Evaluation of extraction buffers using the current approach of detecting multiple allergenic and nonallergenic proteins in food

The detection of food allergens has been a challenge because of the increasing need to ensure the absence of undeclared allergens in foods. The current trend in the detection of some food allergens, like peanuts, is based on the detection of multiple allergenic and nonallergenic proteins, and this is the approach that kit manufacturers have adopted. Because commercial kits differ in their ability to detect allergens, regulatory agencies, the food industry, and kit manufacturers are working together to standardize the detection methods. Three kits for the detection of peanuts have been evaluated for performance by the AOAC Research Institute. For this evaluation, a peanut butter suspension was used as a reference material. Several kit components contribute to between-kit analytical variation, even when the same sample is used. One component of commercial kits, which may be contributing to this variability, is the sample extraction buffer. In this study, differences in extractability of 3 allergenic foods were evaluated by using 4 different extraction buffers. The conclusion is that optimum allergen extractability was buffer-dependent, and no single buffer is appropriate for use as a universal extraction solution for all allergenic foods. Therefore, a thorough evaluation of sample preparation buffers needs to be performed for every individual allergenic food. In light of the results obtained, the current approach used for detection of peanut allergens based on the detection of multiple allergenic and nonallergenic proteins is being analyzed.     

Immunochemical analytical methods for the determination of peanut proteins in foods

Peanut proteins can cause allergenic reactions that can result in respiratory and circulatory effects in the body sometimes leading to shock and death. The determination of peanut proteins in foods by analytical methods can reduce the risk of serious reactions in the highly sensitized individual by allowing for the detection of these proteins in a food at various stages of the manufacturing process. The method performance of 4 commercially available enzyme-linked immunosorbent assay (ELISA) kits was evaluated for the detection of peanut proteins in milk chocolate, ice cream, cookies, and breakfast cereals: ELISA-TEK Peanut Protein Assay, now known as "Bio-Kit" for peanut proteins, from ELISA Technologies Inc.; Veratox for Peanut Allergens from Neogen Corp.; RIDASCREEN Peanut Kit from R-Biopharm GmbH; and ProLisa from Canadian Food Technology Ltd. The 4 test kits were evaluated for accuracy (recovery) and precision using known concentrations of peanut or peanut proteins in the 4 food matrixes. Two different techniques, incurred and spiked, were used to prepare samples with 4 known concentrations of peanut protein. Defatted peanut flour was added in the incurred samples, and water-soluble peanut proteins were added in the spiked samples. The incurred levels were 0.0, 10, 20, and 100 microg whole peanut per g food; the spiked levels were 0.0, 5, 10, and 20 microg peanut protein per g food. Performance varied by test kit, protein concentration, and food matrix. The Veratox kit had the best accuracy or lowest percent difference between measured and incurred levels of 15.7% when averaged across all incurred levels and food matrixes. Recoveries associated with the Veratox kit varied from 93 to 115% for all food matrixes except cookies. Recoveries for all kits were about 50% for cookies. The analytical precision, as measured by the variance, increased with an increase in protein concentration. However, the coefficient of variation (CV) was stable across the 4 incurred protein levels and was 7.0% when averaged across the 4 food matrixes and analytical kits. The R-Biopharm test kit had the best precision or a CV of 4.2% when averaged across all incurred levels and food matrixes. Because measured protein values varied by test kit and food matrix, a method was developed to normalize or transform measured protein concentrations to an adjusted protein value that was equal to the known protein concentration. The normalization method adjusts measured protein values to equal the true protein value regardless of the type test kit or type food matrix.     

The effect of heat treatment on the detection of peanut allergens as determined by ELISA and real-time PCR

Peanut allergic reactions can result from the ingestion of even very small quantities of peanut and represent a severe threat to the health of sensitised individuals. The detection of peanut traces in food products is therefore of prime importance. Peanut traces which can be (unintentionally) present in food products have usually undergone one or more processing steps like roasting and baking. Therefore, methods designed to detect such traces have to be capable of detecting heat-treated peanuts. Commonly used methodologies designed to detect peanut traces in food products are enzyme-linked immunosorbent assays (ELISAs) that detect peanut-specific proteins, and polymerase-chain-reaction (PCR)-based methods targeting peanut-specific DNA. A comparative analysis of such methods was performed and the impact of heat treatment on peanut kernels as well as the impact on a peanut-containing food matrix are investigated. Our results show that heat treatments have a detrimental effect on the detection of peanut with either type of method and that both types of methods are affected in a similar manner.     

