Methods For Detection Of Allergens Research Articles

Performance Evaluation of a Commercial Real-Time PCR Method for the Detection of Lupin Traces in Food.

In accordance with U.S. FDA Foods Program Regulatory Science Steering Committee guidelines, with this study, we optimized and validated a commercial real-time PCR method for the detection of low amounts of lupin in four classes of food matrices: chocolate cookies, ragù, Olivier salad, and barley and rice flour. DNA extracted from blank (true negative) samples artificially contaminated with lupin (Lupinus albus) flour at 1000 ppm underwent dilutions with the DNA extracted from the true negative samples up to 0.5 ppm. The limit of detection for real-time PCR was 0.5 ppm in the complex matrices (range, Ct 26-34), making this a specific, robust, and rapid method for lupin allergen detection and labeling. Our validation data support the suitability of this commercially available real-time PCR method for this purpose.

Assessment of crustacean allergen detection methods: cross reactivity with edible insect samples

Increased interest in consumption of insects in recent years has led to an increased focus on associated food safety concerns, and allergy is one of the most relevant. In the United States, crustacean shellfish are regulated as a major allergenic food group per the Food, Drug, and Cosmetic (FD&C) Act. Insects and crustacean shellfish are both arthropods, and clinical cross-reactivity between the two groups has been demonstrated. The goal of this work was to establish whether that clinical cross-reactivity translates into analytical cross-reactivity with detection assays targeting crustacean shellfish allergens. Edible insect samples were analyzed using four different crustacean allergen detection methods: Multi-Analyte Profiling Food Allergen Detection Assay (xMAP FADA), enzyme-linked immunosorbent assay (ELISA), western blot, and real-time polymerase chain reaction (PCR). Results indicate that the immunoassay-based xMAP FADA, ELISA, and western blot were susceptible to cross-reactivity, while the DNA-based PCR methods had minimal reactivity with insect samples. These results confirm that edible insects show analytical cross-reactivity with the immunoassays which may result in false positive detection of crustacean allergens in insect samples. Confirmation using DNA-based PCR, which shows little to no cross-reactivity, clarifies ambiguous results.

Generation of an Ovomucoid-Immune scFv Library for the Development of Novel Immunoassays in Hen's Egg Detection.

Hen's egg allergy is the second most common food allergy among infants and young children. The possible presence of undeclared eggs in foods poses a significant risk to sensitized individuals. Therefore, reliable egg allergen detection methods are needed to ensure compliance with food labeling and improve consumer protection. This work describes for the first time the application of phage display technology for the generation of a recombinant antibody aimed at the specific detection of hen's ovomucoid. First, a single-chain variable fragment (scFv) library was constructed from mRNA isolated from the spleen of a rabbit immunized with ovomucoid. After rounds of biopanning, four binding clones were isolated and characterized. Based on the best ovomucoid-binding candidate SR-G1, an indirect phage enzyme-linked immunosorbent assay (phage-ELISA) was developed, reaching limits of detection and quantitation of 43 and 79 ng/mL of ovomucoid, respectively. The developed ELISA was applied to the analysis of a wide variety of food products, obtaining a good correlation with a commercial egg detection assay used as a reference. Finally, in silico modeling of the antigen-antibody complex revealed that the main interactions most likely occur between the scFv heavy chain and the ovomucoid domain-III, the most immunogenic region of this allergen.

Standardization of a Mass Spectrometry-Based Workflow for Food Allergen Quantification.

In this chapter, the analytical workflow typically used for the development and validation of an analytical method tailored to food allergen detection and quantification is presented. The main steps defining the workflow are herein described and commented with specific notes about the critical issues that can be faced and common solutions to be adopted. References to guidelines and/or recommendation available from official bodies, as well as main papers from international consortia operating on the specific research field, are also reported, whenever possible. As such, this chapter may represent a practical guide to drive method development in the standardization of analytical methods for food allergen detection.

