ELISA kit for mustard protein determination: interlaboratory study

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.     

Gluten determination by gliadin enzyme-linked immunosorbent assay kit: interlaboratory study

An interlaboratory study with 10 participants was performed to obtain validation and performance data for an enzyme-linked immunosorbent assay (ELISA) kit developed for quantitative gluten determination in foods. The ELISA kit used for this study is based on 2 monoclonal and 1 polyclonal antibody developed by Immunotech, a Beckman Coulter Co. This kit did not show any false positive results or cross-reactivity with oat, rice, maize, and buckwheat. The gliadin standard from the Working Group on Prolamin Analysis and Toxicity was included in the kit as reference material for calibration. All participants obtained a gliadin ELISA kit with Standard Operational Procedure and a form for recording test results. The study included 13 samples labeled as "gluten-free" and 2 samples spiked by wheat flour. Seven samples had gliadin content below the limit of quantitation (LOQ) of the method, and 1 sample exceeded the highest calibration level. Gliadin content in the range from 10 to 157 mg/kg (1st day) and from 11 to 183 mg/kg (2nd day) was found in 7 samples (including 2 spiked samples). Results of these samples were used for further statistical analysis and evaluation. The Cochran, Dixon, and Mandel statistical tests were applied for detection of outliers. The LOQ of the kit was estimated.     

Validation of two commercial lateral flow devices for the detection of peanut proteins in cookies: interlaboratory study

Results are reported for an interlaboratory validation study of 2 commercially available Iateral flow devices (dipstick tests) designed to detect peanut residues in food matrixes. The test samples used in this study were cookies containing peanuts at 7 different concentrations in the range of 0-30 mg peanuts/kg food matrix. The test samples with sufficient and proven homogeneity were prepared in our laboratory. The analyses of the samples (5 times per level by each laboratory) were performed by 18 laboratories worldwide which submitted a total of 1260 analytical results. One laboratory was found to be an outlier for one of the test kits. In general, both test kits performed well. However, some false-negative results were reported for all matrixes containing < 21 mg peanuts/kg cookie. It must be stressed that the test kits were challenged beyond their cut-off limits (> or = 5 mg/kg, depending on the food matrix). One test kit showed fewer false-negative results, but it led to some false-positive results for the blank materials. The sensitivity of the dipstick tests approaches that achieved with enzyme-linked immunosorbent assays.     

Determination of walnut protein in processed foods by enzyme-linked immunosorbent assay: interlaboratory study

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 microg 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: 5.8-9.9% RSDR) and a high level of recovery (81-119%) 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.     

Validation of a new enzyme-linked immunosorbent assay to detect the triggering proteins and peptides for celiac disease: interlaboratory study

The performance of Gluten-Tec (EuroProxima, Arnhem, The Netherlands) was tested through an interlaboratory study in accordance with AOAC guidelines. Gluten-Tec is a competitive ELISA that detects an immunostimulatory epitope of a-gliadin in dietary food for celiacs. Fifteen laboratories, representing 14 different countries, announced their interest in taking part in this study. Of the 12 laboratories that sent the results within the established timeframe, two submitted inappropriate standard curves and were excluded from the statistical analysis. Four different food matrixes (rice-based baby food, maize bread, chocolate cake mix, and beer) were selected for preparing the test samples. Two gliadin extraction procedures were used: the conventional 60% ethanol, and a new method based on the reducing reagent dithiothreitol. The 38 samples (19 blind duplicates) tested in this study were prepared by diluting the different extracts in order to cover a wide range of gliadin levels. Both sample extraction and dilution were performed by EuroProxima; the present interlaboratory study was focused only on testing the ELISA part of the Gluten-Tec kit protocol. Repeatability values (within-laboratory variance), expressed as RSD(r) ranged from 6.2 to 25.7%, while reproducibility values (interlaboratory variance), expressed as RSD(R), ranged from 10.6 to 45.9%. Both statistical parameters were in the acceptable range of ELISAs under these conditions, and the method will be presented to the Codex Alimentarius as a preferred method for gluten analysis.     

