Effect of protein glycation in the presence or absence of wheat proteins on detection of soybean proteins by commercial ELISA

Soybean (Glycine max) is the world's primary provider of protein and oil and is widely used in foodstuffs. However, the use of soybean in foodstuffs might pose a serious threat to allergic consumers since some proteins can cause allergic reactions. To date mostly ELISA methods are used for testing contamination of foodstuffs with soybean. In view of the complexity regarding allergen detection in foodstuffs and appropriate food product labelling, the aim of this study was to investigate the impact of the Maillard reaction on the detectability of soybean proteins using commercial ELISA kits. Accumulation of protein-bound carbonyls, modification of reactive lysine residues and severe aggregation as a result of incubation with glucose, in the presence or absence of soluble wheat proteins, were recorded. Moreover, detection of soybean proteins by means of three commercial ELISA kits was strongly altered and was highly dependent on the type of kit used.     

Detection of hazelnut proteins in foods by enzyme immunoassay using egg yolk antibodies

An enzyme immunoassay (EIA) was developed for the detection of hazelnut proteins in foods. This assay used inexpensive chicken egg yolk antibodies in a sandwich EIA format for the immunospecific capture and detection of hazelnut proteins present in a variety of different food matrices. The assay was able to detect less than 1 ppm of hazelnut protein in most of the foods tested and did not exhibit any appreciable cross-reactivity with other nuts or food matrices. This assay will be a useful tool for the food industry and regulatory agencies that wish to test foods for the presence of undeclared hazelnut allergens.     

Effect of protein glycation in the presence or absence of wheat proteins on detection of soybean proteins by commercial ELISA

Soybean (Glycine max) is the world's primary provider of protein and oil and is widely used in foodstuffs. However, the use of soybean in foodstuffs might pose a serious threat to allergic consumers since some proteins can cause allergic reactions. To date mostly ELISA methods are used for testing contamination of foodstuffs with soybean. In view of the complexity regarding allergen detection in foodstuffs and appropriate food product labelling, the aim of this study was to investigate the impact of the Maillard reaction on the detectability of soybean proteins using commercial ELISA kits. Accumulation of protein-bound carbonyls, modification of reactive lysine residues and severe aggregation as a result of incubation with glucose, in the presence or absence of soluble wheat proteins, were recorded. Moreover, detection of soybean proteins by means of three commercial ELISA kits was strongly altered and was highly dependent on the type of kit used.     

Influence of baking time and matrix effects on the detection of milk allergens in cookie model food system by ELISA

Milk allergens are common allergens occurring in foods, therefore raising concern in allergic consumers. Enzyme-linked immunosorbent assay (ELISA) is, to date, the method of choice for the detection of food allergens by the food industry although, the performance of ELISA might be compromised when severe food processing techniques are applied to allergen-containing foods. In this paper we investigated the influence of baking time on the detection of milk allergens by using commercial ELISA kits. Baked cookies were chosen as a model food system and experiments were set up to study the impact of spiking a matrix food either before, or after the baking process. Results revealed clear analytical differences between both spiking methods, which stress the importance of choosing appropriate spiking methodologies for method validation purposes. Finally, since the narrow dynamic range of quantification of ELISA implies that dilution of samples is required, the impact of sample dilution on the quantitative results was investigated. All parameters investigated were shown to impact milk allergen detection by means of ELISA.     

