ELISA detection of hazelnut proteins: effect of protein glycation in the presence or absence of wheat proteins

Hazelnuts are widely used in the food industry, especially confectionary foods. Nevertheless, these nuts contain several allergenic proteins that may be unexpectedly present as contaminants in various foods and may pose a serious threat to allergic consumers. The enzyme-linked immunosorbent assay (ELISA) is the preferred method to assess the level of hazelnut protein contamination. It is commonly used by both the food industry and enforcement agencies. Several ELISA kits are commercially available. However, protein detectability by ELISA may be affected by severe changes that proteins undergo during processing. The aim of this study is therefore to investigate the impact of processing on the ability to detect hazelnut protein by four commercial ELISA kits. Hazelnut proteins in the presence or absence of soluble wheat proteins were modified with glucose via the Maillard reaction. Changes in hazelnut proteins, such as the formation of protein-bound carbonyls, losses of reactive lysine residues and free amino groups, and severe aggregation dramatically affected the hazelnut protein detection by the commercial kits. The observed impact was highly dependent on the type of ELISA kit used.     

Development of a Competitive ELISA for the Detection of Pecan (Carya illinoinensis (Wangenh.) K. Koch) Traces in Food

Pecan nuts are included among the so-called big-eight food allergens, which are responsible for 90% of food allergies. However, unlike other tree nuts such as Brazil nut and hazelnut, studies on pecan from the point of view of allergies are scarce, and only a few analytical methods to detect its presence in food have been published. The objective of the present study was to develop an enzyme-linked immunosorbent assay (ELISA) method to detect traces of pecan in foods. Therefore, pecan-specific polyclonal antibodies were developed in rabbits and characterized for their specificity to pecan proteins and for their selectivity in challenging studies with other food ingredients and allergens. In addition, the research was complemented with more specific analyses like sodium dodecyl sulfate polyacrylamide gel electrophoresis to characterize the protein standard and western blot to identify the reactive proteins. By applying the selected conditions, the data were fitted to a four parameter logistic curve, which gave a R square of 0.999, and IC50 and IC80 values of 5.0 and 2.2 ng/mL, respectively. This highly sensitive method allows the detection of trace amounts of pecan protein within complex food matrices, which can provide the food industry with a useful tool to comply with the requirements enforced by regulatory agencies.     

Development of a Highly Sensitive Competitive Indirect Enzyme-Linked Immunosorbent Assay for Detection of Acrylamide in Foods and Water

Acrylamide (AA) has been classified as a probable human carcinogen and forms in certain foods, particularly plant-based foods that are rich in carbohydrates and low in proteins, during processing or cooking at high temperatures. In this study, polyclonal antibodies were raised against a hapten derived from acrylamide and 3-mercaptobenzoic acid (3-MBA). An indirect competitive enzyme-linked immunosorbent assay was developed to rapidly quantify AA in complex food matrices and water. The assay was very specific to the AA-3-MBA derivative and showed no cross-reactivity to asparagine, the main precursor of AA formation in foods, aspartic acid, AA, or 3-MBA. The assay was very sensitive with a limit of detection of 5.0 ng/g in model for food matrices to 0.1 μg/L in water. Good recoveries for AA were observed in all matrices tested, and the results using this method were comparable to those obtained from mass spectrometry methods including Food Analysis Performance Assessment Scheme control samples and results for different food products.     

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.     

榛子过敏原双抗夹心酶联免疫吸附方法的建立及应用

目的 建立榛子过敏原双抗夹心酶联免疫吸附法(sandwich enzyme-linked immunosorbent assay,sELISA)快速检测食品中微量榛子的方法.方法 对两株抗体进行配对,确定捕获抗体和检测抗体,利用棋盘法确定捕获抗体、检测抗体、辣根过氧化物酶标记的兔抗大鼠IgG(horseradish p...     

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.     

Detection of sesame seed DNA in foods using real-time PCR

The detection of potentially allergenic foods, such as sesame seeds, in food products is a major concern for the food-processing industry. A real-time PCR method was designed to determine if sesame seed DNA is present in food products. The PCR reaction amplifies a 66-bp fragment of the sesame seed 2S albumin gene, which is detected with a sesame-specific, dual-labeled TaqMan probe. This reaction will not amplify DNA derived from other seeds present in baked goods, such as pumpkin, poppy, and sunflower seeds. Additionally, this assay will not cross-react with DNA from several tree nut species, such as almond, Brazil nut, cashew, hazelnut, and walnut, as well as four varieties of peanut. This assay is sensitive enough to detect 5 pg of purified sesame seed DNA, as well as sesame seed DNA in a spiked wheat cracker sample.     

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.     