Liquid chromatographic determination of deoxynivalenol in baby food and animal feed: interlaboratory study

An interlaboratory study was conducted for the determination of deoxynivalenol in baby food and animal feed by high-performance liquid chromatography with UV detection. The study included 14 participants representing a cross section of industry, official food control, and research facilities. Mean recoveries reported ranged from 89% (at 120 microg/kg) to 85% (at 240 microg/kg) for baby food and from 100% (at 200 microg/kg) to 93% (at 400 microg/kg) for animal feed. On the basis of the results for spiked samples (blind duplicates at 2 levels), as well as those for naturally contaminated samples (blind duplicates at 3 levels), the relative standard deviation for repeatability (RSDr) in analyses of baby food ranged from 6.4 to 14.0% and in analyses of animal feed, from 6.1 to 16.5%. The relative standard deviation for reproducibility (RSDR) in analyses of baby food ranged from 9.4 to 19.5% and in analyses of animal feed, from 10.5 to 25.2%. The HorRat values ranged from 0.4 to 1.0 and from 0.7 to 1.3, for baby food and animal feed, respectively. The method showed acceptable performance for within-laboratory and between-laboratory precision for each matrix, as required by European legislation.     

Interlaboratory evaluation of two enzyme-linked immunosorbent assay kits for the determination of crustacean protein in processed foods

The labeling of foods containing material derived from crustaceans such as shrimp and crab is to become mandatory in Japan because of increases in the number of allergy patients. To ensure proper labeling, 2 novel sandwich enzyme-linked immunosorbent assay (ELISA) kits for the determination of crustacean protein in processed foods, the N kit (Nissui Pharmaceutical Co., Ltd, Ibaraki, Japan) and the M kit (Maruha Nichiro Holdings, Inc., Ibaraki, Japan), have been developed. Five types of model processed foods containing 10 and/or 11.9 microg/g crustacean soluble protein were prepared for interlaboratory evaluation of the performance of these kits. The N kit displayed a relatively high level of reproducibility relative standard deviation (interlaboratory precision; 4.0-8.4% RSDR) and sufficient recovery (65-86%) for all the model processed foods. The M kit displayed sufficient reproducibility (17.6-20.5% RSDR) and a reasonably high level of recovery (82-103%). The repeatability relative standard deviation (RSDr) values regarding the detection of crustacean proteins in the 5 model foods were mostly < 5.1% RSDr for the N kit and 9.9% RSDr for the M kit. In conclusion, the results of this interlaboratory evaluation suggest that both these ELISA kits would be very useful for detecting crustacean protein in processed foods.     

Magnetic particles functionalized with PAMAM-dendrimers and antibodies: a new system for an ELISA method able to detect Ara h3/4 peanut allergen in foods

An innovative enzyme-linked immunosorbent assay (ELISA) format based on antibody-coated magnetic micro-particles (MPs) for the sensitive detection of Ara h3/4 allergen in food is described. The immunosupport is suspended in the incubation solutions and the MPs with the captured allergen can be easily harvested on a magnet, separated from the solutions, and washed using an easy-to-use, fast and selective approach that allows its detection and quantification. Two differently coated MPs, ProteinA-Pn-b and MP-NH₂-PAMAM G 1.5 -Pn-b immunosupports, were tested. The functionalization of the MPs with PAMAM-sodium carboxylate dendrimers elicits a major stability on the immunoglobulin activity resulting in a threefold enhancement of the analytical sensitivity for the assay with respect to a ProteinA immobilization. Validation was carried out on two different matrices: corn flakes and biscuits. In the case of MP-NH₂-PAMAM G 1.5 -Pn-b immunosupport, limit of detection was found to be 0.2 mg peanuts/kg matrix in both matrices; the linear response range was demonstrated from 2.5 to 15 mg peanuts/kg matrix by performing statistical tests (homoscedasticity and Mandel fitting tests). Good accuracy and recovery (>80 ± 2%) were obtained. Different food samples were tested and the results were compared with those obtained with a commercially available ELISA kit. The results obtained in this work demonstrated the applicability of the immunomagnetic ELISA methods on real samples and the possibility to perform the assay with significantly reduced reagent and sample consumption.     

