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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.

Peanut allergy has garnered worldwide attention due to its high incidence rate and severe symptoms, stimulating the demand for the ultrasensitive detection method of peanut allergen. Herein, we successfully developed a novel electrochemical aptasensor for ultrasensitive detection Ara h1, a major allergenic protein present in peanuts. A conductive nickel atoms Anchored Hydrogen-Bonded Organic Frameworks (PFC-73-Ni) were utilized as excellent electrocatalysts toward hydroquinone (HQ) oxidation to generate a readable current signal. The developed electrochemical aptasensor offers wide linear range (1-120 nM) and low detection limit (0.26 nM) for Ara h1. This method demonstrated a recovery rate ranging from 95.00% to 107.42% in standard addition detection of non-peanut food samples. Additionally, the developed electrochemical method was validated with actual samples and demonstrated good consistency with the results obtained from a commercial ELISA kit. This indicates that the established Ara h1 detection method is a promising tool for peanut allergy prevention., Competing Interests: Declaration of competing interest There are no any conflicts of interest regarding of this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Mung bean is an increasingly cultivated legume. This study compared mung bean varieties 'KPS2' from Thailand (Th) and 'Imara' from Tanzania (T) with a focus on protein composition, allergenicity, and techno-functional properties. Two rounds alkaline-acid extraction were performed to produce mung bean protein isolate (MBPI - Th 1 /T 1 and Th 2 /T 2 ), supernatant (S) and protein-poor residue (PPR). Mass spectrometric analysis revealed high abundance of 8 s-vicilin and 11 s-legumin in MBPI and S. Extraction removed considerable amounts of the seed albumin allergen but increased the relative abundance of cupins in MBPI. Higher vicilin levels were found in Th 1 samples, contributed to increased protein solubility above pH 6.5. Th formed stronger gels which were more stable at higher frequencies. In contrast, T proteins were structurally more flexible, leading to its improved foaming ability. This study provides the knowledge and methods for appropriate selection of mung bean varieties for various food applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Walnut represents one of the most allergenic nuts that can be found as a hidden allergen. In this study, sandwich ELISA and lateral flow immunoassay (LFIA), based on the determination of Jug r 1, were developed to detect walnut. Cross-reactivity was only found with Pecan nut among a panel of 88 food ingredients tested. ELISA and LFIA could detect 0.25 and 0.5 µg/g of walnut protein in complex food matrices spiked with walnut extract, respectively. Furthermore, walnut was detected in blended (chocolate) and incurred foods (ice cream and bread) added with ground walnut at levels of 0.5 and 1.5 µg protein/g by ELISA and LFIA, respectively. LFIA could also detect 0.1 μg of walnut protein in working surfaces. ELISA displayed acceptable precision and high recovery (71-97 %) and both tests were robust. This study shows that developed ELISA and LFIA are reliable tools to be applied in allergen control programs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Life-threatening allergic reactions to food allergens, particularly peanut protein Ara h1, are a growing public health concern affecting millions of people worldwide. Thus, accurate and rapid detection is necessary for allergen labeling and dietary guidance and ultimately preventing allergic incidents. Herein, we present a novel ratiometric fluorescence aptasensor based on multivalent aptamer-encoded DNA flowers (Mul-DNFs) for the high-stability and sensitive detection of allergen Ara h1. The flower-shaped Mul-DNFs were spontaneously packaged using ultralong polymeric DNA amplicons driven by a rolling circle amplification reaction, which contains a large number of Ara h1 specific recognition units and has excellent binding properties. Furthermore, dual-color fluorescence-labeled Mul-DNFs probes were developed by hybridizing them with Cy3- and Cy5-labeled complementary DNA (cDNA) to serve as a ratiometric fluorescence aptasensor platform based on fluorescence resonance energy transfer. Benefiting from the combined merits of the extraordinary synergistic multivalent binding ability of Mul-DNFs, the excellent specificity of the aptamer, and the sensitivity of the ratiometric sensor to avoid exogenous interference. The developed ratiometric aptasensor showed excellent linearity (0.05-2000 ng mL -1 ) with a limit of detection of 0.02 ng mL -1 . Additionally, the developed ratiometric fluorescence aptasensor was utilized for quantifying the presence of Ara h1 in milk, infant milk powder, cookies, bread, and chocolate with recoveries of 95.7-106.3%. The proposed ratiometric aptasensor is expected to be a prospective universal aptasensor platform for the rapid, sensitive, and accurate determination of food and environmental hazards.

Peanut allergy is a prevalent and concerning food allergy. Roasting can introduce structural changes to peanut allergens, affecting their allergenicity, but the structure on the primary structure is unclear. Here, the breakage sites were identified by mass spectrometry and software tools, and structural changes were simulated by molecular dynamics and displayed by PyMOL software. Results revealed that the appearance frequencies of L, Q, F, and E were high at the N-terminal of the breakage site, while S and E were dominant at the C-terminal. In the conformational structure, breakage sites were found close to disulfide bonds and the Cupin domains of Ara h 1 and Ara h 3. The breakage of allergens destroyed linear epitopes and might change the conformation of epitopes, which could influence peanuts' potential allergenicity.

The incorporation of nanomaterials into consumer products has substantially increased in recent years, raising concerns about their safety. The inherent physicochemical properties of nanoparticles allow them to cross epithelial barriers and gain access to immunocompetent cells. Nanoparticles in cosmetic products can potentially interact with environmental allergens, forming a protein corona, and together penetrate through damaged skin. Allergen-nanoparticle interactions may influence the immune response, eventually resulting in an adverse or beneficial outcome in terms of allergic reactivity. This study determines the impact of silica nanoparticle-allergen interactions on allergic sensitization by studying the major molecular mechanisms affecting allergic responses. The major birch pollen allergen Bet v 1 was chosen as a model allergen and the birch pollen extract as a comparator. Key events in immunotoxicity including allergen uptake, processing, presentation, expression of costimulatory molecules and cytokine release were studied in human monocyte-derived dendritic cells. Using an in vivo sensitization model, murine Bet v 1-specific IgG and IgE levels were monitored. Upon the interaction of allergens with silica nanoparticles, we observed an enhanced uptake of the allergen by macropinocytosis, improved proteolytic processing, and presentation concomitant with a propensity to increase allergen-specific IgG2a and decrease IgE antibody levels. Together, these events suggest that upon nanoparticle interactions the immune response is biased towards a type 1 inflammatory profile, characterized by the upregulation of T helper 1 (Th1) cells. In conclusion, the interaction of the birch pollen allergen with silica nanoparticles will not worsen allergic sensitization, a state of type 2-inflammation, but rather seems to decrease it by skewing towards a Th1-dominated immune response.