Can commercial peanut assay kits detect peanut allergens?

Peanut is the food group mostly associated with severe and fatal allergic reactions. In the United States, more than 90% of peanut-allergic individuals' serum IgE recognized peanut proteins Ara h 1 and Ara h 2, thus establishing these proteins as major peanut allergens. The amount of Ara h 1 and Ara h 2 in 3 varieties of peanut cultivars that are commonly processed in the industrialized countries was determined to be 12-16 and 6-9%, respectively. Current commercial peanut test kits use polyclonal peanut-specific antibodies to detect soluble or buffer extractable peanut proteins. Because the 2 major peanut allergens Ara h 1 and Ara h 2 are isolated from soluble peanut proteins, it is generally assumed that these commercial kits can detect peanut allergens, although none of these kits claims to detect peanut allergen. This study showed for the first time that the peanut test kits could, in fact, detect major peanut allergens Ara h 1 and Ara h 2 in both native or heat-denatured structures; therefore, these kits qualified to be classified as peanut allergen enzyme-linked immunosorbent assays.     

Sensitive and specific detection of potentially allergenic almond (Prunus dulcis) in complex food matrices by Taqman(®) real-time polymerase chain reaction in comparison to commercially available protein-based enzyme-linked immunosorbent assay

Currently, causative immunotherapies are lacking in food allergy. The only option to prevent allergic reactions in susceptible individuals is to strictly avoid the offending food. Thus, reliable labelling of allergenic constituents is of major importance, but can only be achieved if appropriate specific and sensitive detection techniques for foods with allergenic potential are available. Almond is an allergenic food that requires mandatory labelling on prepackaged foods and belongs to the genus Prunus. Species of this genus are phylogenetically closely related. We observed commercially available almond specific ELISA being highly cross-reactive with other foods of the Prunoideae family, resulting in a false-positive detection of up to 500,000 mg kg(-1) almond. Previously published PCR methods were reported to be cross-reactive with false positive results >1200 mg kg(-1). We describe the development of a novel almond specific real-time PCR, based on mutated mismatch primers and sequence specific Taqman(?) probe detection, in comparison with two quantitative commercially available ELISA. PCR sensitivity was investigated with chocolate, chocolate coating and cookies spiked between 5 and 100,000 mg kg(-1) almond. In all matrices almond was reproducibly detected by real-time PCR at the lowest spike level of 5 mg kg(-1). Further, between 100 and 100,000 mg kg(-1) spiked almond, the method featured good correlation between quantified copy numbers and the amount of spiked almond. Within this range a similar relation between detectable signal and amount of almond was observed for both PCR and ELISA. In contrast to ELISA the Taqman(?) real-time PCR method was highly specific in 59 food items with negligible cross-reactivity for a very limited number of Prunoideae foods. The real-time PCR analysis of 24 retail samples was in concordance with ELISA results: 21% (n=5) contained undeclared almond. This is the first completely disclosed real-time PCR method for a specific and potentially quantitative almond detection. This PCR method detects almond at a level where severe allergic reactions should not be expected for the majority of the almond allergic individuals.     

Competitive inhibition ELISA for quantification of Ara h 1 and Ara h 2, the major allergens of peanuts

Allergies to peanuts are becoming an increasingly important health problem as a result of the persistence and severity of the reaction in allergic individuals. Because no treatment currently is available, avoidance is the only option for peanut-allergic individuals. Avoidance of an abundant and often disguised food such as peanuts, however, is very difficult; therefore, competitive inhibition ELISAs were developed to detect and quantitate each of the major peanut allergens, Ara h 1 and Ara h 2. Under optimal conditions for each assay, the sensitivity of the Ara h 1 and Ara h 2 detection assays were 12 and 0.5 ng/mL, respectively. These assays were primarily devised to effectively compare the levels of Ara h 1 and Ara h 2 in a wide variety of peanuts or peanut products but can also be used to identify cross-reactive antigens. The method is simple and rapid, requiring only one allergen-specific antibody and, therefore, could be adapted specifically to detect the presence of these individual allergens in different foods.     