Commercial Milk Enzyme‐Linked Immunosorbent Assay (ELISA) Kit Reactivities to Purified Milk Proteins and Milk‐Derived Ingredients

Numerous commercial enzyme‐linked immunosorbent assay (ELISA) kits exist to quantitatively detect bovine milk residues in foods. Milk contains many proteins that can serve as ELISA targets including caseins (α‐, β‐, or κ‐casein) and whey proteins (α‐lactalbumin or β‐lactoglobulin). Nine commercially‐available milk ELISA kits were selected to compare the specificity and sensitivity with 5 purified milk proteins and 3 milk‐derived ingredients. All of the milk kits were capable of quantifying nonfat dry milk (NFDM), but did not necessarily detect all individual protein fractions. While milk‐derived ingredients were detected by the kits, their quantitation may be inaccurate due to the use of different calibrators, reference materials, and antibodies in kit development. The establishment of a standard reference material for the calibration of milk ELISA kits is increasingly important. The appropriate selection and understanding of milk ELISA kits for food analysis is critical to accurate quantification of milk residues and informed risk management decisions.     

Towards a Comprehensive Validation of ELISA Kits for Food Allergens: Case 1—Egg

There are several enzyme-linked immunosorbent assay (ELISA) kits in the market that have been proven to be useful for the determination of egg in foods. However, inconsistent results that are obtained when different kits are used make the selection of one kit over another very difficult. Two different approaches were used to help understand why results vary among kits. Different kits were used to analyze spiked egg material [NIST reference material (RM) 8445] in wheat flour (raw ingredients) and cookies containing egg as an ingredient baked for different periods of time (processed food). These results were compared with immunoblotting using conjugated antibodies from the commercial kits to determine the antibody specificity and sample extraction efficiency. ELISA results can be difficult to compare because reporting units differ among kits. Results from both ELISA and immunoblotting are in agreement regarding the decreased detection of proteins in baked cookie extracts. Moreover, immunoblotting showed that this reduction is due to reduced protein content in these extracts. However, a properly selected extraction solution may help improve the solubility of certain egg proteins in processed foods. Harmonization of the reporting unit system along with the use of a common reference material is recommended as the path forward in the standardization of detection methods for food allergens. This would assist the end user in making an informed decision regarding the selection of the most appropriate kit for his or her purpose.     

Towards a Comprehensive Validation of ELISA Kits for Food Allergens. Case 2—Milk

There are several enzyme-linked immunosorbent assay (ELISA) kits in the market that have been proven to be useful for the determination of egg in foods. However, inconsistent results that are obtained when different kits are used make the selection of one kit over another very difficult. Two different approaches were used to help understand why results vary among kits. Different kits were used to analyze spiked egg material [NIST reference material (RM) 8445] in wheat flour (raw ingredients) and cookies containing egg as an ingredient baked for different periods of time (processed food). These results were compared with immunoblotting using conjugated antibodies from the commercial kits to determine the antibody specificity and sample extraction efficiency. ELISA results can be difficult to compare because reporting units differ among kits. Results from both ELISA and immunoblotting are in agreement regarding the decreased detection of proteins in baked cookie extracts. Moreover, immunoblotting showed that this reduction is due to reduced protein content in these extracts. However, a properly selected extraction solution may help improve the solubility of certain egg proteins in processed foods. Harmonization of the reporting unit system along with the use of a common reference material is recommended as the path forward in the standardization of detection methods for food allergens. This would assist the end user in making an informed decision regarding the selection of the most appropriate kit for his or her purpose.     

Effect of roasting history and buffer composition on peanut protein extraction efficiency

Peanut is a major allergenic food. Undeclared peanut (allergens) from mis-formulation or contamination during food processing pose a potential risk for sensitized individuals and must be avoided. Reliable detection and quantification methods for food allergens are necessary in order to ensure compliance with food labelling and to improve consumer protection. The extraction of proteins from allergenic foods and complex food products is an important step in any allergen detection method. In this study, the protein extraction efficiency of various buffers prepared in-house and some extraction buffers included in some commercial allergen enzyme-linked immunosorbent assay (ELISA) test kits for peanut determination in food products were tested. In addition, the effect of roasting history on the extractability of peanut protein was investigated by the biuret and the bicinchoninic acid (BCA) assays. Elevated roasting temperatures in food processing were found to have a major impact on protein extraction efficiency by reducing protein yields of oil and dry roasted peanuts by 50-75% and 75-80%, respectively, compared with the raw material. Extraction buffers operating in the higher pH range (pH 8-11) showed best yields.     

