Allergens In Products Research Articles

Improved quality control of allergen products: Assessing the molecular allergen composition by mass spectrometry.

Natural allergen sources contain a variety of allergens, against which allergic subjects have developed individual sensitization profiles. Ideal allergen products for skin prick testing (SPT) and allergen immunotherapy (AIT) should contain the complete set of allergens of the respective allergen sources to cover all sensitization profiles. However, commercially available allergen products were shown to vary regarding their allergen composition. The qualitative allergen composition of different SPT and AIT products produced from pollen of grasses, birch, mugwort and from house dust mites was assessed by a consistent high-resolution liquid chromatography-coupled tandem mass spectrometry method (LC-MS/MS). All major, mid-tier and most minor allergens were detected in each of the investigated three batches of SPT and AIT products, demonstrating the completeness of the allergen composition and a high degree of batch-to-batch consistency. This is the first study using a single consistent high-resolution LC-MS/MS method to provide solid data on the qualitative allergen composition of SPT and AIT products manufactured from various common allergen sources. The applied method showed high reliability in qualitative batch-to-batch consistency testing and can be performed fast and with high throughput. High-resolution LC-MS/MS is applicable for process development and quality control to ensure market availability of allergen products corresponding to the composition of the respective natural allergen sources.

Microarray-based evaluation of selected recombinant timothy grass allergens expressed in E. Coli and N. Benthamiana

BackgroundTimothy grass (Phleum pratense) is a significant source of allergens, and recombinant allergens are increasingly used for diagnostic purposes. However, the performance of different recombinant allergen production systems in diagnostic assays needs further investigation to optimize their use in clinical settings.ObjectiveThe main objective of this study was to analyze and compare the diagnostic performance of recombinant timothy grass allergens produced in E. coli and N. benthamiana using a custom-made microarray chip.MethodsRecombinant timothy grass allergens Phl p 1, Phl p 2, Phl p 5, Phl p 6, Phl p 11, and Phl p 12 were produced in E. coli and/or N. benthamiana. A total of 113 patient serum samples were tested to evaluate the diagnostic sensitivity, specificity, inter-assay variability, and correlation of allergen-specific IgE detection compared to commercial multiplex tests (ALEX and ISAC). Additionally, the prevalence of sIgE to these allergens was assessed.ResultsPhl p 1, Phl p 2, Phl p 5, Phl p 6 and Phl p 11 showed high or very high positive correlation in immunoreactivity with other commercial multiplex tests. Notably, Phl p 11 fused with maltose-binding protein (MBP) demonstrated high diagnostic specificity and sensitivity, with a 0.3 arbitrary cut-off value. However, a high intra-assay variation was observed. The study also assessed specific IgE prevalence to timothy grass allergens within the tested patient cohort.ConclusionsRecombinant allergens from both E. coli and N. benthamiana demonstrated strong diagnostic potential on the microarray platform, with Phl p 11 (MBP-fused) showing particularly high performance. High intra-assay variation highlights the need for further optimization in allergen formulation and microarray storage conditions. These results highlight the potential of recombinant allergens for diagnostic applications, despite challenges with allergen stability in microarray formats. Specific IgE prevalence to timothy allergens revealed a sensitization profile consistent with findings from multiple studies.Graphical

DNA Meets Protein─Development, Characterization, and Application of Aptamers against Peanut Allergen.

Allergen detection methods support food labeling and quality assessment at the allergen component level of allergen preparations used for allergy diagnosis and immunotherapy (AIT). Commonly applied enzyme-linked immunosorbent assay (ELISA) requires animal antibodies but potentially shows batch variations. We developed synthetic aptamers as alternative binders in allergen detection to meet the replacement, reduction, and refinement (3R) principle on animal protection in science. ssDNA aptamers were specifically selected against the major peanut allergen Ara h 1 and identified by next-generation sequencing. Application in various detection systems (ELISA-like assays, western blot, and surface plasmon resonance) was demonstrated. The ELISA-like assay comprised a sensitivity of 10 ng/mL Ara h 1, comparable to published antibody-based ELISA, and allowed Ara h 1 detection in various peanut flours, similar to those used in peanut AIT as well as in processed food. This ELISA-like aptamer-based assay proofs antibody-free allergen detection for food labeling or quality assessment of diagnostic and therapeutic allergen products.