Polymerase chain reaction coupled with peptide nucleic acid high-performance liquid chromatography for the sensitive detection of traces of potentially allergenic hazelnut in foodstuffs

A new DNA extraction method suitable for a wide variety of complex food matrices has been devised and applied in combination with a new polymerase chain reaction (PCR) method for the sensitive detection of a specific DNA sequence univocally identifying the presence of potentially allergenic hazelnut (Corylus avellana). A 156 base pair amplicon corresponding to an internal region of the complementary DNA of the major hazelnut allergen (Cor a 1) was designed, and was found to be highly specific; the method was tested on both pure and complex food matrices and was found to be able to confirm the presence of hazelnut down to 5 pg of its DNA. The sequence of the amplicon was further confirmed by high-performance liquid chromatography (HPLC) analysis with a specific peptide nucleic acid (PNA) probe. A 15-mer PNA probe was expressly designed and synthesized to hybridize an internal sequence of the previously described amplicon. Given its reported sequence specificity and high hybridization efficiency, the PNA probe was used to develop an anion-exchange HPLC method allowing for a fast and reliable confirmation of the identity of the amplified products. The PCR–HPLC method was successfully tested on commercial samples, allowing for the detection of the presence of potentially hidden allergens even in products where the presence of hazelnut as an ingredient or possible contaminant was not reported.     

Effect of food matrices on the biological activity of ricin

A cell-free translation assay was applied for the quick detection of ricin in food samples. Three economically important foods-ground beef, low-fat milk, and liquid chicken egg--were tested. The results indicated that ground beef had very little matrix effect on the assay, whereas low-fat milk and liquid chicken egg showed clear interference on the protein translation. A simple dilution in phosphate-buffered saline (PBS) effectively eliminated the translational inhibition from these foods. The concentrations inhibiting 50% of luciferase translation derived from the current study were 0.01 nM for the pure ricin A chain, 0.02 nM for pure ricin, and 0.087 nM for crude ricin in PBS. In most cases, the half inhibitory concentration values for ricin in food matrices were significantly lower than for those in PBS buffer, suggesting that some components in these food matrices might potentiate the activity of ricin. Thermal stability tests indicated that the ricin A chain was the least stable among the three forms of ricin in all matrices measured. The thermal stability of pure and crude ricins varied depending on the matrices. The specific activities of ricin in PBS buffer were confirmed by a neutralization test with ricin-specific and nonspecific antibodies. This study demonstrates that the cell-free translation assay is a rapid and sensitive method for detection of biologically active ricin toxin in ground beef, low-fat milk, and liquid chicken egg and that food matrices can greatly affect the thermal stability of ricin.     

An Improved Enzyme Immunoassay for the Detection of Salmonella

An enzyme immunoassay (EIA) was developed for the detection of salmonellae in foods or other samples. This assay detected Salmonella contamination within two working days after initiation, and the EIA required <2 hr to perform. The test was sensitive and specific, showing virtually no cross reactivity to other organisms. No selective enrichment was found to be necessary, as the presence of other organisms, even in much greater numbers, had no effect on the reaction. The sensitivity is such that <10⁶Salmonella/mL were detected, and the food samples studied showed no effect on the background values and did not cause false readings.     

MALDI-based identification of stable hazelnut protein derived tryptic marker peptides

Food allergy is an important health problem especially in industrialised countries. Tree nuts, among which are hazelnuts (Corylus avellana), are typically causing serious and life-threatening symptoms in sensitive subjects. Hazelnut is used as a food ingredient in pastry, confectionary products, ice cream and meat products, therefore undeclared hazelnut can be often present as a cross-contaminant representing a threat for allergic consumers. Mass spectrometric techniques are used for the detection of food allergens in processed foods, but limited information regarding stable tryptic peptide markers for hazelnut is available. The aim of this study was to detect stable peptide markers from modified hazelnut protein through the Maillard reaction and oxidation in a buffered solution. Peptides ³⁹⁵Gly-Arg⁴⁰³ from Cor a 11 and ²⁰⁹Gln-Arg²¹⁷, ³⁵¹Ile-Arg³⁶³, ⁴⁶⁴Ala-Arg⁴⁷⁸ and ⁴⁰¹Val-Arg⁴¹⁷ from Cor a 9 hazelnut allergens proved to be the most stable and could be detected and confirmed with high scores in most of the modified samples. The identified peptides can be further used as analytical targets for the development of more robust quantitative methods for hazelnut detection in processed foods.     

