Traces Of Allergens Research Articles

Purified Clinoptilolite-Tuff as an Efficient Sorbent for Food-Derived Peanut Allergens.

The avoidance of allergen intake is crucial for persons affected by peanut allergy; however, the cross-contamination of food is common and leads to unpredictable consequences after the consumption of supposedly "safe" food. The aim of the present study was to eliminate harmful traces of peanut allergens from food using purified clinoptilolite-tuff (PCT)-a specially processed zeolite material. Analyses were performed using a peanut ELISA and a Coomassie blue (Bradford) assay. Mimicking conditions of the human gastrointestinal tract demonstrated a higher efficacy of PCT in the intestine (pH 6.8) than in the stomach (pH 1.5). Adsorption rates were fast (<2 min) and indicated high capacities (23 µg and 40 µg per 1 mg of PCT at pH 1.5 and pH 6.8, respectively). Allergenically relevant peanut protein concentrations were sorbed in artificial fluids (32 µg/mL by 4 mg/mL of PCT at pH 1.5 and 80.8 µg/mL by 0.25 mg/mL of PCT at pH 6.8) when imitating a daily dose of 2 g of PCT in an average stomach volume of 500 mL. Experiments focusing on the bioavailability of peanut protein attached to PCT revealed sustained sorption at pH 1.5 and only minor desorption at pH 6.8. Accompanied by gluten, peanut proteins showed competing binding characteristics with PCT. This study therefore demonstrates the potential of PCT in binding relevant quantities of peanut allergens during the digestion of peanut-contaminated food.

Reactivity to allergenic food contaminants: A study on products on the market.

The frequency and severity of reactions in food-allergic consumers exposed to unintentional food allergen contamination during production is unknown. To warn allergic consumers, it has been suggested for pre-packaged foods to be precautionary labelled when the food allergen contamination may exceed the amount to which 1%-5% of the population could react (ED01-ED05). ED01 for hazelnut and milk have been estimated at 0.1 and 0.2mg, respectively, by the Voluntary Incidental Trace Allergen Labelling (VITAL) initiative. The respective reference doses recommended by the FAO/WHO Codex consultation are 3 and 2mg. We evaluated the reactivity to potential traces of milk and hazelnut allergens in allergen-free pre-packaged products by children affected by severe allergies to milk and hazelnuts. Oral Food Challenges with commercially available hazelnut-free wafer biscuits and milk-free chocolate pralines were administered to patients with severe food allergies to hazelnut and cow's milk, respectively. Contamination levels of milk or hazelnut allergens were measured using chromatographic separation interfaced with triple quadrupole mass spectrometry. No hazelnut allergic patient showed allergic reactions to exposure to biscuits, nor any milk allergic patient displayed allergic reactions to the dark chocolate praline. While no hazelnut trace was detected in biscuits, the praline was found to be contaminated by milk at concentrations ranging between 8 and 35mg total protein/kg food. In our dose model, these amounts exceeded 1.5-10 times the VITAL ED01 and reached the threshold suggested by the FAO/WHO Codex consultation. Upon the consumption of food products available on the market, many patients with severe food allergies tolerate significantly higher doses of allergen than reference doses indicated in the VITAL system used for precautionary allergen labelling. These doses support the safety of the FAO/WHO recommended reference doses.

Knowledge, Attitudes, and Practices of Consumers on Food Allergy and Food Allergen Labeling: A Case of Lebanon.

The epidemiology of food allergies is increasing worldwide. International labeling standards were developed to enhance consumers' awareness of allergen-free foods. The main objective of the present study is to assess the characteristics of allergen labeling and consumers' knowledge, attitudes, and purchasing habits of food products with allergens in Lebanon. We evaluated the allergen labeling of 1000 food products form Lebanese supermarkets. A random sample of 541 consumers was recruited through an online survey (November 2020-February 2021). Descriptives and regression analysis were conducted. Results showed that wheat represents the largest group of food allergens on food labels, followed by milk and soybean. Furthermore, 42.9% of supermarket food products had a precautionary allergen labeling with "may contain traces of allergens". The majority of food products complied with local regulations for locally manufactured and imported products. One-quarter of survey respondents had a food allergy or were caregivers of food-allergic individuals. Regression analyses showed that "previous experience of a severe reaction" was negatively associated with food allergy-related knowledge and attitude scores respectively (β = -1.394, 95% CI: (-1.827, -1.034) and β = -1.432, 95% CI: (-2.798, -0.067)). The findings of this study provide practical insights on food allergy labeling issues for stakeholders and policymakers in the food supply chain.