Recombinant production of Morus alba lipid transfer protein and prediction of its allergenic epitopes

BackgroundWhite mulberry (Morus alba) pollen grains trigger allergic diseases including asthma, allergic rhinitis, and allergic conjunctivitis. Non-specific lipid transfer proteins (nsLTPs) are robust allergens found in higher plants. Although it was isolated and immunologically characterized in the fruits of Morus nigra, it has not yet been determined whether nsLTP is one of the M. alba pollen allergens. ObjectiveThis study aims to examine the possible allergenicity of M. alba pollen nsLTP by producing it recombinantly, followed by immunoblotting. MethodsTotal RNA was isolated from M. alba pollen grains, and the nsLTP gene was obtained by reverse transcriptase polymerase reaction. It was cloned into Escherichia coli using pQE2 plasmid and purified by immobilized metal affinity chromatography following the expression. To confirm the immunoreactivity of the purified r-nsLTP, immunoblotting was performed with the serum of patients allergic to M. alba pollen. B-cell epitopes of nsLTP were also predicted using the bioinformatics tools, namely Bioinformatics Predicted Antigenic Peptides, BepiPred-2.0, and the Immune Epitope Database, whereas T-cell epitopes were estimated by using NetMHCIIpan-4.0 and NetMHCII-2.3 servers. ResultsThe r-nsLTP showed positive results in immunoblotting with sera of 7/7 (100%) of M. alba allergic patients. Five B-cell epitopes (26–31, 43–50, 65–71, 92–100, 105–115) and three T-cell epitopes (57–65, 78–86, 105–113) were predicted by in silico analyses. ConclusionData obtained in this study are expected to support the use of r-nsLTP in solutions for diagnosis and treatment of M. alba allergy and cross-reactivities between nsLTPs, as well as recombinant allergen production for vaccination.

Development of a low allergenic product for patients with milk allergy and assessment of its specific IgE reactivity.

Milk oral immunotherapy is the riskiest and most unpredictable form of oral immunotherapy. We aimed to produce a low allergenic product than conventional once baked-cake/muffin, to develop indirect in-house ELISA to check the tolerance status with milk products and evaluate IgE reactivity of patients' sera via western blotting (WB) and indirect in-house ELISA. A low allergenic product named biscotti-twice baked-cake was developed, and the total protein concentration was determined. The protein content was studied by SDS-PAGE and proteomics. Milk-specific IgE (sIgE) binding assays were performed by WB and indirect in-house ELISA by using patients' sera. Casein band intensity was observed to be lower in the biscotti-twice baked-cake than in the once baked-cake (p = .014). Proteomics analysis and αS1-casein measurement showed that the lowest intensity of casein was found in biscotti. The low binding capacity of milk sIgE to biscotti compared with once baked-cake was shown by WB (p = .0012) and by indirect in-house ELISA (p = .0001). In the ROC analysis, the area under the curve (AUC) of the in-house ELISA IgE was comparable with Uni-CAP milk and casein sIgE. The AUC of the in-house ELISA IgE for cake (0.96) and biscotti (1) was slightly better than Uni-CAP milk sIgE (0.94; 0.97) and casein sIgE (0.96; 0.97), respectively. The low allergenicity of the newly developed low allergenic product "biscotti-twice baked-cake" has been demonstrated by invitro experiments. Biscotti could be a safe treatment option than once baked-cake/muffin in patients who are reactive to once baked-milk products.