Liquid chromatographic method for the quantification of zearalenone in baby food and animal feed: interlaboratory study

An interlaboratory trial for determination of zearalenone (ZON) in baby food and animal feed was conducted. The study involved 39 participants in 16 European Union member states, as well as Turkey, Uruguay, and China, representing a cross-section of industry, and official food control and research institutes. The method is based on immunoaffinity column cleanup followed by high-performance liquid chromatography using fluorimetry (HPLC-FI). The test portion of the sample is extracted with methanol-water (75 + 25, v/v). The sample extract is filtered, diluted, and passed over an immunoaffinity column. ZON is eluted with methanol. The separation and determination of ZON is performed by reversed-phase HPLC-FI with an excitation wavelength of 274 nm and an emission wavelength of 446 nm. Test portions of the samples were spiked at levels of 20 and 30 microg/kg ZON in baby food and at levels of 100 and 150 microg/kg ZON in animal feed. Mean recoveries from each participant ranged from 78 to 119% with an average value of 92% for baby food and from 51 to 122% with an average value of 74% for animal feed. Based on results for spiked samples (blind duplicates at 2 levels), as well as naturally contaminated samples (blind duplicates at 3 levels), the relative standard deviation for repeatability (RSDr) in baby food ranged from 2.8 to 9.0%. For animal feed, this value ranged from 5.7 to 9.5%. The relative standard deviation for reproducibility (RSDR) in baby food ranged from 8.2 to 13.3%, and for animal feed this value ranged from 15.5 to 21.4%. The Horwitz ratio (HorRat) in baby food ranged from 0.3 to 0.4, and for animal feed this value ranged from 0.6 to 0.9. The method showed acceptable within- and between-laboratory precision for each matrix, as required by European legislation.     

Determination of vegetal proteins in milk powder by enzyme-linked immunosorbent assay: interlaboratory study

Eight laboratories participated in a collaborative study to evaluate an enzyme-linked immunosorbent assay (ELISA) to determine soy, pea, and wheat proteins in pasteurized or ultra-high temperature (UHT) milk powders. To perform this assay, polyclonal antibodies for soy, pea, and wheat proteins were obtained from rabbit sera. Collaborators received calibration standards composed of milk powder containing 0-8% (w/w) vegetal protein in total protein and blind test samples containing approximately 1, 2, and 5% (w/w) vegetal protein. An indirect competitive ELISA was performed with a kit prepared by a participating laboratory; the kit contained plates coated with soy, pea, or wheat proteins, the corresponding specific antisera, enzyme-labeled second antibody, and substrate solution. Test samples and calibrants were extracted with phosphate-buffered saline, pH 7.4, containing 0.05% Tween and assayed with the ELISA kits. The degree of adulteration was affected by the type of heat treatment applied to the samples. The estimated percentage of vegetal protein addition was close to the theoretical value for pasteurized samples but much lower for UHT samples. For pasteurized samples, intralaboratory relative standard deviations ranged from 5 to 22% and interlaboratory relative standard deviations ranged from 14 to 34%.     

Analysis of DDT residues in soil by ELISA: an international interlaboratory study

An international interlaboratory study was conducted to determine the performance of a group of laboratories from developing and developed countries. The study used a commercial microwell ELISA on unknown samples spiked with different levels of DDT. The study design was based on Youden pairs and balanced replicates. Two soils, differing in particle size distributions, organic matter content, and cation-exchange capacities and thought to be DDT-free, were spiked at 5 DDT levels between 0.025 and 2 mg/kg. Nineteen laboratories in 17 countries took part in the collaborative trial; of these, the majority were modestly equipped laboratories in developing countries. Samples were analyzed without filtration or cleanup and using standards of pure DDT in methanol. Data were analyzed for repeatability and reproducibility, and average recoveries at the spike levels were calculated. Mean real recoveries for both soils were similar (103% for soil A and 100% for soil B), with values between 0.1 and 2 mg/kg DDT. Precision estimates were best in the linear working range of the assay (0.1-0.5 mg/kg DDT), with reproducibility relative standard deviations (RSDR) typically averaging about 38 and 46% near the upper and lower detection limits, respectively. Corresponding repeatability relative standard deviation (RSDr) values were 20-36% and 36-57%. Thus, even though much of the trial was performed under developing country conditions, performance statistics were similar to other reported results obtained with ELISAs on small molecules of agricultural importance, such as mycotoxins and pesticide and antibiotic residues.     

Detection of hazelnuts and almonds using commercial ELISA test kits

Three commercial sandwich enzyme-linked immunosorbent assay (ELISA) test kits for the detection of hazelnuts and almonds were evaluated. Limits of detection and dynamic ranges were determined for hazelnuts and almonds spiked into cooked oatmeal, dipping chocolate, and muffins (baked). The limit of detection values varied from 1 to 38 μg/g, depending on the food matrix and ELISA test kit. Percent recoveries based on the standards supplied with the test kits varied from 10% to 170%. It is impossible to ascertain whether the percent recoveries reflect the performance of the ELISAs or differences between the protein content of the nuts used to spike the samples and the test kit standards. Unfortunately, reference materials do not exist that can be used to compare the results from different test kits and standardize the test kit standards. Also, insufficient knowledge regarding the epitope specificity of the antibodies used in the ELISAs further hinders interpretation of the results generated by the different test kits.     