Positive rate of wheat allergens in the Chinese allergic population: a systematic review and meta-analysis

In recent years, the prevalence of allergic diseases has increased significantly, causing great concern, and wheat, as one of the top 8 food allergens, is a common allergy trigger. Nevertheless, reliable estimates of the positivity rate of wheat allergens in the allergic population in China are still lacking. The systematic review and meta-analysis aims to evaluate the positive detection rate of wheat allergens in the Chinese allergic population and further provide a reference for the prevention of allergy. CNKI, CQVIP, WAN-FANG DATA, Sino Med, PubMed, Web of Science, Cochrane Library, and Embase databases were retrieved. Related research and case reports about the positive rate of wheat allergen in the Chinese allergic population published from inception to June 30, 2022, were searched, and meta-analysis was performed using Stata software. The pooled positive rate of wheat allergens and 95% confidence interval were calculated by random effect models, and the publication bias was evaluated using Egger’s test. A total of 13 articles were included for the final meta-analysis, in which wheat allergen detection methods involved only serum sIgE testing and SPT assessment. The results showed that the wheat allergen positivity detection rate in Chinese allergic patients was 7.30% (95% CI 5.68–8.92%). Subgroup analysis showed that the positivity rate of wheat allergens was influenced by region, but hardly by age and assessment method. The positive rates of wheat allergy in the population with allergic diseases were 2.74% (95% CI 0.90–4.58%) and 11.47% (95% CI 7.08–15.87%) in southern and northern China, respectively. In particular, the positive rates of wheat allergens were greater than 10% in Shaanxi, Henan and Nei Mongol, all of which belong to the northern region. These results suggest that wheat allergens are an important cause of sensitization in allergic populations from northern China, and therefore attention should be paid to early prevention in high-risk populations.

Soybean (Glycine max) allergens─A Review on an Outstanding Plant Food with Allergenic Potential

Soybean (Glycine max) is the most used vegetarian protein source and a common ingredient of industrially produced foods. In addition to several nutritional and health benefits, soybean is classified as an allergenic source, affecting 0.2–0.4% of allergic individuals worldwide. Soybean allergy can be persistent and impact the life quality of soybean-sensitive patients, even causing severe allergic reactions and anaphylaxis. However, there is no scientific consensus about the classification of major and minor soybean allergens, the severity of soybean allergy itself, risk assessment, and its classification as an obligatory duty to labeling. This review discusses soybean as an outstanding food in human nutrition, identified major and minor soybean allergens, allergen detection methods, the impact of different food processing steps on the allergenicity of soybean-containing food, and how soybean allergic patients are affected and can be protected by proper allergen risk management.

An antifouling polydopamine-based fluorescent aptasensor for determination of arginine kinase

Simple yet efficient detection methods for food allergens are in urgent need to help people avoid the risks imposed by allergenic food. In this work, a polydopamine (PDA)-based fluorescent aptasensor was developed to detect arginine kinase (AK), one of the major allergens in shellfish. The aptamer towards AK was firstly selected via systematic evolution of ligands by exponential enrichment method and labeled with fluorescein amidite (FAM) to build a fluorescence resonance energy transfer (FRET) system with PDA particles. Polyethylene glycol (PEG) was employed to construct an antifouling surface for the aptasensor to eliminate food matrix interferences. With the presence of AK, the PDA-based aptasensor exhibited elevated fluorescent signals as the FAM-labeled aptamer bound to AK and detached from the PDA particles. The aptasensor showed great stability and resistance to nonspecific interference of background proteins and had a limit of detection (LOD) of 0.298 μg/mL. The proposed aptasensor was further proved to be feasible for quantitative analysis of AK in nine species of shrimps and five commercial processed products, which indicated its high potential in tracing the presence of AK in complex aquatic products.

Au@Ag-labeled SERS lateral flow assay for highly sensitive detection of allergens in milk

Casein and α-lactalbumin (α-LA) are the main allergens in cow's milk, which can affect the skin, respiratory system, and gastrointestinal tract, even cause anaphylactic shock. Developing a rapid and sensitive detection method of casein and α-LA is still pursued. Herein, a surface-enhanced Raman scattering based lateral flow assay (SERS-LFA) method for rapid and highly sensitive detection of milk allergens in food was established for effectively preventing allergic symptoms. Gold@silver nanoparticles (Au@Ag NPs) were synthesized as SERS active substrate to prepare the antibody-modified SERS Probe and SERS-LFA strips toward casein and α-LA were assembled according to the sandwich mode. The detection results were calculated according to colorimetric and Raman signal. The introduction of SRES signal significantly increased the sensitivity of detection with the limit of detection (LOD) of 0.19 ng/mL and 1.74 pg/mL, and exhibited an excellent linear relationship within the range of 0.55–791.50 ng/mL and 0.1 pg/mL–100.0 ng/mL for casein and α-LA, respectively. Furthermore, SERS-LFA strips was highly specific with the recovery rates for corresponding 80.36 %–105.12 % and 85.73 %–118.22 % for casein and α-LA, respectively. Therefore, the SERS-LFA could be in great potential to develop a unique allergen detection method.