Quality control of allergen products with mass spectrometry part I: Positioning within the EU regulatory framework.

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.

The Health Impacts of Endocrine Disrupting Chemicals and Climate Change on Children

Throughout the past 20 years, there have been numerous ecological, economic, and public health crises that have rocked the world, frequently exposing low levels of self-sufficiency and extreme unpreparedness on the side of both European and non-European nations. Adverse events have demonstrated their effects and direct detrimental impact mostly on the population aged 0–18 years, with important repercussions for families and communities. Generally, the most vulnerable suffer the consequences. Environmental pollution and climate change rank highly among the natural disasters that have an effect on children's health. Particularly, there has been a growing correlation between environmental pollution events and many health disorders, such as respiratory diseases, cardiovascular diseases, infectious diseases, diabetes, and allergic diseases linked to elevated allergen production. Data from the literature have demonstrated the detrimental effects of chemicals on children's health, with endocrine disruptors receiving particular attention due to the potential harm they may bring to a variety of endocrine and non-endocrine systems. In order to further increase pediatricians and public health authorities' understanding of this crucial issue for the future of children's health, this brief overview examines the significant effects of environmental pollution and climate change on children's health.

IgE Antibody Assay-Based Allergen Database(CLSI-IL/A-37)

There are 8 established allergen databases (IUIS/WHO ANDB, Allergome, AllFam, AllergenOnline, SDAP, COMPARE, IEDB and AllerBase).None of these provide a comprehensive listing of the internationally-accepted nomenclature codes and Linnean system descriptors of the~1000 extract and molecular allergens used world-wide in diagnostic single-and multi-plex IgE antibody assays. The new Diagnostic Allergen DataBase (DADB) has been prepared by an international scientific committee of the Clinical and Laboratory Standards Institute to specifically provide these data together in a readily-searchable Excel database. Using peanut as an example, the DADB lists the allergens’ unique assay working codes (F13), general category (food), principal IgE antibody assay method(s) where the allergen specificity is available, common name (peanut), taxonomical name (Arachis hypogaea), and the NCBI, NPU, LOINC, Allergome and IUIS/WHO descriptor codes for allergens and their assays. The DADB benefits manufacturers of allergen-specific IgE assays by providing unambiguous specificity descriptors. With the DADB, allergen extract manufacturers will have clearly defined specificity targets for allergenic product development. Government regulators will have clarity on the specificity of new allergen reagents that are being submitted for clearance. IgE antibody proficiency testing programs (e.g. CAP) will be able to clearly define the IgE antibody specificities being measured in their challenge sera. The clinician will precisely know the allergen specificity of IgE antibody that is measured in their reference laboratory. Finally, the allergic patient will be assured that their allergic sensitization is being assessed in an assay with a well-characterized allergen reagent.

Diagnostic gap due to missing patch test allergens—status quo and possible scenarios for mitigation

SummaryAccording to European Directive 2001/83/EC, test and therapeutic allergens are medicinal products in all Member States of the European Union. This applies equally to prick test and to patch test (PT) allergens (haptens). All test allergens commercially marketed in Germany are finished medicinal products requiring marketing authorization (MA). Currently, 211 PT substances are authorized in Germany, and an additional 59 are in an ongoing MA process and are marketable under a transitional provision until a decision on MA is made. The regulatory guidance (CMDh/399/2019) of the Co-ordination Group for Mutual Recognition and Decentralized Procedures—Human (CMDh), published in July 2020, specifies the regulatory requirements for different allergen products. Due to differences in origin and exposure, use, mode of action, and safety risks, the guideline clearly differentiates between products with active ingredients of biological origin (allergen extracts from natural source materials) and products with active ingredients of non-biological origin (hapten-based PT substances). Currently, guideline-compliant patch testing is hampered by the lack of numerous commercial PT allergens from the standard and special test series. Background and possible scenarios for mitigation are presented here.

