteration detection by the ELISA method was performed using a commercial Milk Fraud/Bovine ELISA kit. It was found that the FT-NIR spectroscopy method is capable of detecting an admixture of cow's milk in goat cheese as small as 1%. The ELISA method did not return satisfactory results for the detection of adulteration with cow's milk.

A collaborative study in 10 laboratories was performed to validate an ELISA method developed for the quantitative determination of peanut protein in foods. The ELISA kit used for this study is based on rabbit polyclonal antibody. This kit does not produce any false-positive results or cross-reactivity with a broad range of peanut-free food matrixes. All participants obtained the peanut ELISA kit with standard operational procedures, a list of samples, the samples, and a protocol for recording test results. The study included 15 food samples. Three food matrix samples of zero peanut content showed peanut protein content lower than the first standard (0.10 mg/kg). Three samples with peanut declared as an ingredient revealed peanut protein content outside the calibration curve (absorbance was above the highest standard) in all laboratories, and three samples had the peanut content reported either above the highest standard or within the calibration curve, depending on the laboratory. Six samples with peanut declared as an ingredient gave the peanut protein content within the calibration curve. Only these six samples, together with a positive control sample (CS2), were used for statistical evaluation. The statistical tests (Cochran, Grubbs, and Mandel) and analysis of variance were used for the evaluation of the collaborative study results. Repeatability and reproducibility limits, as well as an LOQ (LOQcollaborative 0.22 mg peanut proteins/kg) and an LOD (LODcollaborative 0.07 mg peanut proteins/kg) for the kit were calculated.

An interlaboratory study in 12 laboratories was performed to prove the validation of the ELISA method developed for the quantitative determination of mustard protein in foods. The ELISA kit used for this study is based on rabbit polyclonal antibody. This kit did not produce any false-positive results or cross-reactivity with in-house validation for a broad range of food matrixes with no detectable mustard protein. All participants obtained the Mustard ELISA kit with standard operational procedures, a list of samples, samples, and a protocol for recording test results. The study included 15 food samples and two spiked samples. Seven food matrix samples of zero mustard content and four samples with mustard declared as an ingredient showed mustard protein content lower than that of the first standard (0.42 mg/kg). Four samples with mustard declared as an ingredient revealed mustard protein content above 12.5 mg/kg (the highest standard). The statistical tests (Cochran, Dixon, and Mandel) and analysis of variance were used to evaluate the interlaboratory study results. Repeatability and reproducibility limits, as well as an LOQ (1.8 mg mustard proteins/kg) and LOD (0.5 mg mustard proteins/kg), for the kit were calculated.

The labeling of foods containing ingredients derived from soybean is recommended in Japan because of an increasing number of patients who are allergic to soybeans. To ensure proper labeling, a novel sandwich ELISA kit for the determination of soybean protein in processed foods (FASTKIT Ver. II, Soybean, Nippon Meat Packers, Inc.; soy kit) has been developed. Five types of incurred samples (model processed foods: rice gruel, sausage, sweet adzuki bean soup, sweet potato cake, and tomato sauce) containing 10 g soybean soluble protein/g food were prepared for use in interlaboratory evaluations of the soy kit. The soy kit displayed a sufficient RSDR value (interlaboratory precision: 9.313.4 RSDR) and a high level of recovery (97114) for all the incurred samples. The RSDr value for the incurred samples was mostly

The labeling of 5 major allergenic ingredients (egg, milk, wheat, buckwheat, and peanut) is mandatory in Japan, and 2 series of enzyme-linked immunosorbent assay (ELISA) kits have been established as official screening methods. However, these official methods have not provided the necessary sensitivity, due in part to poor extraction efficiency. To address this need, 2 novel ELISA kits have been developed: the FASTKIT ELISA Ver. II Series and the FASPEK Allergenic Substances Detection Kit. The new kit systems use an improved extraction buffer that can extract insoluble proteins produced by processing and feature new antibodies that bind to the denatured proteins extracted with the new extraction buffer. The analytical performances of the 2 new ELISA kit series were evaluated in an interlaboratory study. Ten laboratories participated in the study and determined the major allergenic ingredients contained in 5 types of model processed food. The 2 ELISAs displayed fairly good reproducibility and sufficient recovery.