A comprehensive overview of emerging processing techniques and detection methods for seafood allergens.

Seafood is rich in nutrients and plays a significant role in human health. However, seafood allergy is a worldwide health issue by inducing adverse reactions ranging from mild to life-threatening in seafood-allergic individuals. Seafood consists of fish and shellfish, with the major allergens such as parvalbumin and tropomyosin, respectively. In the food industry, effective processing techniques are applied to seafood allergens to lower the allergenicity of seafood products. Also, sensitive and rapid allergen-detection methods are developed to identify and assess allergenic ingredients at varying times. This review paper provides an overview of recent advances in processing techniques (thermal, nonthermal, combined [hybrid] treatments) and main allergen-detection methods for seafood products. The article starts with the seafood consumption and classification, proceeding with the prevalence and symptoms of seafood allergy, followed by a description of biochemical characteristics of the major seafood allergens. As the topic is multidisciplinary in scope, it is intended to provide information for further research essential for food security and safety.

Proteomic Analysis of Oil-Roasted Cashews Using a Customized Allergen-Focused Protein Database.

Cashews are one of the most prevalent causes of tree nut allergies. However, the cashew proteome is far from complete, which limits the quality of peptide identification in mass spectrometric analyses. In this study, bioinformatics tools were utilized to construct a customized cashew protein database and improve sequence quality for proteins of interest, based on a publicly available cashew genome database. As a result, two additional isoforms for cashew 2S albumins and five other isoforms for cashew 11S proteins were identified, along with several other potential allergens. Using the optimized protein database, the protein profiles of cashew nuts subjected to different oil-roasting conditions (139 and 166 °C for 2-10 min) were analyzed using discovery LC-MS/MS analysis. The results showed that the cashew 2S protein is most heat-stable, followed by 11S and 7S proteins, though protein isoforms might be affected differently. Preliminary target peptide selection indicated that out of the 29 potential targets, 18 peptides were derived from the newly developed database. In the evaluation of thermal processing effects on cashew proteins, several Maillard reaction adducts were also identified. The cashew protein database developed in this study allows for comprehensive analyses of cashew proteins and development of high-quality allergen detection methods.

Development and Validation of a Quantitative Method for Multiple Allergen Detection in Food Using Concatemer-Based Isotope Dilution Mass Spectrometry.

Accurate food labeling is essential to protect allergic consumers. However, allergen contaminations may occur during the whole food production process. Reliable, sensitive, and robust methods for detecting multiple allergens in food are needed. This work aims to develop and validate an LC coupled to tandem mass spectrometry (MS/MS) method for the detection and quantification of hazelnuts, peanuts, milk, and eggs in processed food products. In-house-produced incurred test materials, cookies and chocolates, were used for the method development and validation. The quantification was based on the standard addition strategy using qualified reference materials as allergen protein standards and an innovative stable isotope-labeled concatemer as an internal standard. A method targeting 19 allergen-specific peptides was developed and validated in two laboratories, which strengthens its robustness. The AOAC INTERNATIONAL performance requirements for repeatability, intermediate precision, reproducibility, and recovery were reached for at least one peptide per allergen across both matrixes, and quantification limits complied with the action levels of the Food Industry Guide to the Voluntary Incidental Trace Allergen Labelling (VITAL®) Program Version 3.0. The combination of incurred test materials, standard addition strategy, and stable isotope-labeled concatemer as an internal standard allowed us to develop and validate a robust method for detecting and quantifying multiple allergens in food with sufficient sensitivity to protect allergic consumers. The combination of characterized incurred test material, calibration with certified reference material, a single stable isotope labelled concatemer and cross-lab validation result in the required standardization and harmonization in food allergen detection according to the stakeholders' group to assess the robustness of our method.