Impact of climate change-related environmental factors on the allergens production and the epidemiology and severity of allergic pathologies.

Allergic disease affects up to 40% of the global adult population, a proportion that is increasing with environmental changes related to global warming. We undertook a systematic review of the literature to identify and evaluate the current evidence of the impact of climate change-related environmental factors on the allergen production and the epidemiology and severity of allergic pathologies. PECO criteria were established and guided the literature searches of the PubMed and Cochrane databases (Jan 1, 2016 to Dec 31, 2021). Study outcomes were categorized and grouped to facilitate data synthesis. Outcomes were classified as significant (statistical significance <0.05), non-significant (p>0.05) or undetermined (p value not reported). Study quality was assessed using MMAT analysis. Of 195 studies, 40 were considered relevant and 9 of them provided data to be included in the data quantitative synthesis. Environmental factors, including the presence of pollutants, temperature, and drought, influenced the type, volume, and timing of exposure to local aeroallergens. The most relevant environmental factor was the presence of environmental pollutants, of which tropospheric ozone was the most frequently associated to changes in allergen production, prevalence, and severity of allergic disease. Also, several publications demonstrated the impact of environmental factors on the healthcare burden. Climate-change related environmental factors increased allergic disease in terms of prevalence, severity, and healthcare burden due to alterations in allergen exposure (volume and type) with the presence of pollutants such as ozone being the most commonly reported driver of such increase.

Regulatory framework for development and marketing authorization of allergen products for diagnosis of rare type I and type IV allergies: The current status.

Development, production, and marketing authorization of allergen products is generally challenging due to several specific characteristics, including the natural source as well as the multitude of allergenic materials. Also, depending on the frequency of sensitization in the population, the number of patients available for inclusion in clinical trials can be a limiting factor for product development. In the development of allergen products for diagnosis of type I and type IV allergies these challenges are particularly demanding because, in contrast to certain products for allergen-specific immunotherapy, no exemptions from marketing authorization are foreseen for this product group in Directive 2001/83/EC. Thus, the regulatory framework is constantly adapted within the legal scope in order to balance necessary regulatory requirements ensuring quality, safety, and efficacy with the clinical need for a comprehensive range of diagnostic allergen products. In this article, we give an overview on the current regulatory framework for development and marketing authorization of allergen products for diagnosis of rare type I and type IV allergies.

Development of allergy to LTP in children and adults

Background. The importance of issue is the rising incidence of allergies to volatile organic compounds, particularly in children and adults. This necessitates understanding the causes, risk factors, and methods for prevention and treatment. The purpose of the study was to clarify the relationship between the development of allergy to lipid transfer proteins (LTP) and various environmental influences, nutrition and the immune state of the body. Materials and methods. To achieve goal, the following research methods were used: content analysis of the database of medical organizations, the method of a representative sample and quantitative examination. In the practical part of the study, we used the Immuno Solid-phase Allergen Chip test. Results. The study results demonstrated adverse reactions in both children and adults with LTP allergies. Peach (Prunus persica) was identified as an allergenic product. It was found that childhood is a particularly sensitive period for the development of allergic reactions to LTP due to incomplete immune development. Additionally, the peculiarities of food allergy were investigated, taking into account its connection with antibodies and cause-and-effect relationships. Conclusions. Sensitization to lipid transfer proteins occurs in various groups, including children and adults, and depends on the sources of exposure.