Detection of Mustard, Egg, Milk, and Gluten in Salad Dressing Using Enzyme-Linked Immunosorbent Assays (ELISAs)

ABSTRACT:  Enzyme-linked immunosorbent assay (ELISA) is a commonly used method for the detection of trace amounts of potentially allergenic protein residues in foods. However, food matrices and processing conditions can affect the detection of protein residues. The effects of acidity on the detectability of several allergenic proteins commonly found in salad dressing using ELISAs was investigated. First, recovery experiments were performed on salad dressing formulated with 0 to 1000 ppm mustard flour (mustard). The mean percent recovery for mustard from the salad dressing was only 7.7%± 1.6%. When the pH of the salad dressing was adjusted to pH 7 prior to spiking with mustard, recovery improved to 94.1%± 7.6%. However, if the pH was adjusted to pH 7 after spiking and extraction, the recovery was only 11.1%± 1.7%. When vinegar was spiked with mustard flour at pH 3, 3.5, and 4, detectability of mustard was lowest at pH 3. Basic extraction of mustard proteins from salad dressing did not improve the mustard detection. Acidic salad dressing matrices reduced the detectability of mustard by the mustard ELISA probably because of acid precipitation of mustard proteins that renders them insoluble and nonextractable. Commercial salad dressings containing 100 ppm (mg/kg) of egg, milk, or gluten were analyzed every 2 to 4 d for 90 d using 3 commercially available ELISAs. A decrease in the detection of the egg, milk, and gluten in the salad dressing upon storage was observed. Our study highlighted the importance of evaluating the utility of various ELISAs for specific food matrices and the recovery as a function of product storage.     

Digestibility of peanut and hazelnut allergens investigated by a simple in vitro procedure

 Stability under digestion is thought to be an important prerequisite determining the allergenicity of food proteins. To test this hypothesis, two important allergenic plant-derived foods were selected for this investigation. Protein extracts from roasted peanuts and unprocessed (native) hazelnuts were digested by a static, two-step in vitro procedure with commercial enzyme tablets containing peptic and pancreatic enzymes, respectively. The food extracts were subjected to gastric digestion for 2 h followed by a 45-min treatment under duodenal conditions. Undigested control samples and the two digests were investigated by analytical sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), by SDS-PAGE immunoblotting, by an enzyme allergosorbent test (EAST) with human IgE, and by a rat basophil leukaemia (RBL) cell mediator release assay that depends on specific IgE raised in mice. Peanut proteins appeared to be more stable under digestion than hazelnut proteins, as shown by electrophoresis. These results were also underlined by immunblot data. The gastric digest from peanut contained various protein fragments that were detected by antibodies from a peanut-specific rabbit antiserum and by IgE from patients allergic to peanuts. These immunoblot reactivities decreased strongly after subsequent pancreatic digestion. In the gastric digest from hazelnuts, a rabbit antiserum with a broad reactivity against native hazelnut proteins exclusively recognized small protein fragments of <15 kDa. This serum showed no binding to blots of the pancreatic digest. Sera from hazelnut-allergic patients presented IgE reactivities to an 18-kDa major allergen with homology to major tree-pollen allergens, to a minor allergen of 12 kDa, and to multiple bands  1 30 kDa in native hazelnut extract. No binding was observed with these sera on blot strips of the two digests prepared from hazelnut extract. Under the native conditions of EAST, both digests from peanuts strongly reacted with human IgE. Their IgE binding capacity persisted at a level of about 50% when compared to undigested peanut. In the case of hazelnuts the IgE reactivity of the untreated samples was reduced to <10% by both gastric and combined gastric/duodenal digestion for a serum pool prepared from four patients and sera from three additional participants. By contrast, a constantly high immunoreactivity of the hazelnut digests was detected with serum from one patient. The results of the EAST were confirmed by dose-related mediator release experiments performed with RBL cells passively sensitized with allergen-specific murine IgE. As a whole, our results indicated that the EAST and the RBL cell assay are superior to immunoblotting for the immunologic testing of digests. In accordance with clinical observations, the allergenicity of peanut proteins was very persistent during digestion, whereas the native birch-pollen-related hazelnut allergens appeared to be relatively labile under identical conditions. Received: 6 July 1998     

Development of a Sandwich Enzyme-Linked Immunosorbent Assay (ELISA) for Detection of Buckwheat Residues in Food