A novel, sensitive and specific real-time PCR for the detection of traces of allergenic Brazil nut (Bertholletia excelsa) in processed foods

Food-allergic individuals have to strictly avoid the offending food because no causative immunotherapies are available. Thus, reliable labelling of allergenic ingredients or precautionary labelling of cross-contacts with potential allergens is of major importance. Verification of compliance with labelling requirements and identification of cross-contacts demand test methods that enable the specific and sensitive detection of the analyte. Brazil nut (Bertholletia excelsa) is such a food commodity with allergenic potential. We describe the development of a novel qualitative real-time polymerase chain reaction (PCR) specific for Brazil nut DNA and its comparison with a qualitative commercially available lateral flow device (LFD) that detects Brazil nut protein. Specificity was investigated with 58 foods, and no false-positive reactions were observed in real-time PCR. The sensitivity was investigated with spiked chocolate and incurred dough samples as well as cookies baked thereof. The simultaneous spiking of matrices with identical amounts of Brazil nut and peanut between 5 and 100,000 mg/kg allowed the verification of the spike quality with two peanut-specific enzyme-linked immunosorbent assay. The real-time PCR detected Brazil nut in all three matrices down to the lowest investigated spike level of 5 mg/kg. The real-time PCR results from the analysis of 15 retail samples were confirmed by LFD results and were in concordance with the labelling of products. The real-time PCR showed unparalleled specificity, and primary data indicated potentially quantitative features in spiked and retail samples. Because of entirely reproducible chemistry of this real-time PCR, this is the first generally available Brazil nut-specific detection method with an appropriate sensitivity to help avoid severe allergic reactions for Brazil nut-allergic individuals.     

Performance Tested Method multiple laboratory validation study of ELISA-based assays for the detection of peanuts in food

Performance Tested Method multiple laboratory validations for the detection of peanut protein in 4 different food matrixes were conducted under the auspices of the AOAC Research Institute. In this blind study, 3 commercially available ELISA test kits were validated: Neogen Veratox for Peanut, R-Biopharm RIDASCREEN FAST Peanut, and Tepnel BioKits for Peanut Assay. The food matrixes used were breakfast cereal, cookies, ice cream, and milk chocolate spiked at 0 and 5 ppm peanut. Analyses of the samples were conducted by laboratories representing industry and international and U.S governmental agencies. All 3 commercial test kits successfully identified spiked and peanut-free samples. The validation study required 60 analyses on test samples at the target level 5 microg peanut/g food and 60 analyses at a peanut-free level, which was designed to ensure that the lower 95% confidence limit for the sensitivity and specificity would not be <90%. The probability that a test sample contains an allergen given a prevalence rate of 5% and a positive test result using a single test kit analysis with 95% sensitivity and 95% specificity, which was demonstrated for these test kits, would be 50%. When 2 test kits are run simultaneously on all samples, the probability becomes 95%. It is therefore recommended that all field samples be analyzed with at least 2 of the validated kits.     

Protein quantification, sandwich ELISA, and real-time PCR used to monitor industrial cleaning procedures for contamination with peanut and celery allergens

In the United States, peanut is one of the main sources of food allergens. Similarly, celery is a common allergenic food in Western Europe. Severe allergic reactions to both foods are common. Unexpected allergic reactions can occur after the consumption of celery- and peanut-free foods as a result of inadvertent cross-contaminations during manufacturing. Therefore, in cooperation with a flavor manufacturer, we monitored the cleaning process of slurry preparation equipment with regard to contaminations of follow-up products with celery and peanut compounds. Washing water samples taken after different cleaning steps and follow-up products were analyzed for the presence of celery and peanut traces with a celery-specific real-time polymerase chain reaction (PCR) and a peanut-specific sandwich enzyme-linked immunosorbent assay (ELISA). PCR and ELISA were compared with a nonspecific protein assay to evaluate whether the detection of protein traces can be a fast and cost-effective method for monitoring the effectiveness of wet cleaning procedures. Additionally, the allergenic potential of the celery and peanut mush, which were used as source material, were measured by a mediator release assay using a rat basophilic leukemia (RBL) cell line. In conclusion, the quantification of total protein in washing water was suitable for monitoring the cleaning process. Our study also revealed evidence that, in cases where wet cleaning is applicable, allergenic traces can be removed with high efficiency.     