Lateral Flow Microimmunoassay (LFµIA) for the Reliable Quantification of Allergen Traces in Food Consumables.

Quality assurance and food safety are of great concern within the food industry because of unknown quantities of allergens often present in food. Therefore, there is an ongoing need to develop rapid, sensitive, and easy to use methods that serve as an alternative to mass spectrometry and enzyme-linked immunosorbent assay (ELISA) for monitoring food safety. Lateral flow immunoassay is one of the most used point-of-need devices for clinical, environmental, and food safety applications. Compared to traditional methods, it appears to be a simple and fast alternative for detecting food allergens. However, its reliability is frequently questioned due to the lack of quantitative information. In this study, a lateral flow microimmunoassay (LFµIA) is presented that integrates up to 36 spots in microarray format in a single strip, providing semi-quantitative information about the level of allergens, positive and negative controls, internal calibration, and hook effect. The LFµIA has been evaluated for the on-site simultaneous and reliable quantification of almond and peanut allergens as a proof of concept, demonstrating high sensitivity (185 and 229 µg/kg, respectively), selectivity (77%), and accuracy (RSD 5-25%) when analyzing commercial allergen-suspicious food consumables.

Threshold of Reactivity and Tolerance to Precautionary Allergen-Labelled Biscuits of Baked Milk- and Egg-Allergic Children

Extremely sensitive food-allergic patients may react to very small amounts of allergenic foods. Precautionary allergen labelling (PAL) warns from possible allergenic contaminations. We evaluated by oral food challenge the reactivity to a brand of PAL-labelled milk- and egg-free biscuits of children with severe milk and egg allergy. We explored the ability of proteomic methods to identify minute amounts of milk/egg allergens in such biscuits. Traces of milk and/or egg allergens in biscuits were measured by two different liquid-chromatography-mass spectrometry methods. The binding of patient’s serum with egg/milk proteins was assessed using immunoblotting. None of the patients reacted to biscuits. Egg and milk proteins were undetectable with a limit of detection of 0.6 µg/g for milk and egg (method A), and of 0.1 and 0.3 µg /g for milk and egg, respectively (method B). The immunoblots did not show milk/egg proteins in the studied biscuits. Milk/egg content of the biscuits is far lower than 4 µg of milk or egg protein per gram of product, the minimal doses considered theoretically capable of causing reactions. With high sensitivity, proteomic assessments predict the harmlessness of very small amount of allergens in foods, and can be used to help avoiding unnecessary PAL.

Proposal of 0.5mg of protein/100g of processed food as threshold for voluntary declaration of food allergen traces in processed food-A first step in an initiative to better inform patients and avoid fatal allergic reactions: A GA²LEN position paper.

Food anaphylaxis is commonly elicited by unintentional ingestion of foods containing the allergen above the tolerance threshold level of the individual. While labeling the 14main allergens used as ingredients in food products is mandatory in the EU, there is no legal definition of declaring potential contaminants. Precautionary allergen labeling such as "may contain traces of" is often used. However, this is unsatisfactory for consumers as they get no information if the contamination is below their personal threshold. In discussions with the food industry and technologists, it was suggested to use a voluntary declaration indicating that all declared contaminants are below a threshold of 0.5mg protein per 100g of food. This concentration is known to be below the threshold of most patients, and it can be technically guaranteed in most food production. However, it was also important to assess that in case of accidental ingestion of contaminants below this threshold by highly allergic patients, no fatal anaphylactic reaction could occur. Therefore, we performed a systematic review to assess whether a fatal reaction to 5mg of protein or less has been reported, assuming that a maximum portion size of 1kg of a processed food exceeds any meal and thus gives a sufficient safety margin. MEDLINE and EMBASE were searched until 24January 2021 for provocation studies and case reports in which one of the 14major food allergens was reported to elicit fatal or life-threatening anaphylactic reactions and assessed if these occurred below the ingestion of 5mg of protein. A Delphi process was performed to obtain an expert consensus on the results. In the 210studies included, in our search, no reports of fatal anaphylactic reactions reported below 5mg protein ingested were identified. However, in provocation studies and case reports, severe reactions below 5mg were reported for the following allergens: eggs, fish, lupin, milk, nuts, peanuts, soy, and sesame seeds. Based on the literature studied for this review, it can be stated that cross-contamination of the 14major food allergens below 0.5mg/100g is likely not to endanger most food allergic patients when a standard portion of food is consumed. We propose to use the statement "this product contains the named allergens in the list of ingredients, it may contain traces of other contaminations (to be named, e.g. nut) at concentrations less than 0.5mg per 100g of this product" for a voluntary declaration on processed food packages. This level of avoidance of cross-contaminations can be achieved technically for most processed foods, and the statement would be a clear and helpful message to the consumers. However, it is clearly acknowledged that a voluntary declaration is only a first step to a legally binding solution. For this, further research on threshold levels is encouraged.