Effect of proteolysis during Cheddar cheese aging on the detection of milk protein residues by ELISA

Cow milk is a common allergenic food, and cow milk-derived cheese retains an appreciable level of allergenicity. The specific and sensitive detection of milk protein residues in foods is needed to protect milk-allergic consumers from exposure to undeclared milk protein residues contained in foods made with milk or milk-derived ingredients or made on shared equipment or in shared facilities with milk or milk-derived ingredients. However, during cheese ripening, milk proteins are degraded by chymosin and milk-derived and bacterial proteases. Commercial allergen-detection methods are not validated for the detection of residues in fermented or hydrolyzed products. The objective of this research was to evaluate commercially available milk ELISA kits for their capability to detect milk protein residues in aged Cheddar cheese. Cheddar cheese was manufactured at a local dairy plant and was aged at 5°C for 24 mo, with samples removed at various time points throughout aging. Milk protein residues and protein profiles were measured using 4 commercial milk ELISA kits and sodium dodecyl sulfate-PAGE. The ELISA data revealed a 90% loss of milk protein residue signal between the youngest and oldest Cheddar cheese samples (0.5 and 24 mo, respectively). Sodium dodecyl sulfate-PAGE analysis showed protein degradation throughout aging, with the highest level of proteolysis observed at 24 mo. Results suggest that current commercial milk ELISA methods can detect milk protein residues in young Cheddar cheese, but the detection signal dramatically decreases during aging. The 4 evaluated ELISA kits were not capable of detecting trace levels of milk protein residues in aged cheese. Reliable detection of allergen residues in fermented food products is critical for upholding allergen-control programs, maintaining product safety, and protecting allergic consumers. Furthermore, this research suggests a novel use of ELISA kits to monitor protein degradation as an indication of cheese ripening.     

酶联免疫吸附法与二喹啉甲酸法检测核桃蛋白浓度的比较研究

目的 探究间接酶联免疫吸附法(enzyme-linked immunosorbent assay, ELISA)和二喹啉甲酸 (bicinchoninic acid, BCA)试剂盒2种方法在核桃蛋白浓度检测中的优缺点和适用范围。方法 对本实验室提取的核桃蛋白进行烘烤、高温高压以及微波加热处理后, 用ELISA法和BCA法2种方法分别检测其蛋白质浓度。结果 在不同的热加工处理条件下, ELISA对蛋白浓度测定结果存在显著性差异, 高度依赖热处理的类型。与之相比, 使用BCA法测定蛋白浓度时, 受到热加工处理影响较小。结果提示, 在食物加工中的一些剧烈条件下, 蛋白质的高级结构很容易遭到破坏, 蛋白质结构的改变会干扰其与抗体结合部位的作用, 导致ELISA法测定的结果不准确。BCA法测定蛋白质浓度的准确性较高, 且受加热影响较小。结论 在剧烈的食品加工条件下, 尤其当这种条件引起蛋白结构发生改变后, 对其蛋白质浓度进行测定时, 可选用BCA法进行测定。     

Development and validation of two dipstick type immunoassays for determination of trace amounts of peanut and hazelnut in processed foods

Two dipstick-type sandwich-ELISAs were developed allowing the detection of trace amounts of peanut and hazelnut in processed foods. The detection limit of both assays was about 10 ng/ml of hazelnut or peanut protein, equivalent to 1 ppm protein in a food sample. The dipstick format allows a fast and cost-effective screening of foods because no specific instrumentation, such as microplate reader and washer, is necessary. The tests can be performed within 3 and 4 hours respectively. Various commercial food samples were tested and the results were compared with previously described and validated quantitative ELISAs for peanut and hazelnut. Results of the dipstick assays and the corresponding microplate ELISAs were in complete concordance. Our results indicated that the dipstick format could be a useful tool for fast and convenient monitoring of food production with regard to the labelling of allergens in food products. This would contribute to improve the protection of consumers from severe allergic reactions.     