Sensitive and specific detection of potentially allergenic almond (Prunus dulcis) in complex food matrices by Taqman real-time polymerase chain reaction in comparison to commercially available protein-based enzyme-linked immunosorbent assay

Currently, causative immunotherapies are lacking in food allergy. The only option to prevent allergic reactions in susceptible individuals is to strictly avoid the offending food. Thus, reliable labelling of allergenic constituents is of major importance, but can only be achieved if appropriate specific and sensitive detection techniques for foods with allergenic potential are available. Almond is an allergenic food that requires mandatory labelling on prepackaged foods and belongs to the genus Prunus. Species of this genus are phylogenetically closely related. We observed commercially available almond specific ELISA being highly cross-reactive with other foods of the Prunoideae family, resulting in a false-positive detection of up to 500,000 mg kg⁻¹ almond. Previously published PCR methods were reported to be cross-reactive with false positive results >1200 mg kg⁻¹.We describe the development of a novel almond specific real-time PCR, based on mutated mismatch primers and sequence specific Taqman^® probe detection, in comparison with two quantitative commercially available ELISA. PCR sensitivity was investigated with chocolate, chocolate coating and cookies spiked between 5 and 100,000 mg kg⁻¹ almond. In all matrices almond was reproducibly detected by real-time PCR at the lowest spike level of 5 mg kg⁻¹. Further, between 100 and 100,000 mg kg⁻¹ spiked almond, the method featured good correlation between quantified copy numbers and the amount of spiked almond. Within this range a similar relation between detectable signal and amount of almond was observed for both PCR and ELISA. In contrast to ELISA the Taqman^® real-time PCR method was highly specific in 59 food items with negligible cross-reactivity for a very limited number of Prunoideae foods. The real-time PCR analysis of 24 retail samples was in concordance with ELISA results: 21% (n = 5) contained undeclared almond. This is the first completely disclosed real-time PCR method for a specific and potentially quantitative almond detection. This PCR method detects almond at a level where severe allergic reactions should not be expected for the majority of the almond allergic individuals.     

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.     

Variation of analytical results for peanuts in energy bars and milk chocolate

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.     

Commercialized rapid immunoanalytical tests for determination of allergenic food proteins: an overview

Food allergies have become an important health issue especially in industrialized countries. Undeclared allergenic ingredients or the presence of “hidden” allergens because of contamination during the food production process pose great health risks to sensitised individuals. The EU directive for food labelling lists allergenic foods that have to be declared on food products by the manufacturers. The list includes gluten-containing cereals, crustaceans, eggs, fish, peanuts, soybeans, milk, various nuts (e.g. almond, hazelnut, and walnut, etc.), celery, mustard, sesame seeds, lupin, and molluscs. Reliable methods for detection and quantification of food allergens are needed that can be applied in a fast and easy-to-use manner, are portable, and need only limited technical equipment. This review focuses on the latest developments in food allergen analysis with special emphasis on fast immunoanalytical methods such as rapid enzyme-linked immunosorbent assays (ELISA), lateral-flow immunochromatographic assays (LFA) and dipstick tests. Emerging technologies such as immunochemical microarrays and biosensors are also discussed and their application to food allergen analysis is reviewed. Finally, a comprehensive overview of rapid immunochemical test kits that are currently available commercially is given in tabular form.     

