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Mass spectrometry (MS) has advanced greatly and many of its applications are ready for utilization within regulatory procedures and could significantly contribute to overcome challenges in standardization of allergen products. It seems sensible to discuss MS within the regulatory framework, before addressing technical questions. While the application to purified proteins is well established from product development to manufacturer's release analytics, its application to complex products such as allergen products is still under development. It needs to be determined where it can complement or replace established methods or where MS offers limited improvement. Despite its technical appeal and versatility, currently MS is mentioned in regulatory guidelines only as one possible measurement method. For example, no specific MS method is given in the European Pharmacopoeia. We discuss applications of MS within the EU regulatory framework. This includes their advantages and disadvantages and their positioning between research, characterization, manufacturer's release analytics and official batch testing. We discuss the qualitative detection of single and multiple allergens as proof of identity, qualitative to semi‐quantitative protein profiles for batch to batch consistency testing, and quantification of allergens to state mass units of allergens. MS may also facilitate standardization of allergen products, reference products and reference standards. [ABSTRACT FROM AUTHOR]

Background: The aim of the BSP090 project is the establishment of European Pharmacopoeia Chemical Reference Substances (CRSs) in combination with corresponding standard ELISA methods for quantification of major allergens in allergen products. Here, we present data of a Phl p 5‐specific sandwich ELISA that proved suitable for the quantification of Phl p 5, one of the major Timothy grass (Phleum pratense) pollen allergens. Methods: A Phl p 5‐specific ELISA system was assessed with respect to accuracy, precision, inter‐assay (within laboratory) and inter‐laboratory variations, in a ring trial including 14 laboratories in Europe and the USA. Model samples containing recombinant Phl p 5a CRS as well as native grass pollen extracts were analysed. Each participant was instructed to perform at least one preliminary assay to familiarise with the protocol, followed by three independent assays. Results: The candidate standard ELISA proved suitable to quantify recombinant and native Phl p 5 with satisfactory precision (93% of results within ±30% acceptance range). Inter‐assay variation (max. GCV 24%) and especially inter‐laboratory variation (max. GCV 13%) showed conclusive results. When assessing accuracy by means of recovery of recombinant spikes from a grass pollen extract matrix, similarly satisfactory spike recovery results were observed for the two spikes with higher concentrations (all within ±30% acceptance range), whereas recovery of the lowest concentration spike was slightly poorer with mean results of six laboratories exceeding acceptance range. Conclusions: Based on the collaborative study results, the assessed Phl p 5‐specific immunoassay is appropriate to be proposed as European Pharmacopoeia standard method. [ABSTRACT FROM AUTHOR]

The last two decades saw a steady increase of high hydrostatic pressure (HHP) used for treatment of foods. Although the science of biomaterials exposed to high pressure started more than a century ago, there still seem to be a number of unanswered questions regarding safety of foods processed using HHP. This review gives an overview on historical development and fundamental aspects of HHP, as well as on potential risks associated with HHP food applications based on available literature. Beside the combination of pressure and temperature, as major factors impacting inactivation of vegetative bacterial cells, bacterial endospores, viruses, and parasites, factors, such as food matrix, water content, presence of dissolved substances, and pH value, also have significant influence on their inactivation by pressure. As a result, pressure treatment of foods should be considered for specific food groups and in accordance with their specific chemical and physical properties. The pressure necessary for inactivation of viruses is in many instances slightly lower than that for vegetative bacterial cells; however, data for food relevant human virus types are missing due to the lack of methods for determining their infectivity. Parasites can be inactivated by comparatively lower pressure than vegetative bacterial cells. The degrees to which chemical reactions progress under pressure treatments are different to those of conventional thermal processes, for example, HHP leads to lower amounts of acrylamide and furan. Additionally, the formation of new unknown or unexpected substances has not yet been observed. To date, no safety‐relevant chemical changes have been described for foods treated by HHP. Based on existing sensitization to non‐HHP‐treated food, the allergenic potential of HHP‐treated food is more likely to be equivalent to untreated food. Initial findings on changes in packaging materials under HHP have not yet been adequately supported by scientific data. [ABSTRACT FROM AUTHOR]

The growing world population and increased pressure on agricultural resources are driving a shortage of dietary protein sources. As a result, industry is developing more sustainable novel food protein sources such as insects, algae and duckweed and using new processing techniques. Consumer exposure to these novel or processed proteins, could cause new food allergies, exacerbating a public health issue which is already directly affecting an estimated 20 million Europeans. Introduction of novel foods should not add to the burden of food allergy and this calls for a reliable, harmonised, evidence-based and validated allergenicity risk assessment strategy. The COST (Cooperation in Science and Technology) Action ImpARAS (Improved Allergenicity Risk Assessment Strategy), a four-year networking project, identified gaps in current allergy risk assessment, and proposed new ideas and plans for improving it. Here, we report on the lessons learned from the ImpARAS network and suggestions for future research. The safe introduction of novel and more sustainable food protein sources, while protecting humans from food allergy, calls for a multidisciplinary approach based on an improved understanding of what determines the relative allergenic potency of proteins, novel testing and assessment methodologies, harmonized decision-making criteria, and a clear ranking approach to express the allergenicity of novel product relative to that of existing known allergenic proteins: (from 'non'/to weakly and to strongly allergenic proteins). [ABSTRACT FROM AUTHOR]