Background and Objectives: Platanus acerifolia (London plane tree) is a deciduous tree of the Platanaceae family. Sensitization to this plant varies with geography. Madrid, located in central Spain, has one of the highest London plane tree pollen concentration levels on the Iberian Peninsula. We evaluated both the clinical characteristics and the molecular sensitization pattern of patients with allergy to London plane tree pollen in the region of Madrid., Methods: Thirty-eight patients allergic to London plane tree pollen were selected according to their clinical symptoms and positive results in skin prick testing and/or specific IgE determination. Serum was collected, and allergen components were evaluated using immunodetection techniques as well as ImmunoCAP. The IgE-binding proteins detected were identified and characterized using mass spectrometry., Results: Analysis of serum samples from allergic patients revealed 9 IgE-binding bands in London plane tree pollen extract. Among these, the 45-kDa protein, which corresponded to Pla a 2, was detected in 76.3% of patients. However, the 18-kDa (Pla a 1) and 9-kDa (Pla a 3) bands were detected in 44.7% and 23.7% of sera, respectively. These results were confirmed using purified proteins. Characterization of the allergen revealed the 27-kDa protein to be glutathione-S-transferase., Conclusions: The molecular profile of patients sensitized to London plane tree pollen differs from that reported in studies from other locations. In the population we studied, the prevalence of Pla a 2 was higher than that of Pla a 1 and Pla a 3. In addition, the minor allergen previously referred to as Pla a 4 was characterized as glutathione-S-transferase.

Aptamers, due to their small size, strong target affinity, and ease of chemical modification, are ideally suited for molecular detection technologies. Here, we describe successful use of aptamer technology in a consumer device for the detection of peanut antigen in food. The novel aptamer-based protein detection method is robust across a wide variety of food matrices and sensitive to peanut protein at concentrations as low as 12.5 ppm (37.5 µg peanut protein in the sample). Integration of the assay into a sensitive, stable, and consumer friendly portable device will empower users to easily and quickly assess the presence of peanut allergens in foods before eating. With many food reactions occurring outside the home, the type of technology described here has significant potential to improve lives for children and families., (© 2022. The Author(s).)

As allergy towards apples is widespread, the evaluation of various cultivation and postharvest influences on the allergenic potential is of great importance. Therefore, the analysis of the Mal d 1 content was the focus of this study, originally dealing with investigating the influence of a selenium biofortification on apple quality. The Mal d 1 content of apples was in most cases reduced when the fruits were biofortified with selenium. Apple variety and climatic conditions were identified as further influencing factors for the Mal d 1 content of the fruits. The separate analysis of the peel and the fruit flesh showed that the content of Mal d 1 in the fruit flesh was significantly lower in the biofortified samples than in the controls. In conclusion, the results indicate that the selenium biofortification of apples and biochemical mechanism behind can reduce the allergenic potential regarding the content of Mal d 1., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Tracking unreported allergens in commercial foods can avoid acute allergic reactions. A 2-step electrochemical immunosensor was developed for the analysis of the peanut allergen Ara h 1 in a 1-h assay (<15 min hands-on time). Bare screen-printed carbon electrodes (SPCE) were used as transducers and monoclonal capture and detection antibodies were applied in a sandwich-type immunoassay. The short assay time was achieved by previously combining the target analyte and the detection antibody. Core/shell CdSe@ZnS Quantum Dots were used as electroactive label for the detection of the immunological interaction by differential pulse anodic stripping voltammetry. A linear range between 25 and 1000 ng·mL -1 (LOD = 3.5 ng·mL -1 ), an adequate precision of the method (V x0 ≈ 6%), and a sensitivity of 23.0 nA·mL·ng -1 ·cm -2 were achieved. The immunosensor was able to detect Ara h 1 in a spiked allergen-free product down to 0.05% (m/m) of peanut. Commercial organic farming cookies and cereal and protein bars were tested to track and quantify Ara h 1. The results were validated by comparison with an ELISA kit.

Biosensors have been widely applied in tests for allergens, but on-site detection remains a challenge. Herein, we proposed a detection procedure for peanut Ara h 1 as a representative allergen, which was extracted from a cookie, thereby minimising the need for any complex pretreatment that was difficult to perform, and enabling the visual detection of the target without the use of analytical equipment. The extraction procedure was performed in less than 30 min using a syringe and filter (0.45 μm). The detection method for Ara h 1 was based on the aggregation of switchable linkers (SL) and gold nanoparticles (AuNP), and the presence of 0.19 mg peanut protein per 30 g of cookie could be confirmed within 30 min based on the AuNP/SL concentration ratio by the precipitation. This proposed procedure could be successfully applied to the detection of a wide range of food allergens., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Summary: The clinical usefulness of two commercial peach extracts for SPT (by Lofarma SpA and ALK-Abellò, respectively) was compared in a multicenter study carried out in Italy. Peach allergic patients were tested with the two extracts in parallel and underwent the detection of IgE specific for all three peach allergens currently available (Pru p1, Pru p3, and Pru p4, respectively). The two extracts were almost identical in terms of sensitivity and specificity, being able to detect virtually all patients sensitized to stable peach allergens (lipid transfer protein (LTP) and, presumably, peamaclein) but scoring negative in patients exclusively sensitive to labile allergens (either PR-10 and/or profilin). Thus, the two extracts represent an excellent tool to carry out a preliminary component-resolved diagnosis of peach allergy at the first patient visit.

The Pannonian Plain is one of the centers of ragweed distribution in Europe. The province of Vojvodina (Serbia) is located on the southern part of the Pannonian Plain, representing a highly infested region. In this study, we have used the SILAM atmospheric dispersion model to simulate ragweed pollen concentrations during the season 2016 in the Vojvodina region. SILAM was tested with three different source maps of ragweed distribution in Vojvodina only: (1) map used in operational SILAM, which was calibrated with the SILAM model and observations, (2) map derived using "top-down" approach with land cover data inventory, and (3) map obtained with "top-down" approach using crop classification from the satellite data. Additionally, the sensitivity studies were done using two modified maps to study the effect of the source strength and long-range transport. Results of simulations were validated with the bi-hourly, daily, and seasonal pollen concentrations measured at five stations in Vojvodina. Overall Pearson correlation coefficients were 0.51 (Map 1), 0.50 (Map 2), and 0.42 (Map 3), while debiased scores were 232.95 pollen m -3 (Map 1), 245.59 pollen m -3 (Map 2), and 258.24 pollen m -3 (Map 3). Even though Vojvodina is in the area of a major European source, regional transport of ragweed pollen from a few hundred kilometers of the surrounding area was important in explaining the presence of pollen in the afternoon hours, although it could not completely explain total pollen quantity. The results confirmed that it is vital to calibrate source maps using atmospheric dispersion model with the observed pollen data.