Immunoreactivity and detection of wheat proteins by commercial ELISA kits

Wheat proteins are responsible for sensitivities, including baker's asthma, immunoglobulin E (IgE)-mediated allergic reaction, wheat-dependent, exercise-induced anaphylaxis, and celiac disease. The detection of gluten/wheat traces in foods is important to safeguard the health of wheat-sensitive individuals and comply with food labeling. Many immunoanalytical-based commercial kits are available for the quantification of gliadin/gluten/wheat proteins. We compared the immunoreactivity of wheat fractions with wheat-allergic human serum IgE and antibody conjugates used in six commercial immunoassay kits. Moreover, the performance of the kits was tested using corn flour spiked with gluten (5, 10, 25, and 50 ppm) and wheat flour (50, 100, 250, and 500 ppm). The albumin, globulin, gliadin, and glutenin fractions reacted with IgE from nine, eight, two, and eight patients' sera, respectively, out of nine wheat allergic patients tested. Among the antibodies from commercial kits, those from R-Biopharm, Morinaga, and Romer Labs reacted strongly with the gliadin fraction, whereas those from BioKits, ALLER-TEK, and ELISA Systems reacted strongly with the glutenin fraction. All kits showed minimal or no reactivity with albumin and globulin fractions. All kits detected the gluten and wheat flour in a corn flour matrix at the lowest spiked levels of 5 and 50 ppm, respectively. However, there was wide variation among the kits when comparing the recovery of gluten and wheat flour. The recovery was also dependent on the source material (gluten or wheat flour) used for spiking the corn flour matrix.     

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.     

Rapid extraction of aflatoxin from creamy and crunchy peanut butter

A rapid extraction technique was developed for the isolation and subsequent liquid chromatographic determination of aflatoxins B1, B2, G1, and G2 in creamy and crunchy peanut butter. Peanut buftter samples were extracted with a methanol 15% sodium chloride (7 + 3) solution followed by a second extraction with methanol. The extract was subjected to a cleanup using a Vicam Aflatest immunoaffinity column. Control samples for both smooth and crunchy peanut butter were fortified at 4 different levels for aflatoxin B1, B2, G1, and G2. The average aflatoxin B1, B2, G1, and G2 recoveries from smooth peanut buffer were 95.2, 89.9, 94.1, and 62.4%, respectively, and 92.4, 84.3, 85.5, and 53.7%, respectively, from crunchy peanut butter. This extraction method and the official AOAC Method 991.31 produced comparable results for peanut butter samples. This method provides a rapid, specific, and easily controlled assay for the analysis of aflatoxins in peanut butter with minimal solvent usage. Organic solvent consumption was decreased by 85% and hazardous waste production was decreased by 80% in comparison with the AOAC method. Along with the decreased solvent consumption, significant savings in time were observed.     

Commercial lateral flow devices for rapid detection of peanut (<Emphasis Type="Italic">Arachis hypogaea)</Emphasis> and hazelnut (<Emphasis Type="Italic">Corylus avellana)</Emphasis> cross-contamination in the industrial production of cookies

Lateral flow devices (LFDs) are qualitative immunochromatographic tests for the rapid and specific detection of target analytes. We investigated commercially available LFDs for their ability to detect potentially allergenic peanut and hazelnut in raw cookie dough and chocolate, two important food matrices in the industrial production of cookies. Each three commercial LFDs for the detection of hazelnut and peanut were performed according to the manufacturers’ instructions. All LFDs had comparably satisfactory specificity that was investigated with a variety of characteristic foods and food ingredients used in the production of cookies. In concordance with hazelnut-specific enzyme-linked immunosorbent assays (ELISAs), walnut was the most cross-reactive food for hazelnut-specific LFD. The sensitivity was verified in raw cookie doughs and chocolates that were either spiked with peanut or hazelnut between 1 and 25 mg/kg, respectively. Two hazelnut-specific LFDs detected hazelnut at a level of 3.5 mg/kg in both matrices, whereas the third LFD detected hazelnut at a level of 3.9 mg/kg in dough and 12.5 mg/kg in chocolate. Two peanut-specific LFDs detected peanut at a level of 1 mg/kg in both matrices. The third LFD detected peanut at a level of 14.2 mg/kg in chocolate and 4 mg/kg in dough. In conclusion, specific and sensitive LFD were identified for each hazelnut and peanut, having a level of sensitivity that is comparable to commercial ELISA for the investigated matrices. Such sensitive, specific, and rapid tests are useful analytical tools for allergen screening and sanitation in the industrial manufacture of foods.     