Background: Food allergy to pea (Pisum sativum) has been rarely studied in children at the clinical and molecular levels. Objective: To elucidate the allergenic relevance and diagnostic value of pea 7S globulin Pis s 1, nsLTP, and 2S albumins PA1 and PA2 in children. Methods: Children with pea‐specific IgE ≥ 0.35 kUA/L and clinical evidence of pea allergy or tolerance were included in the study. IgE binding against pea total protein extract, recombinant (r) rPis s 1, rPA1, rPA2, and natural nsLTP was analysed using IgE immunoblot/inhibition. Mediator release potency was investigated in passively sensitized rat basophil leukaemia (RBL) 2H3‐cells. IgE binding to synthetic overlapping peptides of Pis s 1 was detected on multipeptide microarrays. Results: 19 pea‐sensitized children were included, 14 with doctors' diagnosed allergy and 5 with tolerance to pea (median age 3.5 and 4.5 years, respectively). 11/14 (78%) pea‐allergic and 1/5 (20%) tolerant children were sensitized to Pis s 1. Under the reducing conditions of immunoblot analysis, IgE binding to rPA1 was negligible, sensitization to rPA2 and nsLTP undetectable. Compared to pea total protein extract, rPis s 1 displayed on average 58% IgE binding capacity and a 20‐fold higher mediator release potency. Selected Pis s 1‐related peptides displayed IgE binding in pea‐allergic but not in pea‐tolerant children. Conclusions and Clinical Relevance: In this study group, Pis s 1 is a major immunodominant allergen in pea‐allergic children. Evidence for sensitization to nsLTP and 2S albumins was low but requires further verification with regard to conformational epitopes. Recombinant Pis s 1 and related peptides which were exclusively recognized by pea‐allergic children may improve in vitro diagnosis of pea allergy once verified in prospective studies with larger study groups. [ABSTRACT FROM AUTHOR]

Background: Novel foods may provide new protein sources for a growing world population but entail risks of unexpected food‐allergic reactions. No guidance on allergenicity assessment of novel foods exists, while for genetically modified (GM) crops it includes comparison of sequence identity with known allergens, digestibility tests and IgE serum screening. Objective: As a proof of concept, to evaluate non‐/allergenic tropomyosins (TMs) regarding their potential as new calibrator proteins in functional biological in vitro assays for the semi‐quantitative allergy risk assessment of novel TM‐containing animal foods with mealworm TM as an example. Methods: Purified TMs (shrimp, Penaeus monodon; chicken Gallus gallus; E coli overexpression) were compared by protein sequencing, circular dichroism analysis and in vitro digestion. IgE binding was quantified using shrimp‐allergic patients' sera (ELISA). Biological activities were investigated (skin testing; titrated basophil activation tests, BAT), compared to titrated biological mediator release using humanized rat basophil leukaemia (RBL) cells. Results: Shrimp and chicken TMs showed high sequence homology, both alpha‐helical structures and thermal stability. Shrimp TM was stable during in vitro gastric digestion, chicken TM degraded quickly. Both TMs bound specific IgE from shrimp‐allergic patients (significantly higher for shrimp TM), whereas skin reactivity was mostly positive with only shrimp TM. BAT and RBL cell assays were positive with shrimp and chicken TM, although at up to 100‐ to 1000‐times lower allergen concentrations for shrimp than chicken TM. In RBL cell assays using both TM as calibrators, an activation of effector cells by mealworm TM similar to that by shrimp TM confirmed the already reported high allergenic potency of mealworm TM as a novel protein source. Conclusions & clinical relevance: According to current GM crops' allergenicity assessment, non‐allergenic chicken TM could falsely be considered an allergen on a weight‐of‐evidence approach. However, calibrating allergenic potency in functional BAT and RBL cell assays with clinically validated TMs allowed for semi‐quantitative discrimination of novel food protein's allergenicity. With TM calibration as a proof of concept, similar systems of homologous protein might be developed to scale on an axis of allergenicity. [ABSTRACT FROM AUTHOR]

This document is a list of contributors and acknowledgements for a study on the generation and characterization of monoclonal antibodies for a specific protein. It also includes a conflict of interest statement and information about the availability of the data. The document does not provide a summary of the study's findings or any additional information about the topic Low. [Extracted from the article]