Background: Soybean meal (SBM) is the most common protein source used in the poultry and livestock industries. It has high-quality protein, an excellent amino acid (AA) profile, and positive isoflavone properties. However, the antigen proteins in SBM are unsuitable for young animals. The objective of this study was to identify a Bacillus strain that can degrade soybean antigen proteins, and to evaluate the feasibility of its application in SBM fermentation., Results: Bacillus velezensis DP-2 was isolated from Douchi, a fermented Chinese food. It degraded 96.14% and 66.51% of glycinin and β-conglycinin, and increased the trichloroacetic acid-soluble protein (TCAN) content by 5.46 times in the SBM medium. DP-2 could secrete alkaline protease and neutral protease, with productivities of 5.85 and 5.99 U mL -1 . It had broad-spectrum, antibacterial activities against Rhizopus nigricans HR, Fusarium oxysporum ACCC37404, Penicillium digitatum SQ2, Aspergillus flavus C1, Aspergillus niger ACCC30005, Trichoderma viride YZ1, Candida tropicalis CICC1630, and Salmonella sp. ZY. For SBM fermentation, the optimal inoculum rate, temperature, and fermentation time of DP-2 were 2.21 × 10 7 CFU g -1 , 37 °C, and 48 h, respectively. The fermented soybean meal (FSBM) was cream-colored and glutinous. Its crude protein (CP), soluble protein, and TCA-N content were improved by 13.45%, 12.53%, and 6.37 times, respectively. The glycinin and β-conglycinin content were reduced by 78.00% and 43.07%, respectively, compared with raw SBM., Conclusions: Bacillus velezensis DP-2 has potential as a starter culture for SBM fermentation. © 2020 Society of Chemical Industry., (© 2020 Society of Chemical Industry.)

Background: Since the second half of the 20th century, the cultivation of ancient and local apple cultivars has almost disappeared from orchards in Italy. Some of these ancient apple cultivars often possess high nutraceutical values and display lower allergenicity than the modern ones, supporting the so-called 'green revolution' theory., Results: In this study, the phenolic composition and the antioxidant activity of five ancient apple cultivars ('Belfiore', 'Pomella Genovese', 'Gravenstein', 'Bella del Bosco', and 'Piatlin') were compared with a 'Golden Delicious' commercial cultivar. Additionally, apples were tested for their potential allergenicity by detecting the presence of Mal d 3, a non-specific lipid transfer protein that represents the main apples' allergen. All apples came from northern Italy (Trentino Region) and were organically produced. Results showed that, for all cultivars, the skins contained more polyphenols than the pulps. 'Bella del Bosco' had the highest amount of polyphenols and antioxidant activity, whereas 'Piatlin' had the lowest phenolic content. All ancient cultivars presented a higher amount of pulp phenolic compounds than 'Golden Delicious'. Immunoblotting techniques showed that 'Bella del Bosco' and 'Piatlin' had very low quantities of Mal d 3 allergen; hence, they can be considered hypoallergenic cultivars., Conclusions: The preservation of ancient apple cultivars would be of great importance, not only to maintain the biodiversity but also for their nutritional properties. The hypoallergenic activity of some of these cultivars could be of interest also for the preparation of different apple-based products. © 2020 Society of Chemical Industry., (© 2020 Society of Chemical Industry.)

The presence of undeclared soy proteins in food can cause severe reactions in soy allergic individuals. The extraction of target proteins from processed foods is a crucial step in allergen detection by immunoassays, as only successfully extracted target proteins can be detected by the specific antibodies. The effectiveness was studied of different conditions (type of buffer, temperature and time of incubation) on the extraction of total protein, and concentration of glycinin and β-conglycinin from different food matrices. The yields were determined using a soy protein isolate and three processed foods (sausage, bread and pâté) incurred with soy proteins. The yields were affected by the processing of analysed products and the composition and pH of the extraction buffers. Neutral and alkaline buffers (pH from 7.4 to 10.6) exhibited good protein extraction capacity and detectability of the specific target proteins. Denaturing additives and highly alkaline buffer (pH 12) extracted more crude protein but they were incompatible with the ELISA assay. Overall, the best results were obtained using phosphate (pH 7.4) and Tris/HCl (pH 8.5) buffers in the presence of 0.5 M NaCl. Crude protein yield of food extracts did not correlate with that of glycinin and β-conglycinin, whereas a good relationship was found between the yields of the two proteins.

A rising proportion of the world population suffers from food-related allergies, including incompatibilities to apples. Although several allergenic proteins have been found in apples, the most important proteins that cause allergic reactions to apples in Central-Northern Europe, and North America are the Mal d 1 proteins, which are homologues of the birch pollen allergen Bet v 1. As the demand for hypoallergenic fruits is constantly increasing, we selected apple genotypes with a low total content of Mal d 1 by enzyme-linked immunosorbent assay analysis from segregating populations and tested the tolerability of these fruits through a human provocation study. This tiered approach, which exploited the natural diversity of apples, led to the identification of fruits, which were tolerated by allergic patients. In addition, we found a significant correlation (coefficient >0.76) between the total Mal d 1 content and flavan-3-ol amount and show that the isoform composition of the Mal d 1 proteins, which was determined by LC-MS/MS has a decisive effect on the tolerability of apple genotypes. The approach presented can be applied to other types of fruit and to other allergenic proteins. Therefore, the strategy can be used to reduce the allergen content of other plant foods, thereby improving food safety for allergy subjects.

Hordeins are the major barley seed storage proteins and are elicitors of celiac disease. Attempts to reduce the hordein level in barley have been made; however, the resultant pleiotropic effects are less understood. Here, data-independent acquisition mass spectrometry was used to measure proteome-wide abundance differences between wild-type and single hordein-null barley lines. Using comparative quantitative proteomics, we detected proteome-wide changes (∼59%) as a result of the specific reduction in hordein proteins. The comparative analysis and functional annotation revealed an increase in non-gluten storage proteins, such as globulins and lipid transfer proteins, and proteins rich in essential amino acids in the null lines. This study yields an informative molecular portrait of the hordein-null lines and the underlying mechanisms of storage protein biosynthesis. This study indicates the extent to which protein content can be manipulated without biological consequence, and we envision its wide-scale application for studying modified crops.