Pollen allergen products: current standardisation issues

Scientific relevance. Pollen allergen medicines are in high demand, and their therapeutic benefits directly correlate with their standardisation. Better diagnosis and treatment of allergic diseases require state-of-the-art procedures for assessing the allergenic activity of pollen allergen products using reference standards and physicochemical testing methods.Aim. The study aimed at developing methodological approaches to the standardisation of pollen allergen products in order to shift to measuring their potency in allergenic activity units (AAU) and bring their quality in line with the requirements of the State Pharmacopoeia of the Russian Federation.Materials and methods. The study used pollen allergen reference standards by Microgen, the WHO International Standard for timothy grass (Phleum pratense) pollen extract, a gel filtration standard kit of molecular weight markers ranging from 1.35 to 670 kDa, bovine serum albumin, serum samples with specific IgE obtained from donors sensitised to the study pollen allergens, labelled anti-human IgE antibodies, and reference standards for determining residual volatile solvents by gas chromatography. The identification of in-house reference standards for the potency of pollen allergens involved Western blotting (for allergenic components). The total protein content was determined by Bradford’s assay. In addition, the authors used high-performance liquid chromatography to study protein fractions and gas–liquid chromatography to determine the content of residual organic solvents.Results. To substitute the existing method of non-specific characterisation of allergenic activity in protein nitrogen units (PNU), the authors developed and tested a new method to control allergenic activity in allergenic activity units (AAU) based on an in vitro competitive enzyme-linked immunosorbent assay (ELISA) procedure developed and validated in this study. Furthermore, the authors developed and certified 15 primary in-house reference standards with allergenic activity established in AAU/mL using skin tests in vivo. The experimental data were analysed to standardise the allergenic activity of the pollen allergens manufactured by Microgen. The authors developed physicochemical methods for the certification of in-house reference standards and validated these methods in accordance with the State Pharmacopoeia of the Russian Federation. The study involved selecting chromatographic separation conditions for residual organic solvents (acetone and diethyl ether) and establishing system suitability criteria for the chromatographic system. The allergenic activity of secondary in-house reference standards was certified against that of primary in-house reference standards using competitive ELISA. Thus, the authors managed to shift to the standardisation of pollen allergen products in vitro.Conclusions. The authors developed their competitive ELISA-based method to standardise pollen allergen products by comparing the inhibition of immune responses to a product and a standard. The study demonstrated the feasibility of substituting allergenic activity quantification (in AAU) for protein nitrogen content determination (in PNU) and showed the first example of using AAU for the certification of in-house reference standards. Additionally, the authors developed and validated an analytical procedure for determining the content of residual organic solvents in pollen allergen products by gas–liquid chromatography.

Impact of the standardization unit’s definition on the in vitro biological potency of allergen extracts

In Europe, allergen products from different manufacturers can be labeled using the same unit with yet different definitions of that unit, which may cause confusion, as is the case for the index of reactivity (IR). In this context, house dust mite (HDM) Staloral 300 IR/mL, from Stallergenes Greer, and HDM Osiris 300 IR/mL, from ALK-Abelló, were characterized in vitro. Qualitatively, namely in terms of protein and allergen profiles, the two products were similar. Quantitatively, and despite the same 300 IR/mL labeling, the two products were shown to have different biological potencies, with HDM Staloral 300 IR/mL displaying a 2.4 times higher total allergenic activity (TAA) than HDM Osiris 300 IR/mL. This higher biological potency of HDM Staloral 300 IR/mL was paralleled by higher allergen and protein contents, namely 1.5 times more Der p 1 and Der f 1, 3.0 times more group 2 allergens, 2.7 times more Der p 23, and 1.8 times more protein. In contrast, HDM Staloral 300 IR/mL was shown to contain far fewer culture medium-derived proteins than HDM Osiris 300 IR/mL.

Comparison of two methods for assessing the pollen allergy risk of urban parks in Chengdu City, China