Sesame, Pistachio, and Macadamia Nut: Development and Validation of New Allergenic Systems for Fast Real-Time PCR Application.

Food allergy is a worldwide health problem that concerns infants to adults. The main health risk for sensitised individuals is due to the presence of traces of allergens as the result of an accidental contamination during food processing. The labelling of allergens such as sesame, pistachio, and macadamia nut on food products is mandatory according to Regulation (EU) N. 1169/2011; therefore, the development of suitable and specific analytical methodologies is advisable. The aim of this study was to perform a multi-allergen real-time PCR system that works well in fast mode at the same annealing temperature and with the same thermal profile. The real-time PCR was developed designing new, specific, and efficient primer and probe systems for the 2S albumin gene for sesame and pistachio and for the vicilin precursor gene for macadamia nut. These systems were subjected to a robust intra-laboratory qualitative validation process prior to their application, by DNA extraction and fast real-time PCR, on some real market samples to reproduce a potential allergen contamination along the food chain. The developed system results were specific and robust, with a sensible limit of detection (0.005% for sesame; 0.004% for pistachio; 0.006% for macadamia nut). The performance and the reliability of the target systems were confirmed on commercial food samples. This molecular approach could be used as a screening or as a support tool, in association with the other widespread monitoring techniques (such as ELISA).

Detection and Quantification of Allergens in Foods and Minimum Eliciting Doses in Food-Allergic Individuals (ThRAll)

Abstract Risk-based approaches to managing allergens in foods are being developed by the food industry and regulatory authorities to support food-allergic consumers to avoid ingestion of their problem food, especially in relation to the traces of unintended allergens. The application of such approaches requires access to good quality data from clinical studies to support identification of levels of allergens in foods that are generally safe for most food-allergic consumers as well as analytical tools that are able to quantify allergenic food protein. The ThRAll project aims to support the application of risk-based approaches to food-allergen management in two ways. First, a harmonized quantitative MS-based prototype reference method will be developed for the detection of multiple food allergens in standardized incurred food matrices. This will be undertaken for cow’s milk, hen’s egg, peanut, soybean, hazelnut, and almond incurred into two highly processed food matrices, chocolate and broth powder. This activity is complemented by a second objective to support the development and curation of data on oral food challenges, which are used to define thresholds and minimum eliciting doses. This will be achieved through the development of common protocols for collection and curation of data that will be applied to allergenic foods for which there are currently data gaps.

Geochemical, stable isotopic, palynological characterization of surface dry soils and atmospheric particles over Jodhpur city (Thar Desert, Rajasthan) during peak summer of 2013

Atmospheric dust originating from the Thar Desert (India) acts as the local source of mineral dust in South Asia, spreading over an area of 0.32 × 106 km2. Regional studies conducted during peak boreal summer are required to characterize this mineral dust that blows in form of episodic dust storms towards Indo-Gangetic Plains (IGP), using a multi-tracer approach. To achieve this goal, atmospheric PM10 particles were collected along with surface dry soils between 3 and 11 June, 2013, from in and around the Jodhpur city (26.2389°N, 73.0243°E) to glean elemental composition, stable isotopic and palynological (pollen types) database. Typical crustal elemental ratios, e.g. Si/Al, Ca/Al, Fe/Al, K/Al, Mg/Al, Ti/Al, varied in narrow ranges 8.1 ± 1.21, 1.02 ± 1.53, 0.50 ± 0.14, 0.34 ± 0.06, 0.19 ± 0.06 and 0.06 ± 0.02, respectively. Average Sr/Al, Rb/Al and Zr/Al ratios were found to be 39.70 ± 12.24, 18.00 ± 2.0 and 70.83 ± 13.11 (μg gm−1/wt%), respectively. Average δ13C, δ15N, δ34S values of surface soils were − 10.5‰ ± 4.0, 11.4‰ ± 1.6 and 3.6‰ ± 2.1, while δ13C and δ15N of atmospheric PM10 particles varied in ranges − 25.6‰ ± 0.67 and 9.9‰ ± 1.7, respectively. Observed palynoassemblage indicated the open nature of vegetation that usually grows under warm-humid conditions with traces of few allergens and pathogens. Generated chemical-isotopic-pollen database could be utilized for deciphering origin of dust storms in IGP. Detailed multi-proxy characterization of mineral dust from the Thar Desert can further help to determine its role in influencing air quality and human health.