Quantification of Soy-Derived Ingredients in Model Bread and Frankfurter Matrices with an Optimized Liquid Chromatography–Tandem Mass Spectrometry External Standard Calibration Workflow

The detection and quantification of soy protein is important for food allergen management and identifying the presence of undeclared soy proteins. Heat processing and matrix interactions can affect the accuracy of allergen detection methods. The sensitivity of enzyme-linked immunosorbent assay methods can be compromised if protein epitopes are modified during processing. Therefore, a mass spectrometry (MS)-based method was evaluated for the recovery of total soy protein in incurred matrices. MS-based quantification of total soy protein was assessed by using a combination of external and internal standards. The reproducibility of the standard curves was investigated by comparing within-day and among-day variation. Incurred samples were prepared using bread and frankfurters as model food matrices. Several soy-derived ingredients were used to prepare the matrices with varying levels of soy protein (1, 10, 50, or 100 ppm of total soy protein). A pooled standard curve was used to estimate the total soy protein concentration of the incurred food matrices and the percent total protein recovery. The variation of replicate standard curves between days and among all days was not significant. The differences in slopes obtained from replicate standards run on different days were minimal. The most influential factor on the quantitative protein recovery in incurred samples was the effect of the physical matrix structure on protein extraction. The lowest percent protein recoveries, less than 50%, were calculated for uncooked matrices. The cooked matrices had percentage recoveries between 50 and 150% for all total soy protein levels. Other factors, such as type of ingredient, were determined to be not as impactful on recovery. The MS method described in this study was able to provide sensitive detection and accurate quantification of total soy protein from various soy-derived ingredients present in processed food matrices.

Effect of gluten on the quality of meat products

Article Details: Received: 2020-10-14 | Accepted: 2020-11-27 | Available online: 2021-01-31 https://doi.org/10.15414/afz.2021.24.mi-prap.80-84 The aim of the study was the evaluation of gluten quality of selected meat products. Gluten is a vegetable protein, which, due to its properties, is very often used in various sectors of the food industry and therefore in the production of meat products. Sensory evaluation, microbiological examination and gluten detection were performed in samples of sausages, Poultry frankfurter, Fine salami and Malokarpatská salami. The meat products came from different producers and were purchased from a retail network. Sensory quality was determined by assessing color, aroma, juiciness, fragility and taste using a 5-point rating scale. The microbiological examination included the determination of the total number of microorganisms, the number of psychrotrophic, coliform microorganisms and coagulase-positive staphylococci. The presence of gluten alone was detected using a commercially produced GlutenTox Home test kit as well as a PCR reaction. Individual testing of selected meat products showed minor differences in the overall quality of gluten free and gluten-free meat products. Keywords: meat products, salami, gluten, quality, microbiological examination References Abaffyová, Z. et al. (2015). Cereal grain a little different. Bratislava. Pediatria pre prax . 4, 140–146. Frič, P., Keil, R. (2011). Celiac disease for practice. Olomouc. Medicína pro praxi. 8, 354–359. Hulín, P., Dostálek, P., Hochel, I. (2008). Methods for determination of gluten proteins in food. Metódy stanovení lepkových bílkovin v potravinách. Chemické listy ,102(5), 327–337. https://www.academia.edu/18759054/Methods_for_determination_of_gluten_proteins_in_food Olexová, L. et al. (2006). Detection of gluten-containing cereals in flours and ‘‘gluten-free’’ bakery products by polymerase chain reaction. Food Control , 17(3), 234–237. https://doi.org/10.1016/j.foodcont.2004.10.009 STN EN ISO 4833-1. Microbiology of food chain. Horizontal method for the enumeration of microorganisms. Part 1: Colony count at 30 degrees C by the pour plate technique. (ISO 4833-1: 2013) STN EN ISO 6888-1. Microbiology of food and animal feeding stuffs. Horizontal method for the enumeration of coagulase-positive staphylococci ( Staphylococcus aureus and other species). Part 1: Technique using Baird-Parker agar medium (ISO 6888-1:1999) STN EN ISO 4832. Microbiology. General guidance for the enumeration of coliforms. Colony count technique Taylor, S. (1992). Chemistry and detection of food allergens. Food Technology , 46(5), 148–152. https://doi.org/10.1080/10408399609527761 Toldrá, F. (2002). Fermentation and starter cultures. Dry-cured meat products , Trumbull : Food & Nutrition Press Inc., 89–112. https://doi.org/10.1002/9780470385111 van Hengel, A. J. (2007). Food allergen detection methods and the challenge to protect food-allergic consumers. Analytical and Bioanalytical Chemistry , 389(1), 111–118. https://doi.org/10.1007/s00216-007-1353-5