Evaluation of immunochromatographic test kits for food allergens using processed food models

It has been mandatory to label five allergenic substances (AS; egg, milk, wheat, buckwheat and peanut) in all processed foods, since April 2002 in Japan. Two kinds of ELISA kits have been provided as screening test kits for the Japanese official method. The kits have many advantages but some disadvantages, i.e., the kits are not necessarily suitable for daily monitoring in food manufacturing plants, because they require various analytical equipments and the use of complicated procedures. To overcome these drawbacks, we have developed other diagnostic kits based on immunochromatography that should enable more rapid and simple screening for food allergens. Then we examined the performance of these immunochromatographic test kits (IC kits) in terms of sensitivity, repeatability and cross-reactivity to AS proteins in 11 kinds of food models with various heating conditions and physical properties. We also examined processed food models including AS protein of constant concentration, using the IC kits and ELISA kits, and compared the results. The IC kits detected AS proteins at 5 microg/g in the extracts from processed food models, and provided highly reproducible results. Cross-reactivity among the AS proteins was not observed. The results obtained using the IC kits showed performance equivalent to that of the ELISA kits we examined in unheating processed food models including AS proteins of constant concentration. The IC kits should be more suitable for daily monitoring in food manufacturing plants.     

Validated sandwich enzyme-linked immunosorbent assay for casein and its application to retail and milk-allergic complaint foods

Cows' milk is a commonly allergenic food. Cross-contamination of milk proteins into nondairy, kosher-pareve foods prepared on shared processing equipment can cause severe, life-threatening reactions in milk-allergic individuals. A sandwich-type enzyme-linked immunosorbent assay (ELISA; 96-well plate format) was developed for the detection of undeclared casein in foods. Rabbit anti-casein antibodies were used as the capture reagent. Food samples and standards were ground, extracted in 0.01 M phosphate-buffered saline, clarified by centrifugation, and added to the wells. Goat anti-casein antibodies were employed as the detector antibody, and the amount of antibody bound was determined with a commercial rabbit anti-goat immunoglobulin conjugated to alkaline phosphatase, with subsequent substrate reaction. Antibodies developed were specific to casein, with no cross-reaction observed with 30 foods and food ingredients. Non-milk-containing products such as fruit juices, fruit juice bars, sorbets, and dark and pareve-labeled chocolate were purchased from June 2002 through June 2003. In addition, samples allegedly causing eight milk-allergic consumer complaints were analyzed. The ELISA had a detection limit of less than 0.5 ppm of casein. The casein content in the analyzed foods ranged from less than 0.5 ppm to more than 40,000 ppm casein; undeclared casein residues were found in all of the samples implicated in allergic reactions. The levels of milk contamination in some of the other surveyed products could also be hazardous for milk-allergic consumers. This ELISA method provides a useful quality control tool for the food industry and could also be used as a validation of kosher-pareve status.     

鱼酶和鱼蛋白的性质及其在食品工业中的应用

综述了鱼酶及鱼蛋白的性质及其在食品工业中的应用。与哺乳动物相比,鱼类新陈代谢需要特殊的酶,这些酶在特殊的环境条件下具有最大的活力。由于活力的不同,尤其是适应冷环境的蛋白酶,可利用其特点可应用于某些食品的生产。除了这些酶以外,还描述了性质较为独特的鱼类多肽抗冷冻蛋白,这些蛋白质和多肽已克隆并且应用于食品加工。同时也讨论了鱼质白在食品工业领域的影响及其展望。     

