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Highlights: [•] The novel method allows quantification of species or nucleic acids. [•] It allows verification of a threshold concentration without external standards. [•] One competitor serves for calibration, matrix normalisation, and inhibition control. [•] Mean recovery of 10–1000mg/kg peanut in various food matrices was 87% (21% CV). [•] Quantitative performance was found comparable to that of ELISA tests. [Copyright &y& Elsevier]

Recently, we investigated the impact of shared equipment on cross-contamination of cookies at a pilot plant scale. Based on those findings, this study investigated the extent and subsequent sanitation of hazelnut cross-contamination (HNCC) of cookies at the industrial scale. Similarly, a product change from cookies with hazelnut ingredient to cookies without hazelnut was performed on standard equipment. HNCC in the hazelnut-free follow-up product was quantified by enzyme-linked immunosorbent assay for each production device and the applied cleaning procedure. All experiments were repeated in duplicate. The highest HNCC was found in concordance with previous studies alter mere mechanical scraping: more than 1,000 mg of hazelnut protein per kg was quantified in the follow-up product after processing by a cookie machine. Additional cleaning with hot water decreased the HNCC irrespective of the processing device to levels at or below 1 mg of hazelnut protein per kg. Furthermore, raw materials for cookie production were monitored over a period of 24 months for unwanted preloads of hazelnut and peanut: hazelnut was quantified in 16% of the investigated raw materials as being between 0.26 and 90 mg/kg. Further critical control points at the industrial scale, where cross-contamination might occur, were identified but did not display noteworthy sources of cross-contamination. In conclusion, the quantitative monitoring of the cleaning efficiency at the industrial scale confirmed the procedure of manual scraping plus wet cleaning as a qualified sanitation procedure to effectively reduce the hazelnut protein cross-contamination down to a level at which severe hazelnut-related allergic reactions are unlikely to occur. [ABSTRACT FROM AUTHOR]

Background: Soybean is considered an important allergenic food, but published data on soybean allergens are controversial. Objective: We sought to identify relevant soybean allergens and correlate the IgE-binding pattern to clinical characteristics in European patients with confirmed soy allergy. Methods: IgE-reactive proteins were identified from a soybean cDNA expression library, purified from natural soybean source, or expressed in Escherichia coli. The IgE reactivity in 30 sera from subjects with a positive double-blind, placebo-controlled soybean challenge (n = 25) or a convincing history of anaphylaxis to soy (n = 5) was analyzed by ELISA or CAP-FEIA. Results: All subunits of Gly m 5 (β-conglycinin) and Gly m 6 (glycinin) were IgE-reactive: 53% (16/30) of the study subjects had specific IgE to at least 1 major storage protein, 43% (13/30) to Gly m 5 , and 36% (11/30) to Gly m 6. Gly m 5 was IgE-reactive in 5 of 5 and Gly m 6 in 3 of 5 children. IgE-binding to Gly m 5 or Gly m 6 was found in 86% (6/7) subjects with anaphylaxis to soy and in 55% (6/11) of subjects with moderate but only 33% (4/12) of subjects with mild soy-related symptoms. The odds ratio (P < .05) for severe versus mild allergic reactions in subjects with specific IgE to Gly m 5 or Gly m6 was 12/1. Conclusion: Sensitization to the soybean allergens Gly m 5 or Gly m 6 is potentially indicative for severe allergic reactions to soy. [Copyright &y& Elsevier]