Progress in research of allergen detection methods

Allergic diseases have continued to increase year by year causing serious physical and mental injury to patients, families and individuals. This increase has been driven by conventional environmental and nutritional changes but is also created by the continual introduction of food additives into the diet and novel interior decoration materials into the living space. The causes of allergic diseases are complex and diverse, and the medical laboratory often is not be able to identify the allergic trigger; this creates a difficult environment to identify the appropriate clinical treatment for disease prevention and control. Physicians must be able to identify these triggers to help patients avoid the underlying allergenic cause of their disease. This can only be done by actively knowing a patient's medical history, identifying the clinical manifestations of hypersensitivity and utilizing confirmatory testing as an important clinical tool in identifying the allergic source.

Discovery based high resolution MS/MS analysis for selection of allergen markers in chocolate and broth powder matrices

Peptide marker identification is an important step in development of a mass spectrometry method for multiple allergen detection, since specificity, robustness and sensitivity of the overall analytical method will depend on the reliability of the proteotypic peptides. As part of the development of a multi-analyte reference method, discovery analysis of two incurred food matrices has been undertaken to select the most reliable peptide markers. Six allergenic ingredients (milk, egg, peanut, soybean, hazelnut, and almond) were incurred into either chocolate or broth powder matrix. Different conditions of protein extraction and purification were tested and the tryptic peptide pools were analysed by untargeted high resolution tandem mass spectrometry and the resulting fragmentation spectra were processed via a commercial software for sequence identification. The analysis performed on incurred foods provides both a prototype effective and straightforward sample preparation protocol and delivers reliable peptides to be included in a standardized selected reaction monitoring method.

An exceptionally intense turn-on fluorescence sensor in the far-red region for common milk allergen, β-lactoglobulin

Common milk allergens, such as, β-lactoglobulin (β-LG), poses a major health concern for public, particularly in children. Thus, sensitive and selective methods for detection of common milk allergens are highly warranted. To the best of our knowledge, detection and quantification of β-lactoglobulin, a common milk allergen, by fluorescence based technique has not been reported till date. Herein, we present the first ever fluorescence based report for the selective detection and quantification of milk allergen, β-LG, using LDS-798 as a fluorogenic probe, which registers an exceptionally large fluorescence enhancement of ∼2900 fold in the far-red region upon binding with β-LG with LOD of ∼6.3 (±0.3) nM for β-LG in aqueous buffer solution and 0.67 (±0.04) μM in 5% bovine milk matrix respectively. High sensitivity and selectivity, fast response, high repeatability and reproducibility, easy operation, along with emission in the technologically advantageous far-red region make the fluorometric quantification of β-LG, using LDS-798 as a fluorogenic probe, far more advantageous than the other techniques, generally used for β-LG quantification. Importantly, we have demonstrated the applicability of this probe for quantification of β-LG in real milk samples which has been validated by an established method.

A colorimetric and fluorescent gold nanoparticle-based dual-mode aptasensor for parvalbumin detection

Food allergy has risen as a worldwide food safety concern due to the increasing prevalence over the last decades. It’s in urgent need to develop straight yet sensitive detection methods for food allergens to help people avoid the potential risks imposed by food allergy. Here, we developed a dual-mode aptasensor that gave both colorimetric and fluorescent readouts in response to parvalbumin (PV), a major allergen in fish. The aptasensor was constructed by hybridizing the aptamer modified gold nanoparticles (AuNP-APT), the complementary short chain modified gold nanoparticles (AuNP-CS1) and the fluorescent dye-labeled complementary short chain (FAM-CS2). The presence of PV led to disassembly of the aptasensor through competitive interaction between APT and PV, which resulted in a change in the solution color of the AuNPs and a recovery of the once quenched fluorescence signals of FAM-CS2. The aptasensor exhibited a colorimetric response in the PV concentration range of 2.5–20 μg/mL and a linear fluorescent response in the PV concentration range of 2.38–40 μg/mL. Moreover, the aptasensor showed encouraging specificity, precision, accuracy as well as efficiency in real fish sample detection, which held great potential in recognition and analysis of food allergens.