The urban green space is an important inducer of pollen allergy. In recent years, the incidence of pollen allergy in China has increased from 5 % to 17.8 %, and it still rapidly increases. However, only few assessment methods of pollen allergy risk have been developed and applied in the urban green spaces. The feasibility and the accuracies of the assessment results of these methods has been unknown. Therefore, in this paper, the two best-known methods, total pollen concentration monitoring method and urban green zone allergenicity index (IUGZA) method, were both used to assess the pollen allergy risk of the same urban park (the People’s Park of Chengdu City, China). The impact factors and prediction consistency of both models were compared. The results showed that the proportion of medium and highly allergenic tree species reached up to 75.93 % in People’s Park. The total IUGZA value of this park was 0.69. A total of 36,171 pollen grains were collected here. The average pollen concentration of this park was 125.88 grains/cm2 per day. Both methods showed that the pollen allergy risk of the People's Park was the highest in spring. Correlation analysis showed that no vegetation characteristics of this park were correlated with IUGZA values and total pollen concentration. However, pollen concentration was extremely significantly correlated with air temperature and solar radiation in autumn and winter. Kendall’s concordance coefficient identified a super high consistency between total pollen concentration and IUGZA values in this park. This result means that both methods for assessing the risk of pollen allergy are effective. Based on the total pollen concentration, a polynomial formula was established for succinctly calculating the IUGZA. A three-step greening strategy is proposed to minimize the volume of allergen production and increase the well-being of users of urban parks in Chengdu City.

Comparison of Intact Allergen Extracts and Allergoids For Subcutaneous Immunotherapy: The Effect of Chemical Modification Differs Both Between Species and Between Individual Allergen Molecules.

Allergen products for subcutaneous immunotherapy (SCIT) contain intact allergen extracts or chemically modified allergoids. Chemical modification was introduced to reduce allergenicity while retaining immunogenicity and thereby enable safer and more efficient allergy immunotherapy. Experimental allergoids were produced from intact allergen extract for birch, grass, and house dust mite (HDM) to evaluate the effects of chemical modification. Preparations were compared with commercial allergoids and analyzed using SDS-PAGE/immunoblotting, IgE-inhibition assays, and crossed immunoelectrophoresis (CIE). Dermatophagoides pteronyssinus (Der p) vaccines were also tested for protease activity and immunizing capacity in a mouse model. The composition of IgE-binding epitopes in allergoids differed from that of intact allergen vaccines. Birch and grass allergoids produced smears of protein aggregates on SDS-PAGE, whereas intact allergen preparations showed distinct protein bands as expected. Der p allergoid vaccines, however, showed a distinct protein band corresponding to major allergen Der p 1 in both SDS-PAGE and CIE analysis, and commercial Der p allergoid vaccines showed Der p 1-related cysteine protease activity. Allergoids and intact allergen preparations differ with respect to the composition of IgE-binding epitopes. However, chemical cross-linking does not affect every allergen molecule to the same degree. Der p 1, for example, remains largely unmodified. Furthermore, the investigational HDM allergoid vaccines showed reduced and delayed immune responses when used for immunization of mice.

Allergen analysis and authenticity control: Development of mass spectrometry‐based methods for animal‐derived food products