Development of a Biological Assay to Determine the Allergenic Potential of Foods

Food allergies represent a risk for many people in industrialized countries. Unrecognizable allergenic proteins of foodstuffs may be present as ingredients that are not labeled or as unknown cross-contamination. Such hidden allergens can cause severe reactions in allergics, even at minute quantities, sometimes with fatal outcome. For the verification of the presence of allergenic food constituents, analytical methods such as ELISA and PCR have been developed. However, these tests cannot measure allergenic potential. For this reason, a test system that measures the biological activity of allergens has been developed. It is based on the cellular mechanisms of the type I allergy. Rat basophilic leukemia cells (RBL-2H3) were transfected with the genes of the human high affinity receptor for IgE. The resulting cell line expressed the human receptor α-chain and could bind allergenspecific IgE from allergic subjects, in contrast to the parent cell line. After cross-linking of receptor-bound, allergen-specific human IgE by allergens, the cells released measurable inflammatory mediators. These cells were used for the analysis of a variety of allergen extracts, including extracts prepared from foods containing allergenic hazelnut and peanut. The comparative validation with existing ELISA and PCR for hazelnut and peanut demonstrated similar sensitivity and specificity. The established cell line will be a novel tool in the detection of allergens in complex mixtures, especially to address the issue of their allergenic potential, which cannot be accomplished by classical analytical methods. This will add valuable information about the allergenic potential of food constituents to the risk assessment of foods.     

Detection of Walnut Residues in Foods Using an Enzyme-Linked Immunosorbent Assay

ABSTRACT:  Tree nuts, including walnuts, can be responsible for allergic reactions. Food manufacturers have the responsibility to declare the presence of walnuts on packaged foods even when trace residues may be present from the use of shared equipment or the adventitious contamination of ingredients. The aim of this study was to develop a rapid, sensitive, and specific enzyme-linked immunosorbent assay (ELISA) method for the detection of walnut protein residues. Mixtures of raw and roasted English walnuts of several varieties were defatted, powdered, and used as separate antigens in sheep and New Zealand white rabbits. An ELISA was developed using the sheep antiroasted walnut serum as the capture reagent and rabbit antiroasted walnut serum as the detector reagent followed by addition of commercial goat anti-rabbit IgG antibody labeled with alkaline phosphatase and subsequent substrate addition. The performance of the ELISA was validated by testing known amounts of walnut (0 to 100 ppm) either spiked into or manufactured into milk chocolate, cookies, muffins, or ice cream. Recoveries of 1 to 100 ppm walnut-in-chocolate ranged from 71.6% to 119%± 7% to 16.5%. The walnut ELISA has a detection limit of 1 ppm (1 μg/g) walnut in several food matrices. Substantial cross-reactivity was observed with pecan while minimal cross-reactivity was noted for hazelnut, mustard, mace, and poppy seed among almost 100 foods and food ingredients tested. This walnut ELISA can be used to detect undeclared walnut residues in foods and ingredients and as a tool to validate the effectiveness of allergen control programs for walnuts.     

Influence of different extraction conditions on the detection of glycinin and β-conglycinin in model processed foods by ELISA

ABSTRACT

The presence of undeclared soy proteins in food can cause severe reactions in soy allergic individuals. The extraction of target proteins from processed foods is a crucial step in allergen detection by immunoassays, as only successfully extracted target proteins can be detected by the specific antibodies. The effectiveness was studied of different conditions (type of buffer, temperature and time of incubation) on the extraction of total protein, and concentration of glycinin and β-conglycinin from different food matrices. The yields were determined using a soy protein isolate and three processed foods (sausage, bread and pâté) incurred with soy proteins. The yields were affected by the processing of analysed products and the composition and pH of the extraction buffers. Neutral and alkaline buffers (pH from 7.4 to 10.6) exhibited good protein extraction capacity and detectability of the specific target proteins. Denaturing additives and highly alkaline buffer (pH 12) extracted more crude protein but they were incompatible with the ELISA assay. Overall, the best results were obtained using phosphate (pH 7.4) and Tris/HCl (pH 8.5) buffers in the presence of 0.5 M NaCl. Crude protein yield of food extracts did not correlate with that of glycinin and β-conglycinin, whereas a good relationship was found between the yields of the two proteins.     