Abstract: Buckwheat is a pseudocereal (an eudicot with seed qualities and uses similar to those of monocot cereals, family Poaceae) that is consumed in some Asian countries as a staple, and in some western countries as a health food. Allergic reactions to buckwheat are common in some countries. The objective was to develop a specific and sensitive sandwich enzyme-linked immunosorbent assay (ELISA) to detect traces of buckwheat that might inadvertently contaminate other foods in order to assure accurate labeling and consumer protection. Buckwheat-specific antibodies produced in 3 species of animals were tested for specificity and titer by direct ELISA and immunoblot. A sandwich ELISA was developed utilizing pooled rabbit antibuckwheat sera to capture buckwheat proteins and pooled goat antibuckwheat sera, followed by enzyme-labeled rabbit antigoat immunoglobulin G (IgG), to detect bound buckwheat proteins. The lower limit of quantification (LOQ) of the sandwich ELISA was 2 parts per million (ppm) of buckwheat in the presence of complex food matrices. The ELISA is highly specific with no cross-reactivity to any of 80 food ingredients and matrices tested. Validation studies conducted with buckwheat processed into noodles and muffins showed greater than 90% and 60% recovery, respectively. The percent recovery of buckwheat from noodles was similar to that achieved with a commercial buckwheat ELISA kit (ELISA Systems Pty. Ltd., Windsor, Queensland, Australia) at high buckwheat concentrations. However, the sensitivity of this ELISA was greater than the commercial ELISA. This newly developed ELISA is sufficiently specific and sensitive to detect buckwheat residues in processed foods to protect buckwheat-allergic subjects from potential harm. Practical Application: Buckwheat is becoming a common food ingredient in a number of processed foods due to potentially beneficial nutritional properties, without the celiac disease inducing glutenin proteins of wheat and related cereals. However, buckwheat causes allergy in some individuals and must be labeled and tested accurately to protect those with allergy to buckwheat. We describe the development of a new test assay to help food producers ensure that buckwheat is not present in foods that are not intended to contain buckwheat.     

Food allergy—towards predictive testing for novel foods

The risks associated with IgE-mediated food allergy highlight the need for methods to screen for potential food allergens. Clinical and immunological tests are available for the diagnosis of food allergy to known food allergens, but this does not extend to the evaluation, or prediction of allergenicity in novel foods. This category includes foods produced using novel processes, genetically modified (GM) foods, and foods that might be used as alternatives to traditional foods. Through the collation and analysis of the protein sequences of known allergens and their epitopes, it is possible to identify related groups which correlate with observed clinical cross-reactivities. 3-D modelling extends the use of sequence data and can be used to display eptiopes on the surface of a molecule. Experimental models support sequence analysis and 3-D modelling. Observed crossreactivities can be examined by Western blots prepared from native 2-D gels of a whole food preparation (e.g. hazelnut, peanut), and common proteins identified. IgEs to novel proteins can be raised in Brown Norway rat (a high IgE responder strain), and the proteins tested in simulated digest to determine epitope stability. Using the CSL serum bank, epitope binding can be examined through the ability of an allergen to cross-link the high affinity IgE receptor and thereby release mediators using in vitro cell-based models. This range of methods, in combination with data mining, provides a variety of screening options for testing the potential of a novel food to be allergenic, which does not involve prior exposure to the consumer.     

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 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.     

免疫磁珠捕获结合荧光PCR 技术检测食品中黄曲霉菌的研究

目的：研究利用免疫磁珠对食品中黄曲霉菌进行捕获的技术,为检测食品中污染的黄曲霉菌提供新方法。方法：应用山羊抗黄曲霉菌血清的免疫磁珠捕获食品中的黄曲霉菌,不需要进行增菌培养,应用磁捕获- 荧光聚合酶链式反应(IMC-FPCR)技术快速检测鉴定食品中的黄曲霉菌。结果：IMC-FPCR 方法检测黄曲霉菌的最低检测值为2CFU/mL。应用IMC-FPCR 对从市场上购买的12 种食品进行检测,发现有两份样品含有产毒素相关基因(即为可能的致癌菌株),检出率为16.7%,其中花生中检出1 株黄曲霉菌,面线中检出1 株黄曲霉菌。结论：建立了黄曲霉菌的磁捕获- 荧光聚合酶链式反应(IMC-FPCR)方法,该方法可用于食品中黄曲霉菌的快速检测及鉴定,实物检测结果提示市场上食品有被真菌污染。     

Validating allergen coding genes (Cor a 1, Cor a 8, Cor a 14) as target sequences for hazelnut detection via Real-Time PCR

Hazelnuts have been shown to contain different allergenic proteins. Amongst these, major allergens Cor a 1 and Cor a 8 and the 2S albumin Cor a 14 were selected as targets to comparatively validate three Real-Time PCR protocols. We investigated both on the choice of the amplification target, and on the matrix effect on different sample foods. Applying statistics on the validation parameters obtained from the three protocols, we showed a significant difference in Ct values. This could turn critical when a high sensitivity method is required for the detection of hazelnut traces, confirming how fundamental the choice of the template during primer design phase is. Concluding, statistical approach represents a useful tool for the identification of the best performing primer pairs in Real-Time PCR. Cor a 8 gene permitted the identification of hazelnut based ingredients in complex foods, providing a significantly higher sensitivity in the PCR amplification, when compared to Cor a 1 and Cor a 14.