Food allergy and food fraud involving animal‐derived products are two of the most significant issues in food markets. On one hand, immunoglobulin E (IgE)‐mediated allergic reactions after ingestion of fish, crustaceans, eggs, or milk are among the most prevalent and can happen even after ingestion of trace amounts. On the other hand, new rules regarding product commercialization (e.g., novel food regulation) are more and more created while fraudulent species substitution in fishery products is very common. Sensitive and accurate analytical methods for allergen quantification and species identification in commercial food products are therefore urgently required whether to help food industries inform allergic consumers, to ensure the food compliance with new regulations or to combat food fraud.In the past few years, bottom‐up proteomic techniques, which rely on the detection of peptide biomarkers resulting from a tryptic digest of food proteins using liquid chromatography coupled to tandem mass spectrometry (LC‐MS/MS), have been emerging in this field. The selection of reliable allergen‐specific or species‐specific peptide biomarkers is one of the most crucial steps when developing such methods whether for qualitative protein detection (i.e., screening analysis) or protein absolute quantification.The first part of this dissertation relates therefore to the selection of allergen peptide biomarkers for fish, invertebrates, eggs, and milk in an experimental way using a single chaotropic urea extraction buffer. The allergenic proteins responsible for those severe reactions are mainly parvalbumin, tropomyosin, ovalbumin, and caseins.The protein extraction was first assessed via sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and the major allergens were well extracted. After that, data‐dependent MS/MS spectra which were obtained using digest samples of authentic animals or pure allergenic ingredients were processed against a matching protein database, and identified peptides were filtered according to several criteria such as the sequence length, amino acid composition, specificity, reproducibility, and sensitivity. Myosin proteins were also a target of choice for fish detection due to their high prevalence and sequence homology even if they are non‐allergenic. Contrary to fish and invertebrate databases which included entries for only one specific protein (i.e., parvalbumin, myosin, or tropomyosin), egg and milk databases contained all known allergenic proteins or even additional proteins for egg yolk. Two extra selection steps were achieved for egg and milk markers based on more stringent criteria regarding their sensitivity after targeting them in the corresponding allergenic ingredients. At this point, 17, 13, 10, 10 and 12 potential markers were respectively selected for fish, invertebrates, egg white, egg yolk and milk. An alignment algorithm was used for all those markers to get an idea about their biological specificity. The biological specificity was verified experimentally for fish and invertebrate potential markers by targeted analysis in digest samples of animal species that are relevant in the food industry (vertebrates and invertebrates). This verification was not done for egg and milk markers as it was not regarded as significant due to reported cross‐reactivity among avian eggs and among mammalian milk. Two potential parvalbumin markers were detected in other nonfish vertebrates, while all potential tropomyosin markers except one were specific to at least an invertebrate class belonging to the same phylum. Parvalbumin and myosin markers as well as tropomyosin markers were exclusively found in vertebrates or invertebrates. Marker detectability was checked by analyzing processed fish products as well as cooked fish for potential parvalbumin and myosin markers, while commercial insect‐based food products such as cereals bars or pasta were studied for potential tropomyosin markers. All expected fish and invertebrate markers were detectable in those complex food products. Detectability of egg and milk markers was assessed by analysis of bread, cookies, and chocolate samples contaminated at different stages of the sample preparation with trace amounts (100 pg/g) of eggs and milk (i.e, fortified, spiked, and incurred digest samples). Two egg white markers and seven milk markers were detected in all those samples. The most suitable markers in terms of sensitivity and specificity were finally chosen for each allergenic product. Thus, besides two myosin fish global markers, five parvalbumin markers were retained including at least one of the investigated fish species. In addition, five tropomyosin markers were chosen, their specificity allowing us to distinguish crustacean tropomyosin from that of insects/arachnids, or mollusks. At last, two ovalbumin markers and three casein markers were confirmed to be the most suitable allergen markers respectively for egg white and milk. All those retained markers could be compiled in a single multiplex method. The automation of the sample preparation could also be a promising improvement whether for qualitative or quantitative analysis.

Biological activity of human IgE monoclonal antibodies targeting Der p 2, Fel d 1, Ara h 2 in basophil mediator release assays.

Human Immunoglobulin E monoclonal antibodies (hIgE mAb) are unique tools for investigating IgE responses. Here, the biological activity of hIgE mAb, derived from immortalized B cells harvested from the blood of allergic donors, targeting three allergens (Der p 2, Fel d 1 and Ara h 2) was investigated. Three Der p 2-, three Fel d 1- and five Ara h 2-specific hIgE mAb produced by human B cell hybridomas, were combined in pairs and used to passively sensitize humanized rat basophilic leukemia cells and compared with sensitization using serum pools. Sensitized cells were stimulated with corresponding allergens (recombinant or purified), allergen extracts or structural homologs, having 40-88% sequence similarity, and compared for mediator (β-hexosaminidase) release. One, two and eight pairs of Der p 2-, Fel d 1- and Ara h 2-specific hIgE mAb, respectively, produced significant mediator release (>50%). A minimum hIgE mAb concentration of 15-30 kU/L and a minimum antigen concentration between 0.01-0.1 µg/mL were sufficient to induce a pronounced mediator release. Individual sensitization with one Ara h 2-specific hIgE mAb was able to induce crosslinking independently of a second specific hIgE mAb. Der p 2- and Ara h 2-specific mAb showed a high allergen specificity when compared to homologs. Mediator release from cells sensitized with hIgE mAb was comparable to serum sensitization. The biological activity of hIgE mAb reported here provides the foundation for novel methods of standardization and quality control of allergen products and for mechanistic studies of IgE-mediated allergic diseases, using hIgE mAb.