Application of a liquid chromatography tandem mass spectrometry method for the simultaneous detection of seven allergenic foods in flour and bread and comparison of the method with commercially available ELISA test kits

To protect the allergic consumer, analytical methods need to be capable of detecting allergens in finished products that typically contain multiple allergens. An LC/MS/MS method for simultaneous detection of seven allergens was developed and compared with commercially available ELISA kits. The detection capabilities of this novel method were demonstrated by analyzing incurred material containing milk, egg, soy, peanut, hazelnut, walnut, and almond. Bread was chosen as a model matrix. To assess the influence of baking on the method's performance, analysis was done before and after baking. The same samples were analyzed with ELISA test kits from ELISA Systems, Morinaga, Neogen, and r-Biopharm. Peanut, hazelnut, walnut, and almond could be detected with both ELISA and LC/MS/MS regardless of whether the product was baked or not. LC/MS/MS clearly showed superior detection of milk in processed matrixes compared to ELISA, which exhibited significantly lower sensitivities when analyzing the baked products. Similar results were obtained when analyzing egg; however, one kit was capable of detecting egg in the processed samples as well.     

Molecular Modeling of Metabolism for Allergen-Free Low Linoleic Acid Peanuts

It is necessary to eliminate linoleic acid and allergenic arachins from peanuts for good health reasons. Virginia-type peanuts, harvested from plots treated with mineral salts combinations that mimic the subunit compositions of glutamate dehydrogenase (GDH) were analyzed for fatty acid and arachin compositions by HPLC and polyacrylamide gel electrophoresis, respectively. Fatty acid desaturase and arachin encoding mRNAs were analyzed by Northern hybridization using the homologous RNAs synthesized by peanut GDH as probes. There were 70?C80?% sequence similarities between the GDH-synthesized RNAs and the mRNAs encoding arachins, fatty acid desaturases, glutamate synthase, and nitrate reductase, which similarities induced permutation of the metabolic pathways at the mRNA level. Modeling of mRNAs showed there were 210, 3,150, 1,260, 2,520, and 4,200 metabolic permutations in the control, NPKS-, NS-, Pi-, NH₄Cl-, and PK-treated peanuts, respectively. The mRNA cross-talks decreased the arachin to almost zero percent in the NPKS- and PK-treated peanuts, and linoleate to ??18?% in the PK-treated peanut. The mRNA cross-talks may account for the vastly reported environmentally induced variability in the linoleate contents of peanut genotypes. These results have quantitatively unified molecular biology and metabolic pathways into one simple biotechnology for optimizing peanut quality and may encourage small-scale industry to produce arachin-free low linoleate peanuts.     

Determination of peanut allergens in cereal-chocolate-based snacks: metal-tag inductively coupled plasma mass spectrometry immunoassay versus liquid chromatography/electrospray ionization tandem mass spectrometry

A comparison of two methods for the identification and determination of peanut allergens based on europium (Eu)-tagged inductively coupled plasma mass spectrometry (ICP-MS) immunoassay and on liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) with a triple quadrupole mass analyzer was carried out on a complex food matrix like a chocolate rice crispy-based snack. The LC/MS/MS method was based on the determination of four different peptide biomarkers selective for the Ara h2 and Ara h3/4 peanut proteins. The performance of this method was compared with that of a non-competitive sandwich enzyme-linked immunosorbent assay (ELISA) method with ICP-MS detection of the metal used to tag the antibody for the quantitative peanut protein analysis in food. The limit of detection (LOD) and quantitation of the ICP-MS immunoassay were 2.2 and 5 microg peanuts g(-1) matrix, respectively, the recovery ranged from 86 +/- 18% to 110 +/- 4% and linearity was proved in the 5-50 microg g(-1) range. The LC/MS/MS method allowed us to obtain LODs of 1 and 5 microg protein g(-1) matrix for Ara h3/4 and Ara h2, respectively, thus obtaining significantly higher values with respect to the ELISA ICP-MS method, taking into account the different expression for concentrations. Linearity was established in the 10-200 microg g(-1) range of peanut proteins in the food matrix investigated and good precision (RSD <10%) was demonstrated. Both the two approaches, used for screening or confirmative purposes, showed the power of mass spectrometry when used as a very selective detector in difficult matrices even if some limitations still exist, i.e. matrix suppression in the LC/ESI-MS/MS procedure and the change of the Ag/Ab binding with matrix in the ICP-MS method.