The effectiveness of some non-specific proteases in reducing raw peanut allergenicity was investigated. Peanut kernels were treated by Alcalase, papain, Neutrase and bromelain, respectively. The residues of major peanut allergens Ara h 1, Ara h 2 and Ara h 6 were determined by sandwich ELISA and SDS-PAGE, and the allergenicities of treated peanuts were compared to that of untreated peanuts by western blot. All tested proteases were effective in reducing Ara h 1, but their effectiveness in hydrolyzing Ara h 2 and Ara h 6 varied greatly. The maximal reductions of extractable Ara h 1, Ara h 2 and Ara h 6 were 100%, 100% and 99.8%, respectively, achieved by Alcalase hydrolysis. Alcalase was more effective in overall allergenicity reduction; bromelain and Neutrase were the least effective in reducing Ara h 2 and Ara h 6, respectively. The hydrolysis of original allergens also produced some smaller peptides with strong IgE-binding., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Identification of allergen proteins by using wet-lab technology is a time-consuming and also costly process. In recent years, thanks to the developments in the field of bioinformatics, it is now possible to estimate the allergen proteins by using in silico tools. In the present study, it is aimed to find kiwellin-like proteins from different fruits samples by using bioinformatics tools. According to the results of the study, six proteins from Corchorus olitorius, Cucumis sativus, Capsicum chinense, Carica papaya, Morus notabilis and Jatropha curcas were defined as the allergens. In conclusion, in silico tools developed under the field of bioinformatics can provide a big contribution to the estimation of unknown allergen proteins in different fruits. Based on the in silico results, physicians can suggest people who have allergenicity to kiwellin not to consume the fruits that contain kiwellin-like proteins.

β-Conglycinin is the major storage protein in soybeans. Pre-clinical animal models and human clinical studies have demonstrated the triglyceride-lowering effect of this protein, suggesting that it could be put into practical use as a functional food material. To date, however, there are no accurate and simple assays for quantification of β-conglycinin. In this study, samples were pretreated by mixing them with rice flour powder prior to extraction of proteins. Then, we used commercially available ELISA kits for detection of allergens that could be present in any contaminating soybean residue. This enabled accurate and highly reproducible quantitation of β-conglycinin content in several processed soybean foods.

Large-scale synoptic conditions are able to transport considerable amounts of airborne particles over entire continents by creating substantial air mass movement. This phenomenon is observed in Europe in relation to highly allergenic ragweed (Ambrosia L.) pollen grains that are transported from populations in Central Europe (mainly the Pannonian Plain and Balkans) to the North. The path taken by atmospheric ragweed pollen often passes through the highly industrialised mining region of Silesia in Southern Poland, considered to be one of the most polluted areas in the EU. It is hypothesized that chemical air pollutants released over Silesia could become mixed with biological material and be transported to less polluted regions further North. We analysed levels of air pollution during episodes of long-distance transport (LDT) of ragweed pollen to Poland. Results show that, concomitantly with pollen, the concentration of air pollutants with potential health-risk, i.e. SO 2 , and PM 10 , have also significantly increased (by 104% and 37%, respectively) in the receptor area (Western Poland). Chemical transport modelling (EMEP) and air mass back-trajectory analysis (HYSPLIT) showed that potential sources of PM 10 include Silesia, as well as mineral dust from the Ukrainian steppe and the Sahara Desert. In addition, atmospheric concentrations of other allergenic biological particles, i.e. Alternaria Nees ex Fr. spores, also increased markedly (by 115%) during LDT episodes. We suggest that the LDT episodes of ragweed pollen over Europe are not a "one-component" phenomenon, but are often related to elevated levels of chemical air pollutants and other biotic and abiotic components (fungal spores and desert dust)., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Common ragweed is a highly allergenic invasive species in Europe, expected to become widespread under climate change. Allergy to ragweed manifests as eye, nasal and lung symptoms, and children may retain these throughout life. The dose-response relationship between symptoms and pollen concentrations is unclear. We undertook a longitudinal study, assessing the association between ragweed pollen concentration and allergic eye, nasal and lung symptoms in children living under a range of ragweed pollen concentrations in Croatia. Over three years, 85 children completed daily diaries, detailing allergic symptoms alongside daily location, activities and medication, resulting in 10,130 individual daily entries. The daily ragweed pollen concentration for the children's locations was obtained, alongside daily weather and air pollution. Parents completed a home/lifestyle/medical questionnaire. Generalised Additive Mixed Models established the relationship between pollen concentrations and symptoms, alongside other covariates. Eye symptoms were associated with mean daily pollen concentration over four days (day of symptoms plus 3 previous days); 61 grains/m 3 /day (95%CI: 45, 100) was the threshold at which 50% of children reported symptoms. Nasal symptoms were associated with mean daily pollen concentration over 12 days (day of symptoms plus 11 previous days); the threshold for 50% of children reporting symptoms was 40 grains/m 3 /day (95%CI: 24, 87). Lung symptoms showed a relationship with mean daily pollen concentration over 19 days (day of symptoms plus 18 previous days), with a threshold of 71 grains/m 3 /day (95%CI: 59, 88). Taking medication on the day of symptoms showed higher odds, suggesting responsive behaviour. Taking medication on the day prior to symptoms showed lower odds of reporting, indicating preventative behaviour. Different symptoms in children demonstrate varying dose-response relationships with ragweed pollen concentrations. Each symptom type responded to pollen exposure over different time periods. Using medication prior to symptoms can reduce symptom presence. These findings can be used to better manage paediatric ragweed allergy symptoms., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The present work studied the effect of the year of harvest, the genotype and the cultivation method on the nutritional quality and the allergen content of three plum cultivars. The common quality parameters and the phytochemical content strongly varied with the year and the cultivar, while the system of cultivation had a minor influence. In particular, ascorbic acid greatly decreased in 2016 compared to 2015, while polyphenols were higher in 2016. The health-promoting compounds, and particularly phenolics, were significantly correlated with the antioxidant capacity. Finally, the allergen content was strongly dependent on the content of flavan-3-ols, suggesting that this class of phenolics is determinant in influencing the allergen content in plums. Results showed that the major factor affecting the quality and the concentration of natural metabolites of plum, in addition to the diversity among genotypes, is the year-to-year variation, whereas the system of cultivation plays a marginal role.