The effects of climate change and exposure to endocrine disrupting chemicals on children's health: A challenge for pediatricians

The natural, economic and public health crises that have periodically struck the world over the past two decades have often revealed a low degree of self-sufficiency and a high degree of unpreparedness on the part of European and non-European countries. It is generally the most vulnerable who suffer the consequences, and adverse events have shown their effects and direct negative impact mainly on the population aged 0–18 years, with major implications for families and communities. Climate change and environmental pollution are certainly prominent among the natural disasters impacting children's health. In particular, environmental pollution events have been increasingly correlated with health conditions, including allergic diseases related to increased allergen production, infectious diseases, diabetes, respiratory diseases, and cardiovascular diseases. Data from the literature have shown how chemicals can interfere with children's health, and special attention has been paid to endocrine disruptors because of their possible damage on various endocrine and nonendocrine systems. This brief overview explores the important impacts of climate change and environmental pollution on children's health, with the goal of further raising awareness among pediatricians and public health authorities on this critical issue for the future of children's health.

Introduction of Complementary Foods and the Risk of Sensitization and Allergy in Children up to Three Years of Age.

Allergy is known to be influenced by both diet and the immune system. In addition, the time of first exposure to food allergens and their type appear to play a particularly important role in the development of allergies. To determine the influence of the time of exposure, and the type, degree of processing, and frequency of supply of complementary foods and the development of sensitization and allergies in children up to three years of age. Materials and metods: The study was conducted prospectively in two stages. The first stage included 106 children aged 6-18 months, while the second stage included 86 children selected from the first stage, after a further 12 months. A questionnaire based on validated FFQ sheets was created for the purpose of the study. The following were assessed: nutrition in the first year of life (time, type, degree of processing), frequency of supply of complementary foods and allergic symptoms, sIgE concentration against 10 foods and 10 inhalant allergens. Four groups of patients were formed. This paper presents the results of the second stage of the study. For all participants, allergenic products, viz. hen's egg, milk, peanuts, wheat, soybean, fish, tree nuts and shellfish were typically introduced at an age of 7 to 12 months. During this period, egg white was introduced in 47 (85.5%) children with allergy (p = 0.894), in 29 (82.9%) with allergy and sensitization (p = 1.00), and in 38 (82.6%) children with sensitization alone (p = 0.533). Milk was introduced at 7 to 12 months in 35 (64.8%) children with allergy (p = 0.64), 22 (64.7%) with both allergy and sensitization (p = 0.815), and 26 (57.8%) children with sensitization alone (p = 0.627). For other foods, the time of introduction appeared not to significantly influence the presence of allergies or sensitization. Heat-treated peanuts were introduced significantly more often to children without allergies and without sensitization (n = 9; 56.2%) than those without allergies but with sensitization (n = 6; 54.5%) (p = 0.028). Fish was consumed significantly more often by children with allergies, i.e., 1-3x/week (n = 43; 79.6%) than children without allergies, i.e., 1-3x/month (n = 9; 30%) (p = 0.009). No relationship was observed between the introduction time of complementary foods, including allergenic ones, or their type, and the development of allergies and sensitization in children up to three years of age. The degree of processing and the frequency of supply of products may affect the development of allergies and sensitization.