Evaluation of a Real Time PCR Assay and a ELISA Method for the Detection of Walnuts and Almonds Allergen Traces in Food Products

Food allergens are a well acknowledged issue in food industry and are regulated by legislation. The presence of allergens can either origin from the raw material or due to contamination during production. Allergen information on packaging is mandatory although it cannot be accurate in the case of contamination therefore warnings are used. The purpose of the study is the development and validation of a SYBR Green Real Time Polymerase Chain Reaction method using specific primer pairs based on Jug r 1, Jug r 3, and Jug r 4 allergen-coding sequences to improve the sensitivity of Real Time Polymerase Chain Reaction techniques for detection of walnut and almond traces in commercial food products and its comparison with ELISA methodology in terms of detection ability. A total of 100 samples were collected from local markets and were analyzed by Real Time Polymerase Chain Reaction (RT-PCR) and ELISA methods. The results indicated that 16 samples (16%) were found positive in walnut traces and 18 samples (18%) were found positive in almond traces by Real Time Polymerase Chain Reaction of which Elisa identified 14 samples (14%) positive in walnut traces and 15 samples (15%) positive in almond traces. Among them, 4 samples (25%) that contained walnut traces and 6 samples (33.3%) that contained almond traces had no allergen declaration on their label. The improved accuracy of Real Time Polymerase Chain Reaction underlines the importance of this method for allergen detection and quantification in the food industry

Detection and Quantification of Allergens in Foods and Minimum Eliciting Doses in Food-Allergic Individuals (ThRAll).

Risk-based approaches to managing allergens in foods are being developed by the food industry and regulatory authorities to support food-allergic consumers to avoid ingestion of their problem food, especially in relation to the traces of unintended allergens. The application of such approaches requires access to good quality data from clinical studies to support identification of levels of allergens in foods that are generally safe for most food-allergic consumers as well as analytical tools that are able to quantify allergenic food protein. The ThRAll project aims to support the application of risk-based approaches to food-allergen management in two ways. First, a harmonized quantitative MS-based prototype reference method will be developed for the detection of multiple food allergens in standardized incurred food matrices. This will be undertaken for cow's milk, hen's egg, peanut, soybean, hazelnut, and almond incurred into two highly processed food matrices, chocolate and broth powder. This activity is complemented by a second objective to support the development and curation of data on oral food challenges, which are used to define thresholds and minimum eliciting doses. This will be achieved through the development of common protocols for collection and curation of data that will be applied to allergenic foods for which there are currently data gaps.

Novel low-abundance allergens from mango via combinatorial peptide libraries treatment: A proteomics study

Mango allergy is a rare condition, which may cause severe hypersensitivity reactions, such as anaphylaxis, angioedema, asthma and contact dermatitis. By exploiting the combinatorial peptide ligand library (CPLL) technology, mango proteomes have been extracted and the presence of traces of allergens assessed via Western blot analysis two-dimensional maps. Upon reactive spot elution and mass spectrometry analyses, four major mango allergens could be identified for the first time and shown to be in common with three of the five known banana species. These allergens include: Mus a 1, Mus a 2 and Mus a 5. Additional mango allergens detected do not seem to be in common with the banana species. In particular, a pectinesterase and a superoxide dismutase, both widely described as allergens, could be identified in mango extracts. Conversely, plain mango extracts not treated with CPLLs did not exhibit any reactive spots in Western blot analysis.