Development of an automated on-line pre-column derivatization procedure for sensitive determination of histamine in food with high-performance liquid chromatography-fluorescence detection

An improved sensitive method was developed and validated for the determination of histamine in food samples by using automated on-line pre-column derivatization coupled with high performance liquid chromatography and fluorescence detection (HPLC-FLD). o-Phthaldialdehyde (OPA) was adopted as derivatization reagent, and a "sandwich" (OPA+histamine+OPA) aspiration mode for the automated on-line pre-column derivatization was found to efficiently enhance the method sensitivity and precision. Histamine in food samples was efficiently extracted with a methanol-phosphate buffer solution (50:50, v/v) at 60 degrees C for 30 min, and purified with Waters Oasis MCX solid-phase extraction (SPE) cartridge. The limit of quantification for this method is 0.2 mg/kg, which is very sensitive for histamine determination. With the "sandwich" injection program, 3.7% of relative standard deviation (RSD) was achieved by five replicative determinations of a sample blank spiked with 0.25 mg/kg histamine standard. Histamine in food samples such as fumitory skipjack and mackerel was analyzed with relative recoveries over 95% at spiking level of 150 mg/kg, as well as canned tuna fish and cheese with relative recoveries up to 98% at spiking levels of 0.50 and 5.0 mg/kg, respectively. The proposed method was validated with a sample from the Food Analysis Performance Assessment Scheme (FAPAS) as a standard certified material; and the results (140+/-6 mg/kg) agreed well with the assigned value (139 mg/kg).     

Interlaboratory evaluation of two enzyme-linked immunosorbent assay kits for the detection of egg, milk, wheat, buckwheat, and peanut in foods

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.     

Determination of arsenic in seafood by electrothermal atomic absorption spectrometry after microwave digestion: NMKL collaborative study

Eight laboratories participated in an interlaboratory method performance (collaborative) study of a method for the determination of arsenic in foodstuffs of marine origin by electrothermal atomic absorption spectrometry after wet digestion using a microwave oven technique. The study was preceded by a practice round of familiarization samples. The method was tested on 8 materials (cod roe, krill, blue mussel, saithe, scampi, cod fillet, shrimp, and cod extract) ranging in As content from 2 to 75 mg/kg. The materials were sent to participants in the study as blind duplicates, and the participants were asked to perform single determinations on each sample. Repeatability relative standard deviations (RSDr) for As ranged from 6.8 to 17.4%. Reproducibility relative standard deviations (RSDR) ranged from 7.6 to 24%. The highest RSDR value was found for the sample with the highest concentration of As.     

Method of determination of appropriate heat treatment of animal meal by immunoassay developed for detection of cooked beef: interlaboratory study.

An interlaboratory trial was conducted for the validation of an enzyme-linked immunosorbent assay (ELISA) method for determination of appropriate heat treatment of animal meal. A commercially available ELISA test kit developed for the identification of beef in cooked food was used in the study. Twelve laboratories from 7 European countries examined 2 different analytical protocols to establish the most appropriate analytical method. Three different samples were used, 2 animal waste materials sterilized at 129 and 134 degrees C (wet conditions), respectively, and a meat and bone meal material processed at dry conditions (maximum temperature, 140 degrees C). Statistical evaluation applying t-statistics showed that the animal meal treated according to European legislation (>133 degrees C) was clearly distinguishable from the 2 other test materials at a 99% confidence level using both analytical protocols. This method can be considered as a complementary test to the immunoassay developed for the detection of pork in cooked food that is already applied in routine analysis for the surveillance of rendering plants.     