Shared equipment in industrial food manufacture has repeatedly been described as a potential source of unlabeled food allergens, i.e., hidden allergens. However, the impact of shared equipment on allergen cross-contamination is basically unknown. Therefore, we sought to investigate systematically the extent of hazelnut cross-contamination in fine bakery wares as a model. A product change from cookies with 10% hazelnut to cookies without hazelnuts was simulated on pilot plant equipment. The extent of hazelnut cross-contamination (HNCC) was analyzed by enzyme-linked immunosorbent assay (ELISA) for each production device (kneaders, rotary molder, wire cutting machine, and steel band oven) and various cleaning procedures used between products. The experiments were performed repeatedly with finely ground hazelnuts and with roughly chopped hazelnut kernels. Cross-contamination from chopped kernels was distributed statistically but not homogeneously, and sampling and analysis with the ELISA was therefore not reproducible. Further analysis concentrated on homogenously distributed HNCC from ground hazelnut. Apart from product changes without intermediate cleaning, the highest HNCC was found after mechanical scraping: Up to 100 mg/kg hazelnut protein was found in the follow-up product after processing by one machine. After additional cleaning with hot water, the HNCC decreased regardless of the processing device to levels at or below 1 mg/kg hazelnut protein. In our pilot plant study, the application of an appropriate wet cleaning procedure in combination with quantitative monitoring of the cleaning efficiency reduced the hazelnut protein cross-contamination to a level at which severe hazelnut-related allergic reactions are unlikely to occur. [ABSTRACT FROM AUTHOR]

Background: Soybean is a relevant allergenic food, but little is known about individual threshold doses in soy allergy. Objective: We sought to determine the clinical characteristics of soy allergy in Europe, including a dose-response curve. Methods: Patients with a history of soy allergy underwent a titrated, double-blind, placebo-controlled food challenge. A statistical model was used to calculate the risk of allergic consumers to experience an allergic reaction to soy. Sera were analyzed for specific IgE to soy, peanut, Bet v 1, and Gly m 4. Results: All patients but one responded primarily with subjective symptoms to the challenge followed by objective symptoms in 11 subjects, ranging from rhinitis up to a decrease in blood pressure. Cumulative threshold doses for allergic reactions ranged from 10 mg to 50 g for subjective symptoms and from 454 mg to 50 g for objective symptoms. The pattern of IgE reactivity against proteins with molecular weights of between approximately 10 and 70 kd was highly individual among the patients and did not correlate with the severity of symptoms. Conclusions: When data are fitted by using a normal distribution statistical model, they predict that 1% of patients with soy allergy would react subjectively and objectively with 0.21 and 37.2 mg of soy protein, respectively. Clinical implications: Both the clinical and immunologic basis of soy allergy in Europe are highly complex, which affects the diagnosis of soy allergy and the advice given to patients with soy allergy in regard to risk management. [Copyright &y& Elsevier]

Food allergy affects up to 6% of Europeans. Allergen identification is important for the risk assessment and management of the inadvertent presence of allergens in foods. The VITAL® initiative for voluntary incidental trace allergen labeling suggests protein reference doses, based on clinical reactivity in food challenge studies, at or below which voluntary labelling is unnecessary. Here, we investigated if current analytical methodology could verify the published VITAL® 2.0 doses, that were available during this analysis, in serving sizes between 5 and 500 g. Available data on published and commercial ELISA, PCR and mass spectrometry methods, especially for the detection of peanuts, soy, hazelnut, wheat, cow's milk and hen's egg were reviewed in detail. Limit of detection, quantitative capability, matrix compatibility, and specificity were assessed. Implications by the recently published VITAL® 3.0 doses were also considered. We conclude that available analytical methods are capable of reasonably robust detection of peanut, soy, hazelnut and wheat allergens for levels at or below the VITAL® 2.0 and also 3.0 doses, with some methods even capable of achieving this in a large 500 g serving size. Cow's milk and hen's egg are more problematic, largely due to matrix/processing incompatibility. An unmet need remains for harmonized reporting units, available reference materials, and method ring-trials to enable validation and the provision of comparable measurement results. • Methods that detect clinically relevant reference doses of allergens are required. • Methods: Peanuts, soy, hazelnut, wheat, cow's milk, hen's eggs reviewed in detail. • ELISA, qPCR, and MS revealed method specific advantages and limitations. • Sensitive methods for the verification of VITAL® 2.0/3.0 reference doses exist. • Comparability demands harmonized units, reference materials and method ring-trials. [ABSTRACT FROM AUTHOR]