Detection of Peanut Allergen Traces with a Real Time PCR Assay - The Challenge to Protect Food-Allergic Consumers

The aim of the present study was the implementation of molecular techniques in the detection and quantification of allergic substances of peanut in various kinds of food products, e.g., breakfast cereals, chocolates and biscuits that are frequently related to allergies. In some cases, the presence of peanuts can be due to contamination during production and are not declared on the label. A total of 152 samples were collected from supermarkets and were analysed by a Real Time PCR method. The results indicated that 125 samples (83,3%) were found positive in peanut traces but the most important finding is that from the 84 samples that had no allergen declaration for peanuts, 48 (57,1%) of them were found positive. In conclusion, Real Time PCR can be a very important tool for the rapid detection and quantification of food allergens.

Development of an LC-MS/MS method for the detection of traces of peanut allergens in chili pepper.

The recent detection of nuts (including peanut) in spices across the globe has led to enormous recalls of several spices and food products in the last two years. The lack of validated detection methods specific for spices makes it difficult to assess allergen presence at trace levels. Because of the urgent need for confirmation of possible peanut presence in chili peppers, an LC-MS/MS method was optimized and developed for this particular food matrix. Although several studies optimized LC-MS detection strategies specific for peanuts, the presence of complex components in the spices (e.g., phenolic components) makes method optimization and validation necessarily. Focus was laid on validation of the method with real incurred chili peppers (whereby a known amount of peanut is added) at low concentrations, to deal with possible matrix interferences. LC-MS/MS proves to be a good alternative to the currently most applied methods (ELISA and RT-PCR) and can be used as a complementary method of analysis when results are unclear. Peanut marker peptides were selected based on their abundancy in digested incurred chili peppers. The limit of detection was determined to be 24ppm (mg peanut/kg), a level whereby the risk for potential allergic reactions is zero, considering the typical portion size of spices. The chili pepper powder under investigation proved to contain low levels of peanuts after LC-MS/MS, ELISA, and RT-PCR testing. Graphical abstract Standard curve of the detected peanuts in chili pepper samples using the novel LC-MS/MS method.

Safety evaluation of the food enzyme β-amylase obtained from soybean (Glycinemax).

The food enzyme considered in this opinion is a β‐amylase (EC 3.2.1.2) from soybean submitted by Nagase (Europa) GmbH. This β‐amylase is intended to be used in the starch processing for maltose syrup production and the manufacture of a Japanese rice cake type. Based on the maximum use levels recommended for the respective food processes, dietary exposure to the food enzyme–total organic solids (TOS) was estimated on the basis of Japanese consumption data. Conservative average infant formula consumption, as reported in the EFSA Draft Guidance on risk assessment of substances present in food intended for infants below 16 weeks of age, was used to estimate the exposure to a fraction of soybean comparable to the food enzyme–TOS, resulting from the consumption of soybean‐derived foods. The exposure estimate to the food enzyme–TOS was found to be lower than the comparable fraction from the source material. Potential allergenicity of the β‐amylase was evaluated by searching for similarity of the amino acid sequence to those of known allergens, and no match was found. The β‐amylase is produced from soybean, which is a known allergenic food. Japanese rice cake, consequently, may contain traces of soybean allergens, which may give rise to safety concerns in soybean‐allergic consumers. Based on the origin of the food enzyme from edible parts of soybean, the manufacturing process, the compositional and biochemical data provided and the dietary intake estimates, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use, except that Japanese rice cake produced with this food enzyme may contain traces of soybean allergens.

Enzyme-Linked Immunosorbent Assay (ELISA).

Food allergy is a public health concern especially after recognizing its constantly increased prevalence and severity. Despite careful reading of food ingredient statements, food allergic individuals may experience reactions caused by "hidden", "masked", or "contaminated" proteins that are known major allergens. Many techniques have been developed to detect even small traces of food allergens, for clinical or laboratory purposes. Enzyme-linked immunosorbent assay (ELISA) is one of the best validated and most routinely used immunoassay in allergy research, in allergy diagnosis in allergy-related quality control in various industries. Although as a technique it has been implemented for the last 45 years, the evolution in biochemistry allowed the development of ultrasensitive ELISA variations that are capable of measuring quantities in the scale of picograms, rendering ELISA attractive, robust, and very famous.