Peanut allergic consumers rely on food package labels to avoid foods containing peanut. The inadvertent presence of peanut in foods due to cross-contact can be fatal if ingestion of such food leads to an allergic reaction. Analytical methods are available to detect undeclared peanut in foods. However, depending on the type of food matrix and food processing parameters, method performance can be adversely affected due to reduction in the extraction efficiency of peanut proteins. Temperature and probe sonication were used as a preincubation treatment for peanut flour slurries to assess their effect on the total peanut protein solubility from raw, light-roasted, and dark-roasted peanut flours. The effect of these treatments on the immunoreactivity of peanut allergens (Ara h 1, Ara h 2, Ara h 3, and Ara h 6) was determined by an indirect enzyme-linked immunosorbent assay using antibodies raised against these individual peanut proteins. Preincubation at 50 °C did not significantly improve the peanut protein solubility, whereas an increase in protein solubility was observed when light- and dark-roasted peanut flour slurries were preincubated at 90 °C or sonicated. The immunoreactivity of peanut allergens varied depending on the degree of peanut flour roasting and type of preincubation treatment. Overall, the immunoreactivity of peanut allergens from most peanut flour slurries was unaffected when preincubated at 50 °C for up to 60 min or sonicated with a probe for up to 5 min, whereas preincubation at 90 °C resulted in a time-dependent reduction in immunoreactivity of peanut allergens. Sonication treatment may improve peanut protein extraction without markedly affecting their immunoreactivity. PRACTICAL APPLICATION: Extraction of peanut proteins is vital for developed analytical methods to estimate peanut allergens in foods. The manuscript describes the effect of two different temperatures (50 and 90 °C) and probe-type sonication on peanut protein solubility. The findings suggest sonication can improve peanut protein solubility without markedly affecting their immunoreactivity., (Published 2019. This article is a U.S. Government work and is in the public domain in the USA.)

Approximately 50 million Americans have allergic diseases. Airborne plant pollen is a significant trigger for several of these allergic diseases. Ambrosia (ragweed) is known for its abundant production of pollen and its potent allergic effect in North America. Hence, estimating and predicting the daily atmospheric concentration of pollen (ragweed pollen in particular) is useful for both people with allergies and for the health professionals who care for them. In this study, we show that a suite of variables including meteorological and land surface parameters, as well as next-generation radar (NEXRAD) measurements together with machine learning can be used to estimate successfully the daily pollen concentration. The supervised machine learning approaches we used included random forests, neural networks, and support vector machines. The performance of the training is independently validated using 10% of the data partitioned using the holdout cross-validation method from the original dataset. The random forests (R= 0.61, R 2 = 0.37), support vector machines (R= 0.51, R 2 = 0.26), and neural networks (R= 0.46, R 2 = 0.21) effectively predicted the daily Ambrosia pollen, where the correlation coefficient (R) and R-squared (R 2 ) values are given in brackets. Three independent approaches-the random forests, correlation coefficients, and interaction information-were employed to rank the relative importance of the available predictors.

Millions of people have an allergic reaction to pollen. The impact of pollen allergies is on the rise due to increased pollen levels caused by global warming and the spread of highly invasive weeds. The production, release, and dispersal of pollen depend on the ambient weather conditions. The temperature, rainfall, humidity, cloud cover, and wind are known to affect the amount of pollen in the atmosphere. In the past, various regression techniques have been applied to estimate and forecast the daily pollen concentration in the atmosphere based on the weather conditions. In this research, machine learning methods were applied to the Next Generation Weather Radar (NEXRAD) data to estimate the daily Ambrosia pollen over a 300 km × 300 km region centered on a NEXRAD weather radar. The Neural Network and Random Forest machine learning methods have been employed to develop separate models to estimate Ambrosia pollen over the region. A feasible way of estimating the daily pollen concentration using only the NEXRAD radar data and machine learning methods would lay the foundation to forecast daily pollen at a fine spatial resolution nationally.

Olive pollen is the main cause of pollinosis in Mediterranean countries. The immunological analysis of Ole e 1, the major allergen of Olea europaea, has usually carried out by means of ELISA (Enzyme-Linked ImmunoSorbent Assay). However, most published works only specify the methodology related to antigen quantifications, but not the related to protein extraction. Furthermore, the results obtained are not compared with different buffers or modifications of them. The main aim of this study is to obtain an optimized and reproducible ELISA protocol for quantifications of Ole e 1 in the atmosphere. The study of Ole e 1 allergen and olive pollen in the atmosphere of Malaga (Spain) was carried out by means of an automatic multi-vial cyclonic sampler and a Hirst volumetric pollen trap, respectively. ELISA was tuned up on the basis of previously published protocols to quantify this allergen. Variations in the concentrations of capture and detection antibodies, as well as in the buffers used to carry out the extraction, were evaluated. The highest protein extraction was obtained when a modified buffer was applied. The correlation analysis between daily pollen concentrations and allergen quantifications showed highly significant values. The ELISA protocol, together with the buffer combination proposed in this work, considerably reduced the concentrations of capture and detection antibodies used for quantifying Ole e 1 in the atmosphere, allowing detect this allergen even in days in which the airborne pollen concentration was very low or null., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

To understand the allergenicity of rice bran, the distribution of rice allergens in brown rice grains was analysed and the allergenicity of cosmetics and health foods containing rice bran determined. RAG2 and a 19-kDa globulin were localized in polished rice, while a 52-kDa globulin was localized in rice bran. The 52-kDa globulin was also identified as the most likely causative allergen of rice bran allergy. Several products containing intact rice bran were found to contain the 52-kDa globulin. Our study provides the first data regarding cosmetics and health foods containing potential rice bran allergens. Western blot analysis using a rice-bran-allergic patient's plasma showed that 52-kDa globulin was detected as an IgE-binding protein of rice bran and some rice bran-containing cosmetics and health foods. Our results indicate that patients with rice bran allergy need to be careful about using products containing intact rice bran as a constituent., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Pollen represents the male sexual lineage in flowering plants. At maturity, pollen grains are composed of a companion vegetative cell with embedded sperm. During pollen development, these two cell types acquire vastly differing cell fates. Underlying this differential fate acquisition is dramatic reconfiguration of pollen chromatin that is highly evident at a cytological level. The precise link between histone mark deposition and fate acquisition remains largely unexplored, which in part has been hindered by the difficulty in working with pollen in model plant species like Arabidopsis. Here, we describe a simple and robust protocol to isolate Arabidopsis pollen nuclei and immunostain for histone marks. Plant growth aside, the protocol can be performed over 2 days with few Arabidopsis plants, thus allowing multiple genotypes to be analysed in parallel. We also describe a method to de-mask epitopes through antigen retrieval, which vastly improves the signal for antibodies that target heterochromatic histone marks.

Shuang Huang Lian Injection (SHLI) has been used in China for over 30 years as an effective and widely used Chinese herbal prescription to treat acute respiratory infectious. SHLI has, however, caused many severe anaphylactoid reactions. It is important to identify the potential anaphylactoid components of SHLI. Spectrum-effect relationships were used to explore potentially anaphylactoid components. Based on the original herbal formula, honeysuckle, Fructus Forsythiae and Radix Scutellariae extracts were prepared and combined in appropriate proportions. The preparations were then injected into the caudal vein of rats to obtain in vivo serum samples for pharmacological evaluation and fingerprint analysis. The release rate of β-hexosaminidase from RBL-2H 3 cells and plasma histamine level was used as the pharmacological index. Chromatographic fingerprint analysis identified 22 common peaks. Regression analysis and correlation analysis were used to calculate the relationships between the peaks and the pharmacological effects and identified peaks 5, 6, 11, 12 and 17 as likely anaphylactoid agents. The correlated peaks were identified by comparing the fingerprints with in vitro samples and reference standard samples and the structure was identified by UPLC-TOF-MS. This study established a prospective method to clarify the anaphylactoid components in SHLI, which would provide guidances for screening anaphylactoid components in other traditional Chinese medicine injections., (© 2018 John Wiley & Sons, Ltd.)