Peanuts contain proteins that can cause severe allergic reactions in some sensitized individuals. Studies were conducted to determine the percentage of recovery by an enzyme-linked immunosorbent assay (ELISA) method in the analysis for peanuts in energy bars and milk chocolate and to determine the sampling, subsampling, and analytical variances associated with testing energy bars and milk chocolate for peanuts. Food products containing chocolate were selected because their composition makes sample preparation for subsampling difficult. Peanut-contaminated energy bars, noncontaminated energy bars, incurred milk chocolate containing known levels of peanuts, and peanut-free milk chocolate were used. A commercially available ELISA kit was used for analysis. The sampling, sample preparation, and analytical variances associated with each step of the test procedure to measure peanut protein were determined for energy bars. The sample preparation and analytical variances were determined for milk chocolate. Variances were found to be functions of peanut concentration. Sampling and subsampling variability associated with energy bars accounted for 96.6% of the total testing variability. Subsampling variability associated with powdered milk chocolate accounted for >60% of the total testing variability. The variability among peanut test results can be reduced by increasing sample size, subsample size, and number of analyses. For energy bars the effect of increasing sample size from 1 to 4 bars, subsample size from 5 to 20 g, and number of aliquots quantified from 1 to 2 on reducing the sampling, sample preparation, and analytical variance was demonstrated. For powdered milk chocolate, the effects of increasing subsample size from 5 to 20 g and number of aliquots quantified from 1 to 2 on reducing sample preparation and analytical variances were demonstrated. This study serves as a template for application to other foods, and for extrapolation to different sizes of samples and subsamples as well as numbers of analyses.

An interlaboratory study was conducted in 11 laboratories to validate an ELISA method developed for the quantitative determination of egg white proteins (EWPs) in foods. The ELISA kit used for this study is based on sheep polyclonal antibody. It does not produce any false-positive results or cross-reactivity in a broad food matrix range with zero EWP content. All participants obtained the Egg ELISA Kit-native with standard operational procedure and the list of samples, as well as the samples and a protocol for recording test results. The study included 10 food samples. Four samples of food matrix with zero EWP content showed EWP content lower than the first standard (EWP content 0.5 mg/kg). One sample of food matrix with zero EWP content revealed EWP content higher than standard 3 (1.5 mg EWP/kg). Five food samples containing EWP as an ingredient tested positive and one negative. The statistical tests (Cochran, Dixon, and Mandel) and analysis of variance were used to evaluate the interlaboratory study results. Repeatability and reproducibility limits, as well as LOQ (1.4 mg EWP/kg) and LOD (0.43 mg EWP/kg), were calculated for the kit.

Because food allergens from tree nuts, including walnuts, are a frequent cause of adverse food reactions for allergic patients, the labeling of foods containing ingredients derived from tree nuts is required in numerous countries. According to Japanese regulations, the labeling of food products containing walnuts is recommended. To ensure proper labeling, a novel sandwich ELISA kit for the determination of walnut protein in processed foods (Walnut Protein [2S-Albumin] Kit; Morinaga Institute of Biological Science, Inc.; walnut kit) has been developed. We prepared seven types of incurred samples (model processed foods: biscuits, bread, sponge cake, orange juice, jelly, chicken meatballs, and rice gruel) containing 10 g walnut soluble protein/g of food for use in interlaboratory evaluations of the walnut kit. The walnut kit displayed sufficient reproducibility relative standard deviations (interlaboratory precision: 5.89.9 RSDR) and a high level of recovery (81119) for all the incurred samples. All the repeatability relative standard deviation (RSDr) values for the incurred samples that were examined were less than 6.0. The results of this interlaboratory evaluation suggested that the walnut kit could be used as a precise and reliable tool for determination of walnut protein in processed foods.

An interlaboratory study was performed in six laboratories to prove the validation of the ELISA method developed for quantitative determination of beta-lactoglobulin (BLG) in foods. The ELISA kit used for this study is based on rabbit polyclonal antibody. In-house validation of the kit did not produce false-positive results or cross-reactivity in a broad range of food matrixes containing no milk proteins. All participants obtained the BLG kit with a standard operational procedure, the list of the samples, samples, and a protocol for recording test results. The study included 14 food samples (extruded breakfast cereals, bread, two soy desserts, butter, chicken ham, chicken meat, wheat flour, long grain rice, jelly, two whey drinks, crackers, and bitter chocolate) and six spiked samples (two rice, two wheat flour, and two chicken meat). Nine samples of food matrixes containing no milk proteins showed BLG content lower than the first standard (0.15 mg/kg). Two samples of food matrixes with no milk proteins revealed BLG content higher than standard 3 (1.5 mg/100 g) and standard 4 (5.0 mg/100 g). Three food samples containing milk were tested as positive, and all spiked samples were evaluated as positive. The statistical tests (Cochran, Dixon, and Mandel) and analysis of variance were used to evaluate the interlaboratory study results. Repeatability and reproducibility limits, as well as LOQ (0.22 mg BLG/kg) and LOD (0.07 mg BLG/kg), for the kit were calculated.