Liquid chromatographic method for quantitation of patulin at 10 ng/mL in apple-based products intended for infants: interlaboratory study

An interlaboratory trial for the determination of patulin in apple juice and fruit puree was conducted, involving 17 participants representing a cross section of industry, official food control, and research facilities. Mean recoveries reported ranged from 74 (10 ng/g) to 62% (25 ng/g) for apple juice and from 72 (25 ng/g) to 74% (10 ng/g) for fruit puree. Based on results for spiked samples (blind pairs at 2 levels), as well as naturally contaminated samples (blind pairs at 3 levels), the relative standard deviation for repeatability (RSDr) in juice ranged from 8.0 to 14.3% and in puree from 3.5 to 9.3%. The relative standard deviation for reproducibility (RSD(R)) in juice ranged from 19.8 to 39.5% and in puree from 12.5 to 35.2%, reflecting HORRAT values from 0.6 to 1.0 for juice and 0.4 to 0.9 for puree. The method showed acceptable within-laboratory and between-laboratory precision for each matrix, as required by current European legislation.     

石墨炉原子吸收光谱法测定压水反应堆硼酸介质中钙

以甘露醇为基体改进剂,建立石墨炉原子吸收光谱法测定压水反应堆硼酸介质中钙含量的方法。考察基体改进剂用量、灰化温度、原子化温度、基体干扰以及共存离子干扰对测定结果的影响,确定最佳测定条件:以硼含量为1000 mg/kg的硼酸为背景基体,加入适量甘露醇,样品作酸化处理,灰化温度为1700℃,原子化温度为2450℃。钙含量在8~32 μg/kg范围内与光谱强度成良好的线性关系,相关系数为0.9996,方法检出限为1.79 μg/kg。对于硼基体含量为0~2500 mg/kg的样品,测定结果的相对标准偏差为0.5%~7.5%(n=6),相对误差不大于13.2%,加标回收率为93.9%~113.3%。该方法检测速度快,结果准确,能满足实际生产要求。     

Proficiency testing as a tool for implementing internal quality control: the case of ochratoxin A in cocoa powder

This paper describes the interlaboratory study aimed at assessing the performance of 18 laboratories (14 national and 4 European) for Ochratoxin A (OTA) determination in cocoa powder samples. The study was tested at three levels of OTA covering the range in which presumably European regulatory limits could fall in the near future. For the extraction step, almost all laboratories used an aqueous solution of sodium hydrogen carbonate with the exception of one laboratory using dichloromethane consistently with the ELISA procedure adopted in the study. The clean-up step was performed by utilizing the immunoaffinity columns by the two main manufacturers (R-Biopharm Rhone and VICAM) and for the quantitative analysis, HPLC was used by all the participants except one using ELISA. From the output of the study, it can be concluded that at low level (0.19 μg/kg) 10 out of 18 (56%), at medium level (0.45 μg/kg) 11 out of 18 (61%), and at high level (1.45 μg/kg) 12 out of 18 (67%) results fell within the satisfactory ranges. This interlaboratory study provides an estimate of the performance of national and European laboratories involved in OTA determination in cocoa powder samples, which sounds extremely valuable in view of potential future legislation by the European Commission.     

Determination of egg proteins in snack food and noodles

Egg is one of the 5 major allergenic foods that are responsible for more than 3/4 of food allergies in children. Food-allergic responses can be controlled by avoidance of the offending foods. The applicability of a commercial enzyme-linked immunosorbent assay (ELISA) kit for the detection of egg in food products such as cookies, crackers, pretzels, salad dressings, and raw and cooked noodles was evaluated. A preliminary evaluation of an antibody-based biosensor was also performed. A National Institute of Standards and Technology (NIST) whole dried egg powder reference material, SRM 8415, was used as a standard. A homogeneous and stable aqueous egg suspension was prepared for the evaluation of the performance of the Veratox for Egg Allergen Test (Neogen Corp., Lansing, MI). This test does not detect egg yolk proteins. Each gram of the aqueous dried egg suspension contained 643 microg whole dried egg, 0.5 mg thimerosal, and 2.5 mg bovine serum albumin. When cookies, crackers, salad dressings, noodles, and ice cream were spiked at a level of 24 mg/kg SRM 8415, recoveries for whole egg averaged about 28%. All foods containing egg as indicated on the ingredient label were found positive by the Veratox test. No false positives occurred in samples that did not contain eggs. Similar results were obtained using the Naval Research Laboratory (NRL) array biosensor, an evanescent wave fluoroimmunosensor. Results for cooked noodles showed that they contained <1% of the egg found in uncooked noodles. A comparison of extracts from cooked and uncooked noodles by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed differences in protein profiles. The boiling of the noodles could have reduced the immunoreactivity of the egg proteins to the antibodies used in the kit or rendered the egg proteins nonextractable.