Gly m 5.0101, the alpha subunit of β-conglycinin, is one of the major allergens found in soybeans that has been identified as causing an allergic reaction. Here, we developed a quantification method of Gly m 5.0101 with multiple reaction monitoring using the synthetic peptide 194 NPFLFGSNR 202 as the external standard. Firstly, the ground soybean was defatted and extracted with a protein extraction buffer. Then the crude extract was on-filter digested by trypsin and analyzed by liquid chromatography-tandem mass spectrometry. The selected peptide exhibited a detection limit of 0.48 ng/mL and a linear relationship in a concentration range from 1.6 to 500 ng/mL (r² > 0.99). The developed method was successfully applied to quantify the Gly m 5.0101 level in dozens of soybean varieties from different sources and soybean products derived from different processing techniques. The developed method could be used to further analyze β-conglycinin in soybean seeds combined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis.

Lupine is widely used as an ingredient in diverse food products, but it is also a source of allergens. This work aimed at proposing a method to detect/quantify lupine as an allergen in processed foods based on a normalised real-time PCR assay targeting the Lup a 4 allergen-encoding gene of Lupinus albus. Sensitivities down to 0.0005%, 0.01% and 0.05% (w/w) of lupine in rice flour, wheat flour and bread, respectively, and 1 pg of L. albus DNA were obtained, with adequate real-time PCR performance parameters using the ΔCt method. Both food matrix and processing affected negatively the quantitative performance of the assay. The method was successfully validated with blind samples and applied to processed foods. Lupine was estimated between 4.12 and 22.9% in foods, with some results suggesting the common practice of precautionary labelling. In this work, useful and effective tools were proposed for the detection/quantification of lupine in food products., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: The peanut protein Arachis hypogaea (Ara h) 6 is one of the most serious food allergens that contributes to food-related, life-threatening problems worldwide. The extremely low allergic dose demands for more selective and rapid methods for detecting Ara h 6., Objective: The goal of this study was to develop a single-walled carbon nanotube (SWCNT)-based biosensor for the rapid detection of Ara h 6 in commercial food products., Methods: The detection principle of this biosensor was based on the binding of Ara h 6 to the anti-Ara h 6 antibody (pAb) through 1-pyrenibutanoic acid succinimidyl ester. The resistance difference (ΔR) was calculated via linear sweep voltammetry using a potentiostat., Results: The ΔR increased as the Ara h 6 concentrations increased above the range of 100-107 pg/L. A specificity analysis showed that the anti-Ara h 6 pAb selectively interacted with Ara h 6 molecules in the buffer solution (pH 7.4)., Conclusions: This research proposes that an SWCNT-based biosensor in self-assembly with antibodies could be an effective tool for the rapid detection of allergen proteins in food., Highlights: The developed biosensor exhibited higher sensitivity and selectivity. Application studies resulted in precise Ara h 6 detection in peanut-containing processed food.

The goals of this research were to develop a rapid single-walled carbon nanotube (SWCNT)-based biosensor and to employ it to commercial food products for Ara h1 detection. The SWCNT-based biosensor was fabricated with SWCNTs immobilized with antibody (pAb) through hybridization of 1-pyrenebutanoic acid succinimidyl ester (1-PBASE) as a linker. The resistance difference (ΔR) was calculated by measuring linear sweep voltammetry (LSV) using a potentiostat. Resistance values increased as the concentration of Ara h1 increased over the range of 1 to 10 5  ng/L. The specific binding of anti-Ara h1 pAb to antigen including Ara h1 was confirmed by both indirect ELISA kit and biosensor assay. The biosensor was exposed to extracts prepared from commercial processed food containing peanuts, or no peanuts, and could successfully distinguish the peanut containing foods. In addition, the application of present biosensor approach documented the precise detection of Ara h1 concentrations in commercially available peanut containing foods.

The paper reports synthesis of gold-palladium nanowaxberries(AuPd NWs)/dodecylamine-functionalized graphene quantum dots(D-GQDs)-graphene micro-aerogel(GMA). D-GQDs was used as a solid particle surfactant for stabilizing Pickering emulsion of toluene-in-graphene oxide aqueous dispersion. Graphene oxide sheets in the aqueous phase are reduced by hydrazine hydrate, diffused into the toluene droplet and self-assembled into graphene oxide micro-gels. Followed by freeze-drying, thermal annealing and hybridized with AuPd NWs. The as-prepared AuPd NWs/D-GQDs-GMA shows an unique three-dimensional structure with the size of microns. The small size and strong polarity make it can be directly dispersed in ethanol to form stable dispersion for sensor preparation. The hybrid of GMA, D-GQDs and AuPd NWs greatly improves the electron transfer, electroactive surface area and ion diffusion. The architecture of conductor/semiconductor/conductor achieves to a significant amplification of detection signal. The DNA biosensor based on the AuPd NWs/D-GQDs-GMA exhibits ultrasensitive differential pulse voltammetric (DPV) response towards peanut allergen Ara h 1. The DPV signal linearly increases with increasing DNA concentration in the range of 1.0 × 10 -22 -1.0 × 10 -17  M with the detection limit of 4.7 × 10 -23  M (S/N = 3). The analytical method was successfully applied to voltammetric determination of peanut allergen Ara h 1 in peanut milk beverage., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Tyrosine (Tyr) residues of the major pollen allergen of birch Betula verrucosa, Bet v 1a, were nitrated by peroxynitrite. This modification enhances the allergenicity. Modified tyrosines were identified by analyzing intact allergen variants in combination with top-down and bottom-up approaches. Therefore, a laboratory-built sheath-liquid assisted ESI interface was applied for hyphenation of CE to an Orbitrap mass spectrometer to localize individual nitration sites. The major focus was on identification of primary nitration sites. The top-down approach unambiguously identified Tyr 5 as the most prominent modification site. Fragments from the allergen core and the C-terminal part carried up to three potential nitration sites, respectively. Thus, a bottom-up approach with tryptic digest was used as a complementary strategy which allowed for the unambiguous localization of nitration sites within the respective peptides. Nitration propensity for individual Tyr residues was addressed by comparison of MS signals of nitrated peptides relative to all cognates of homolog primary sequence. Combined data identified surface exposed Tyr 5 and Tyr 66 as major nitration sites followed by less accessible Tyr 158 whereas Tyr 81, 83 and 150 possess a lower nitration tendency and are apparently modified in variants with higher nitration levels., (© 2018 The Authors. Electrophoresis published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Here, we produce Ambrosia pollen source inventories for Italy that focuses on the periods before and after the accidental introduction of the Ophraella communa beetle. The inventory uses the top-down approach that combines the annual Ambrosia pollen index from a number of monitoring stations in the source region as well as Ambrosia ecology, local knowledge of Ambrosia infestation and detailed land cover information. The final inventory is gridded to a 5 × 5-km resolution using a stereographic projection. The sites with the highest European Infection levels were recorded in the north of Italy at Busto Arsizio (VA3) (European Infection level 2003-2014 = 52.1) and Magenta (MI7) (European Infection level 2003-2014 = 51.3), whereas the sites with the lowest (i.e. around 0.0) were generally located to the south of the country. Analysis showed that the European Infection level in all of Italy was significantly lower in 2013-2014 compared to 2003-2012, and this decrease was even more pronounced at the sites in the area where Ophraella communa was distributed. Cross-validations show that the sensitivity to the inclusion of stations is typically below 1% (for two thirds of the stations) and that the station Magenta (MI7) had the largest impact compared to all other stations. This is the first time that pollen source inventories from different temporal periods have been compared in this way and has implications for simulating interannual variations in pollen emission as well as evaluating the management of anemophilous plants like Ambrosia artemisiifolia.