Use of IHF-QD Microscopic Analysis for the Detection of Food Allergenic Components: Peanuts and Wheat Protein.

The aim of the study was to analytically evaluate quantum dots in immunohistofluorescence (IHF-QD) microscopic imaging as detectors of food allergens—peanut and wheat. The experiment was designed as two in silico experiments or simulations: (a) models of pastry samples were prepared with the addition of allergenic components (peanut and wheat protein components) and without the addition of allergenic components, and (b) positive and negative commercial samples underwent food allergen detection. The samples from both simulations were tested by the ELISA and IHF-QD microscopic methods. The primary antibodies (secondary antibodies to a rabbit Fc fragment with labeled CdSe/ZnS QD) were labelled at 525, 585, and 655 nm emissions. The use of quantum dots (QDs) has expanded to many science areas and they are also finding use in food allergen detection, as shown in the study. The study indicated that differences between the ELISA and IHF-QD microscopic methods were not observable among experimentally produced pastry samples with and without allergenic components, although differences were observed among commercial samples. The important value of the study is certainly the differences found in the application of different QD conjugates (525, 585, and 655). The highest contrast was found in the application of 585 QD conjugates that can serve for the possible quantification of present food allergens—peanuts and wheat. The study clearly emphasized that QD can be used for the qualitative detection of food allergens and can represent a reliable analytical method for food allergen detection in different food matrixes.

Chestnut allergen detection in complex food products: Development and validation of a real-time PCR method

Chestnut is gaining importance as food allergen, but a specific detection method has not been performed so far. For the first time, the development of a real-time PCR assay for chestnut allergen detection in processed foods is described. Initially, three sets of novel primers specific for allergen-encoding genes (Cas s 5, Cas s 9 and Cas s TLP) were designed and evaluated for chestnut detection. Primers for Cas s 9 and a TaqMan probe were finally selected for a sensitive and specific detection of chestnut in binary mixtures with a practical limit of detection (LOD) of 100 ppm. Effect of processing has been evaluated and sensitivity slightly reduced after boiling treatment. Amplification of Cas s 9 was importantly affected when heat and pressure were applied (autoclaving). Applicability of the method was tested by analysis of commercial food products. Consequently, the results support the development and validation of a real-time PCR method for accurate chestnut allergen detection and labelling.

Potential Transfer of Peanut and/or Soy Proteins from Poultry Feed to the Meat and/or Eggs Produced.

Previous studies have demonstrated that allergenic feed proteins from peanuts in the diets of layer hens are not detected in the eggs produced. Hence, in this study, we aimed to determine if soy and/or peanut proteins in poultry feed rations of broiler chickens or layer hens would be transferred or detectable in the meat or eggs produced. To meet this objective, 99 layer hens and 300 broiler chickens were equally divided into treatment groups and fed one of three experimental diets: control soybean meal and corn diet, whole unblanched high-oleic peanut and corn diet (HO PN), or a control diet spiked supplemented with oleic acid (OA) oil. At termination, broiler chickens were processed, and chicken breast samples of the left pectoralis muscle were collected, and eggs were collected from layers. Total protein extracts from pooled egg samples and chicken breast samples were subjected to enzyme-linked immunosorbent assay (ELISA) methods and immunoblotting analysis with rabbit antipeanut agglutinin antibodies and rabbit antisoy antibodies for the detection of peanut and soy proteins. Peanut and soy proteins were undetected in all pooled egg samples and individual chicken breast meat samples using immunoblotting techniques with rabbit antipeanut agglutinin and rabbit antisoy antibodies. Moreover, quantitative ELISA allergen detection methods determined all pooled egg samples and individual meat samples as “not containing” peanut or soy allergens. Therefore, this study helps to evaluate the risk associated with the potential transfer of allergenic proteins from animal feed to the products produced for human consumption.