Background: Analysis of IgE antibody binding to epitopes provides information for food allergy diagnosis and management and construction of hypoallergenic candidate vaccines, but the contribution of sequential epitopes to functionally relevant IgE binding is not fully understood. Objectives: We sought to study the impact of IgE-binding peptides described as major sequence epitopes in the literature on IgE-binding capacity of 2 selected food allergens. Methods: IgE-binding peptides of the food allergens Ara h 2 (peanut) and Pen a 1 (shrimp) were identified. Synthetic soluble peptides representing the identified sequences were assessed for their capacity to inhibit IgE binding to the parent allergens by means of ELISA and in mediator release assay. The IgE-binding capacity of unfolded recombinant (r) Ara h 2 was analyzed. A hybrid tropomyosin carrying the IgE-binding regions of Pen a 1 grafted into the structural context of the nonallergenic mouse tropomyosin was applied in ELISA inhibition experiments and ImmunoCAP analysis. Results: Although IgE-binding peptides representing sections of the allergen sequences were detected, no relevant capacity to inhibit the IgE binding to the parent allergen in ELISA or basophil activation test was observed. Unfolded rAra h 2 showed reduced IgE-binding capacity compared with folded rAra h 2 and failed to elicit mediator release. Hybrid tropomyosin bound less IgE than rPen a 1 in ImmunoCAP analysis and revealed marginal inhibitory capacity. Conclusion: Peptides identified as major sequence epitopes on Pen a 1 and Ara h 2 show little contribution to the IgE binding of the allergens studied. [Copyright &y& Elsevier]

Abstract: There is detailed guidance on how to perform bioinformatic analyses and enzymatic degradation studies for genetically modified crops under consideration for approval by regulatory agencies; however, there is no consensus in the scientific community on the details of how to perform IgE serum studies. IgE serum studies are an important safety component to acceptance of genetically modified crops when the introduced protein is novel, the introduced protein is similar to known allergens, or the crop is allergenic. In this manuscript, we describe the characteristics of the reagents, validation of assay performance, and data analysis necessary to optimize the information obtained from serum testing of novel proteins and genetically modified (GM) crops and to make results more accurate and comparable between different investigations. [Copyright &y& Elsevier]

• Cell-permeable VLPs as antigen carrier loaded with HBV-specific antigen were made. • Formation of neutralizing antibodies against HBV after immunization of mice. • Induction of robust T cell response with increased cytotoxicity. • Membrane permeability of antigen carriers enables oral and transdermal vaccination. There are currently about 257 million people suffering from chronic HBV infection worldwide. In many cases, an insufficient T cell response is causative for establishment of a chronic infection. To ensure a robust cellular immune response and induction of neutralizing antibodies a novel vaccine platform based on modified cell–permeable HBV capsids was utilized. Cell permeability was achieved by fusion of the membrane–permeable TLM-peptide to HBV core monomers, assembling the capsids. Insertion of a Strep-tagIII into the spike tip domain that protrudes from the capsid surface enables flexible loading with antigens that are fused to streptavidin. In this study, HBV surface antigen-derived PreS1PreS2 domain, fused to monomeric streptavidin, served as cargo antigen. Binding between antigen and capsids was characterized by surface plasmon resonance spectroscopy, electron microscopy and density gradient centrifugation. Confocal immunofluorescence microscopy and in vivo imaging of immunized mice demonstrated membrane permeability of cargo-loaded carriers and spread of antigen over the whole organism. Immunization experiments of mice revealed a robust induction of a specific cellular immune response, leading to destruction of HBV-positive cells and induction of HBV-specific neutralizing antibodies. Membrane permeability of these carriers allows needle-free application of antigen-loaded capsids as evidenced by induction of an HBV-specific CTL response and HBV-specific B cell response after oral or transdermal vaccination. These data indicate that cell–permeable antigen carriers, based on HBV capsids and loaded with HBV antigen, have the capacity to induce a cellular and a neutralizing humoral immune response. In addition, cell permeability of the vaccine platform enables antigen transfer across several cell layers, that could allow oral or transdermal immunization. [ABSTRACT FROM AUTHOR]