Development of two immunoassay formats to detect beta-lactoglobulin: influence of heat treatment on beta-lactoglobulin immunoreactivity and assay applicability in processed food

Milk proteins are commonly used as ingredients in the food industry because of their functional properties, but they can cause severe reactions in milk-allergic individuals. In this work, two enzyme-linked immunosorbent assay (ELISA) formats were developed to detect bovine beta-lactoglobulin. The indirect competitive ELISA involved the use of anti-beta-lactoglobulin antisera, and the sandwich ELISA involved the use of specific antibodies isolated using a beta-lactoglobulin immunosorbent material. The effect of heat treatment on immunoreactivity of the protein in buffer and in milk was determined with both assays. The amount of immunoreactive protein in buffer and in milk decreased as determined by the sandwich ELISA, whereas the amount increased when measuring with the competitive ELISA. Several food products, including meat, bakery products, sauces, and snacks, were analyzed. With both assays, 10 of 11 products in which the ingredient list included the terms "powdered milk" or "milk proteins" contained beta-lactoglobulin. However, the beta-lactoglobulin concentration in these products obtained with the competitive ELISA were much higher than those obtained with the sandwich ELISA. These differences could be explained by the fact that the determination of beta-lactoglobulin concentration by immunoassay is influenced by differences in antibody recognition of the protein present in highly processed foods. Therefore, the antigen-binding properties of antibodies used in a particular immunoassay are important for a correct interpretation of results obtained in food processed at high temperature.     

Hazelnut Allergens: Molecular Characterization,Detection, and Clinical Relevance

In last few years, special attention has been given to food-induced allergies, in which hazelnut allergy is highlighted. Hazelnut is one of the most commonly consumed tree nuts, being largely used by the food industry in a variety of processed foods. It has been regarded as a food with potential health benefits, but also as a source of allergens capable of inducing mild to severe allergic reactions in sensitized individuals. Considering the great number of reports addressing hazelnut allergens, with an estimated increasing trend, this review intends to assemble all the relevant information available so far on the following main issues: prevalence of tree nut allergy, clinical threshold levels, molecular characterization of hazelnut allergens (Cor a 1, Cor a 2, Cor a 8, Cor a 9, Cor a 10, Cor a 11, Cor a 12, Cor a 14, and Cor a TLP) and their clinical relevance, and methodologies for detection of hazelnut allergens in foods. A comprehensive overview of the current data about the molecular characterization of hazelnut allergens is presented, relating to biochemical classification and biological function with clinical importance. Recent advances in hazelnut allergen detection methodologies are summarized and compared, including all the novel protein-based and DNA-based approaches.     

A critical review of the specifications and performance of antibody and DNA-based methods for detection and quantification of allergens in foods

Despite the availability of a large number of antibody and DNA based methods for detection and quantification of allergens in food there remain significant difficulties in selecting the optimum technique to employ. Published methods from research groups mostly contain sufficient detail concerning target antigen, calibration procedures and method performance to allow replication by others. However, routine allergen testing by the food industry relies upon commercialised test kits and frequently the suppliers provide disappointingly little specification detail on the grounds that this is proprietary information. In this review we have made a critical assessment of the published literature describing the performance of both commercial and non-commercial test kits for food allergens over the period 2008–2018. Mass spectrometric methods, which have the potential to become reference methods for allergens, are not covered in this review. Available information on the specifications of commercial ELISA and LFD test kits are tabulated for milk, egg and peanut allergens, where possible linking to publications concerning collaborative studies and proficiency testing. For a number of commercial PCR test kits, specifications provided by manufacturers for detection of a small selection of allergen are tabulated. In conclusion we support the views of others of the critical need for allergen reference materials as the way forward to improve the comparability of different testing strategies in foods.