Introduction: Respiratory allergies induced by allergenic plants pollen represent an important public health problem with increasing prevalence and severity. Aerobiologic study of allergenic pollens is performed in many countries on regular basis and correlated with health data from allergists in the frame of national aerobiology networks. Romania has no aerobiology network and pollen measurements have been done between 1999-2012 in West region only. In the frame of COST Action called Sustainable management of Ambrosia artemisiifolia in Europe (SMARTER FA 1203), three years collaboration with Reseau National de Surveillance Aerobiologique (RNSA) from France and the first pollen monitoring center in Bucharest were established.The aim of this paper is to present results of first pollen monitoring in Bucharest, activities of Romanian SMARTER group and collaboration with European aerobiology specialists., Material and Method: We used a Hirst-type pollen trap placed on the roof of the Research Center from "Colentina" Clinical Hospital and the pollen monitoring method based on European Aeroallergen Network (EAN) standardized requirements. Monthly results during the pollen seasons 2014-2016 were sent to RNSA and EAN and posted on the European pollen information site., Results: We found high amounts of allergenic pollen, mainly grasses from May to September and Ambrosia artemisiifolia during September. Conlcusions. We concluded that SMARTER offered access to aerobiology training, improved multidisciplinary collaboration and perspectives to further develop national and international projects. More coordinated efforts to develop national aerobiology network and to recuperate the gap comparing to other European countries in the field of aerobiology and respiratory allergology are needed.

A soybean experimental line (BSH-3) devoid of a subset of seed storage proteins was developed by crossing a mutant donor line "HS99B" with a Chinese cultivar "Dongnong47" (DN47). One-dimensional and high-resolution 2-D gel electrophoresis revealed the absence of G1 (A1 a B 2) , G2 (A 2 B1 a) , and G4 (A 5 A 4 B 3) glycinin and the α' subunit of β-conglycinin in BSH-3 seeds. Despite the lack of these abundant seed proteins, BSH-3 seeds still accumulated 38% protein. BSH-3 seeds also accumulated high levels of free amino acids as compared with DN47 seeds, particularly arginine, and the amount of several essential amino acids were significantly elevated in BSH-3 seeds. Elevated accumulation of α and β-subunit of β-conglycinin, G5 glycinin, Kunitz trypsin inhibitor, and Bowman-Birk protease inhibitor indicates seed proteome rebalancing in BSH-3 seeds. Immunoblot analysis using sera from soybean allergic patients demonstrated the complete lack of a major allergen (α' subunit of β-conglycinin) in BSH-3 seeds. However, elevated levels of other allergens were found in BSH-3 seeds due to proteome rebalancing. Transmission electron microscopy observation of mature seeds of BSH-3 revealed striking differences in the appearance of the protein storage vacuoles when compared with DN47.

In the Mediterranean area, lipid transfer proteins (LTPs) are important causes of plant-food allergies often associated with severe allergic reactions. There, peach LTP (Pru p 3) seems to be the primary sensitizer, whereas in Central Europe, little is known about the importance of LTP sensitization. In this region, allergen extract-based diagnosis is often complicated by co-sensitization to Bet v 1, the major birch pollen allergen, its cross-reactive food allergens, and profilins. We investigated the role of LTP sensitization in Central European patients displaying strong allergic reactions to plant-derived food. Analysis of IgE reactivity revealed that ten of thirteen patients were sensitized to Pru p 3, nine to Bet v 1, and two to profilin. Our results showed that LTP sensitization represents a risk factor for severe allergic symptoms in Central Europe. Furthermore, the strong IgE reactivity detected in immunoblots of plant-food extracts indicated that Pru p 3 can be used as a marker allergen for LTP sensitization also in Central European patients., (© 2017 The Authors Allergy Published by John Wiley & Sons Ltd.)

Pollen grains from Phoenix sylvestris (date palm), a commonly cultivated tree in India has been found to cause severe allergic diseases in an increasing percentage of hypersensitive individuals. To unearth its allergenic components, pollen protein were profiled by two-dimensional gel electrophoresis followed by immunoblotting with date palm pollen sensitive patient sera. Allergens were identified by MALDI-TOF/TOF employing a layered proteomic approach combining conventional database dependent search and manual de novo sequencing followed by homology-based search as Phoenix sylvestris is unsequenced. Derivatization of tryptic peptides by acetylation has been demonstrated to differentiate the 'b' from the 'y' ions facilitating efficient de novo sequencing. Ten allergenic proteins were identified, out of which six showed homology with known allergens while others were reported for the first time. Amongst these, isoflavone reductase, beta-conglycinin, S-adenosyl methionine synthase, 1, 4 glucan synthase and beta-galactosidase were commonly reported as allergens from coconut pollen and presumably responsible for cross-reactivity. One of the allergens had IgE binding epitope recognized by its glycan moiety. The allergenic potency of date palm pollen has been demonstrated using in vitro tests. The identified allergens can be used to develop vaccines for immunotherapy against date palm pollen allergy., The Significance of the Study: Identification of allergenic proteins from sources harboring them is essential in developing therapeutic interventions. This is the first comprehensive study on the identification of allergens from Phoenix sylvestris (date palm) pollen, one of the major aeroallergens in India using a proteomic approach. Proteomic methods are being increasingly used to identify allergens. However, since many of these proteins arise from species which are un-sequenced, it becomes difficult to interpret those using conventional proteomics. Date palm being an unsequenced species, the IgE-reactive proteins have been identified using a stratified proteomic workflow incorporating manual de novo sequencing and homology-based proteomics. This study also gives an insight into the presence of glycan nature of the IgE binding epitopes. Five proteins have been found to be common with coconut pollen allergens and presumably responsible for cross-reactivity. These can be used in diagnostics to differentiate patient cohorts allergic to both coconut and date palm pollen from true date palm pollen allergic subjects. This would also determine better specific immunotherapy regimes between the two cohorts. The allergens identified herein have potential towards vaccine development in date palm pollen allergy as well as in enriching the existing catalogue of allergenic proteins., (Copyright © 2017. Published by Elsevier B.V.)