A systematic nomenclature for allergens originated in the early 1980s, when few protein allergens had been described. A group of scientists led by Dr. David G. Marsh developed a nomenclature based on the Linnaean taxonomy, and further established the World Health Organization/International Union of Immunological Societies (WHO/IUIS) Allergen Nomenclature Sub-Committee in 1986. Its stated aim was to standardize the names given to the antigens (allergens) that caused IgE-mediated allergies in humans. The Sub-Committee first published a revised list of allergen names in 1986, which continued to grow with rare publications until 1994. Between 1994 and 2007 the database was a text table online, then converted to a more readily updated website. The allergen list became the Allergen Nomenclature database ( www.allergen.org ), which currently includes approximately 880 proteins from a wide variety of sources. The Sub-Committee includes experts on clinical and molecular allergology. They review submissions of allergen candidates, using evidence-based criteria developed by the Sub-Committee. The review process assesses the biochemical analysis and the proof of allergenicity submitted, and aims to assign allergen names prior to publication. The Sub-Committee maintains and revises the database, and addresses continuous challenges as new “omics” technologies provide increasing data about potential new allergens. Most journals publishing information on new allergens require an official allergen name, which involves submission of confidential data to the WHO/IUIS Allergen Nomenclature Sub-Committee, sufficient to demonstrate binding of IgE from allergic subjects to the purified protein. [ABSTRACT FROM AUTHOR]

Background The working group “Food Technology and Safety” of the DFG Senate Commission on Food Safety (SKLM) deals with new technologies which are being developed or used to treat foods. Ohmic heating is a new process for heating food by means of direct application of current to the food. Compared to conventional heating methods, this process can achieve shorter heating times while avoiding hot surfaces and can reduce temperature gradients. The electrical, thermophysical and rheological properties of the products play an important role in achieving uniform heating. In addition to the product parameters, process parameters such as the current frequency used, the electrode material and the geometry of the treatment chamber are also relevant. Scope and approach On June 22nd 2015, the SKLM issued an assessment of the process for Ohmic heating of food in German. The English version was issued on December 14th, 2015. The objective of this statement was to describe the state of the research, to draw attention to critical points in the application and science-based further development of the process, and to define the need for research. Key findings and conclusions As with conventional heating, the effectiveness of ohmic heating as a preservation process depends on reaching and maintaining a certain temperature at each point of the food for a sufficient period of time to inactivate microorganisms. The physicochemical product properties are extremely important for achieving heating conditions that are as uniform as possible. Because the electric field strengths applied are low, mainly thermal effects come into play. However, some studies discuss potential additional synergistic or non-thermal inactivation effects of the electric field. As with other processing methods, the structure and concentration of ingredients and contaminants in foods may be altered during ohmic heating. Besides the thermal effects of ohmic heating, it is also necessary to pay attention to potential electrochemical reactions at the contact surface between electrodes and food as well as potential non-thermal effects of the electric field, depending on the process conditions. Therefore, process control becomes particularly important to prevent such effects, which are sometimes undesirable. Compared to conventional heating methods, the primary requirement in evaluating ohmic heating is a standardised means of acquiring the process control parameters. This includes, first and foremost, a space- and time-resolved temperature measurement that takes into account the product and electric field properties. It is absolutely necessary to carry out systematic studies while paying attention to the comparability with respect to product and process parameters as well as the system design. Consequently, the existing gaps in the data records are in part due to the insufficient comparability of the available studies. Moreover, it is necessary to analyze thermal and non-thermal as well as additional process-induced changes in the food and its ingredients. This applies particularly to the effect on the potential allergenicity of the food components. Thermal and non-thermal effects can be studied in a differentiated manner in simulation models. This is regarded as a promising approach for providing a model-like description of combination processes and for optimising process conditions as well. [ABSTRACT FROM AUTHOR]