Development of a mass spectrometry immunoassay for unambiguous detection of egg allergen traces in wines

A mass spectrometry immunoassay (MSIA) specifically designed for the detection of egg allergens in wines is described. MSIA is based on an immunoaffinity enrichment procedure combined with targeted MS/MS detection of selected egg peptide markers. Polyclonal antibodies raised against native ovalbumin, chosen as the target protein tracing for egg powder, were immobilized onto low backpressure monolithic MSIA customized disposable tips. Ovalbumin-free wine samples were fortified with standard protein at different concentrations in the low microgram-per-milliliter range. A simple protocol was devised consisting of a 1:4 dilution of the wine sample with a basic solution for pH adjustment, followed by a semi-automated purification/enrichment step on MSIA customized disposable tips fitted on a multichannel electronic pipette. Among the main figures of merit, LOD and LOQ values as low as 0.01 and 0.03μg/mL, respectively, and within-day precision of 18% should be noticed. Noteworthy, the developed assay outperformed current MS-based methods for the detection of allergenic protein in wine matrices, thanks to the immunoaffinity enrichment. In addition, compared to other immunoassays, the present approach boasts the unquestionable advantage of providing an unambiguous identification of the target protein by simultaneous detection of three unique peptide markers each giving three specific MS/MS transitions.

Streamlining the analytical workflow for multiplex MS/MS allergen detection in processed foods.

Allergenic ingredients in pre-packaged foods are regulated by EU legislation mandating their inclusion on labels. In order to protect allergic consumers, sensitive analytical methods are required for detect allergen traces in different food products. As a follow-up to our previous investigations, an optimized, sensitive, label-free LC-MS/MS method for multiplex detection of five allergenic ingredients in a processed food matrix is proposed. A cookie base was chosen as a complex food matrix and home-made cookies incurred with whole egg, skimmed milk, soy flour, ground hazelnut and ground peanut were prepared at laboratory scale. In order to improve the analytical workflow both protein extraction and purification protocols were optimized and finally a sensitive streamlined SRM based analytical method for allergens detection in incurred cookies was devised. The effect of baking on the detection of selected markers was also investigated.

Coupling SPE on-line pre-enrichment with HPLC and MS/MS for the sensitive detection of multiple allergens in wine

Mass spectrometry (MS) represents an essential tool in proteomics studies, in the last years also exploited for monitoring allergens contamination in food products. Milk and egg are renowned allergens often used as fining agents to promote clarification of wines, therefore any residual amount in the end-products could represent a menace for allergic individuals. In view of this, it is of paramount importance to have at disposal sensitive analytical methods able to detect traces of milk and egg allergens in food. In this work we describe the upgrade and the optimization of an analytical workflow based on the use of a pre-enrichment column coupled with HPLC separation and MS/MS detection for the selective and sensitive detection of milk and egg allergens in white wine. Two different sample pre-treatments based on the use of mass cut-off filters or size exclusion cartridges were evaluated and compared, before tryptic digestion and LC-SRM-MS/MS analysis of the resulting peptides mixture. The devised UF based method coupled with peptide on-line pre-enrichment enabled to reach the lowest LODs down at 0.036 μg/mL and 0.05 μg/mL for egg and milk allergens respectively, proving to be the most sensitive strategy for monitoring allergens contamination in wine.

Advances in ultra-high performance liquid chromatography coupled to tandem mass spectrometry for sensitive detection of several food allergens in complex and processed foodstuffs

Sensitive detection of food allergens is affected by food processing and foodstuff complexity. It is therefore a challenge to detect cross-contamination in food production that could endanger an allergic customer's life. Here we used ultra-high performance liquid chromatography coupled to tandem mass spectrometry for simultaneous detection of traces of milk (casein, whey protein), egg (yolk, white), soybean, and peanut allergens in different complex and/or heat-processed foodstuffs. The method is based on a single protocol (extraction, trypsin digestion, and purification) applicable to the different tested foodstuffs: chocolate, ice cream, tomato sauce, and processed cookies. The determined limits of quantitation, expressed in total milk, egg, peanut, or soy proteins (and not soluble proteins) per kilogram of food, are: 0.5mg/kg for milk (detection of caseins), 5mg/kg for milk (detection of whey), 2.5mg/kg for peanut, 5mg/kg for soy, 3.4mg/kg for egg (detection of egg white), and 30.8mg/kg for egg (detection of egg yolk). The main advantage is the ability of the method to detect four major food allergens simultaneously in processed and complex matrices with very high sensitivity and specificity.