As part of the regulatory approval process in Europe, comparison of endogenous soybean allergen levels between genetically engineered (GE) and non-GE plants has been requested. A quantitative multiplex analytical method using tandem mass spectrometry was developed and validated to measure 10 potential soybean allergens from soybean seed. The analytical method was implemented at six laboratories to demonstrate the robustness of the method and further applied to three soybean field studies across multiple growing seasons (including 21 non-GE soybean varieties) to assess the natural variation of allergen levels. The results show environmental factors contribute more than genetic factors to the large variation in allergen abundance (2- to 50-fold between environmental replicates) as well as a large contribution of Gly m 5 and Gly m 6 to the total allergen profile, calling into question the scientific rational for measurement of endogenous allergen levels between GE and non-GE varieties in the safety assessment.

Background: Oral immunotherapy (OIT) is an investigational therapeutic approach for the treatment of food allergies. Characterization of the drug product used in oral immunotherapy trials for peanut allergy has not been reported., Objective: To quantify relative amounts of the major peanut allergens and microbial load present in peanut flour used in OIT trials and assess whether these parameters change over a 12-month period. We also anticipate that this report will serve as a guide for investigators seeking to conduct OIT trials under Food and Drug Administration-approved Investigational New Drug applications., Methods: Densitometric scanning of Ara h 1 and Ara h 2 resolved on SDS-PAGE gels was used to assess allergen content in peanut flour extracts. Microbial testing was conducted on peanut flour under US Pharmacopeia guidelines for the presence of Escherichia coli, salmonella, yeast, mold, and total aerobic bacteria. In addition, aflatoxin was quantified in peanut flour. Reported results were obtained from 4 unique lots of peanut flour., Results: Relative amounts of the major peanut allergens were similar between different lots of peanut flour and remained stable over a 12-month period. E coli and salmonella were absent from all lots of flour. Yeast, mold, total aerobic bacteria, and aflatoxin were within established US Pharmacopeia guidelines on all lots tested and remained within the criteria over a 12-month period., Conclusions: Peanut flour used as a drug product contains the major peanut allergens and has low levels of potentially harmful microbes. Both these parameters remain stable over a 12-month period., (Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

An analysis technique using LC with tandem MS (MS/MS) has been developed for the determination of buckwheat proteins, including major allergens. A protein solution extracted from buckwheat was reduced, alkylated, and digested by trypsin. Peptide spectra were obtained using full-scan LC-MS/MS analysis, and peptide sequences were determined through a protein search. Nine peptides of the 13S globulin seed storage protein and one peptide of a 16 kDa allergen were selected as the marker peptides, and multiple reaction monitoring conditions were optimized. Using the conditions, different kinds of buckwheat noodles, powders, and other food ingredients were analyzed. As a result, buckwheat samples present all the fragment peaks, whereas other foods, including Sesamum indicum, wheat, and soybeans, are not detected at all. These findings indicate that LC-MS/MS analysis may be applied to the detection of buckwheat food allergens.

Common ragweed (Ambrosia artemisiifolia L.) is a highly allergenic annual ruderal plant and native to Northern America, but now also spreading across Europe. Air pollution and climate change will not only affect plant growth, pollen production and duration of the whole pollen season, but also the amount of allergenic encoding transcripts and proteins of the pollen. The objective of this study was to get a better understanding of transcriptional changes in ragweed pollen upon NO 2 and O 3 fumigation. This will also contribute to a systems biology approach to understand the reaction of the allergenic pollen to air pollution and climate change. Ragweed plants were grown in climate chambers under controlled conditions and fumigated with enhanced levels of NO 2 and O 3 . Illumina sequencing and de novo assembly revealed significant differentially expressed transcripts, belonging to different gene ontology (GO) terms that were grouped into biological process and molecular function. Transcript levels of the known Amb a ragweed encoding allergens were clearly up-regulated under elevated NO 2 , whereas the amount of allergen encoding transcripts was more variable under elevated O 3 conditions. Moreover transcripts encoding allergen known from other plants could be identified. The transcriptional changes in ragweed pollen upon elevated NO 2 fumigation indicates that air pollution will alter the transcriptome of the pollen. The changed levels of allergenic encoding transcripts may have an influence on the total allergenic potential of ragweed pollen., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Approximately 1% of the U.S. population has a peanut allergy. Previous studies that measured peanut protein in house dust support the hypothesis that household peanut consumption may lead to clinical sensitization through transdermal exposure., Objective: The aim of this pilot study was to characterize Ara h2 levels in house dust from homes with and without individuals with peanut allergy., Methods: Household dust was obtained from homes with an individual with peanut allergy and from homes with no individual with peanut allergy. Ara h2 levels were determined by using a monoclonal antibody-based immunoassay with a level of determination of 150 ng per gram of dust. Peanut consumption information was obtained by questionnaire., Results: A total of 85 dust samples were collected: 38 from homes with a individual with peanut allergy and 47 from control homes. The median Ara h2 level in homes with an individual with peanut allergy was 1236 ng/g (interquartile range [IQR], 256-1342 ng/g), whereas the median Ara h2 level in homes without an individual with peanut allergy was 650 ng/g (IQR, 163-2201 ng/g). Ara h2 levels in dust from homes of individuals with peanut allergy were not significantly lower than in dust from control homes. Of the homes with an individual with peanut allergy, 15 reported complete avoidance of peanut in the home (39%). Ara h2 levels in homes that completely avoided peanuts were not significantly lower than Ara h2 levels in homes that did not restrict peanuts (p = 0.531)., Conclusion: Although families may restrict peanuts and peanut products in the home, there was still detectable Ara h2 levels found in homes. Each subject's definition of restriction may vary, there seemed to be peanut protein entering the home, although the protein origin is not known. Possibilities include cross-reactivity with another antigen or transport into the home on some vector. Further investigation of hypotheses regarding cross-reactivity and environmental exposure to Ara h2 is necessary.