Food processing can have many beneficial effects. However, processing may also alter the allergenic properties of food proteins. A wide variety of processing methods is available and their use depends largely on the food to be processed. In this review the impact of processing (heat and non-heat treatment) on the allergenic potential of proteins, and on the antigenic (IgG-binding) and allergenic (IgE-binding) properties of proteins has been considered. A variety of allergenic foods (peanuts, tree nuts, cows' milk, hens' eggs, soy, wheat and mustard) have been reviewed. The overall conclusion drawn is that processing does not completely abolish the allergenic potential of allergens. Currently, only fermentation and hydrolysis may have potential to reduce allergenicity to such an extent that symptoms will not be elicited, while other methods might be promising but need more data. Literature on the effect of processing on allergenic potential and the ability to induce sensitisation is scarce. This is an important issue since processing may impact on the ability of proteins to cause the acquisition of allergic sensitisation, and the subject should be a focus of future research. Also, there remains a need to develop robust and integrated methods for the risk assessment of food allergenicity. [ABSTRACT FROM AUTHOR]

Highlights: [•] Quality control material for allergen analysis developed using a clinically validated matrix. [•] Comparison of allergen egg and milk ELISA kit showed effective detection of allergen at low levels. [•] Quantification of allergens was generally poor. [•] Egg kits were the most precise, those based on “casein” being more consistent than other milk kits. [Copyright &y& Elsevier]

Abstract: Background: Published data on crustacean allergens are incomplete. The identification of tropomyosin (TM), arginine kinase (AK), sarcoplasmic Ca-binding protein (SCP) and myosin light chain (MLC) as shrimp allergens are all important contributions but additional allergens are required for the development of a complete set of reagents for component resolved diagnosis and the exploration of novel vaccination strategies. Methods: The North Sea shrimp (Crangon crangon), which is frequently consumed in Europe, served as a model organism in this study. TM and AK were directly cloned from mRNA based on sequence homology and produced as recombinant proteins. Additional IgE-reactive proteins were isolated by preparative SDS-PAGE and identified by mass spectrometry and corresponding cDNAs were cloned and expressed in E. coli. The relevance of the 6 cloned crustacean allergens was confirmed with sera of 31 shrimp-allergic subjects, 12 of which had a positive double-blind, placebo-controlled food challenge (DBPCFC) to shrimp and 19 a convincing history of food allergy to shrimp, including 5 cases of anaphylaxis. Quantitative IgE measurements were performed by ImmunoCAP. Results: Six recombinant crustacean proteins: TM, AK, SCP, a novel MLC, troponin C (TnC), and triosephosphate isomerase (TIM) bound IgE in ImmunoCAP analysis. Specific IgE to at least one of these single shrimp allergens was detected in 90% of the study population, thus the in vitro diagnostic sensitivity was comparable to that of shrimp extract (97%). In 75% of the subjects, the combined technical sensitivity was similar to or greater with single shrimp allergens than with natural shrimp extract. Conclusions: We identified six IgE-binding proteins from C. crangon, three of which have not before been described as allergens in crustaceans. This extensive panel of shrimp allergens forms a valuable asset for future efforts towards the identification of clinically relevant biomarkers and as a basis to approach patient-tailored immunotherapeutic strategies. [Copyright &y& Elsevier]

Abstract: For better understanding the cross-reactivity between the major birch pollen and celery allergens, Bet v 1 and Api g 1, respectively, putative epitope areas and structurally important positions for IgE-binding of the isoforms Api g 1.01 and Api g 1.02 were point mutated. The IgE binding capacities were measured in ELISA, the IgE cross-reactivity between the isoforms, mutants and Bet v 1 investigated by ELISA-inhibition experiments with serum pools from patients with confirmed celery allergy (DBPCFC). Api g 1.01 displayed a clearly higher frequency and capacity of IgE binding than Api g 1.02. In Api g 1.01, substitution of lysine against glutamic acid at amino acid position 44, a key residue of the Bet v 1 “P-loop”, increased the IgE-binding properties. Structural instability due to proline insertion at position 111/112 resulted in loss of IgE binding of Api g 1.01, but not of Api g 1.02. Between Api g 1.01 and Api g 1.02 only partial cross-reactivity was seen. The data suggest that the IgE epitopes of the two isoforms are distinct and that in contrast to Api g 1.01, the “P-loop” region plays an important role for IgE binding of celery allergic subjects to Api g 1.02. Understanding and investigation of the molecular mechanisms in celery allergy is an important step to generate hypoallergenic proteins for safe and efficacious immunotherapy of food allergy. [Copyright &y& Elsevier]