Investigation of reduced ELISA recovery of almond and hazelnut traces from roasted nut samples by SDS-PAGE and mass spectrometry.
Western society is facing an increase in the number of food-allergic individuals, with rising incidence in the past years. Therefore, allergen-free food and accurate and reliable analysis of allergen contamination are essential for the protection of consumers. Yet, there is limited understanding on the effect of food processing on allergenicity and on the ability of available methods to detect trace contamination in processed food. Available studies addressing this have relied on sample processing on a laboratory scale. In this study, industry-like processing under precisely defined conditions (ranging from 110 to 150°C roasting temperatures) was employed to better understand the limitations of state-of-the-art methods for detecting traces of hazelnut and almond in processed food. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated an overall reduction in extracted proteins from roasted nut samples, and with matrix-assisted laser desorption ionization time-of-flight Cor a 9 and Prunin, were identified as majorly expressed proteins for hazelnut and almond, respectively. A commercial ELISA kit detected nut traces only up to a 130°C roasting temperature. Untargeted MS (Orbitrap) analysis was able to detect traces of nuts roasted up to 150°C while also confirming Cor a 9 and Prunin as the major expressed proteins for hazelnut and almond, respectively. Preparing cookie dough spiked with roasted nut samples, a complex food matrix was simulated. Analysis by ELISA showed the same limitations encountered for pure nuts samples, hardly detecting traces of nuts roasted above 130°C. Targeted MS (linear ion trap) using multiple reaction monitoring methods for one proteotypic peptide for Cor a 9 and Prunin, respectively, enabled a detection of nut traces up to 150°C. The results indicated that a reduced extractability because of temperature-related effects (e.g., protein denaturation, cross-linking, poor solubility) caused the significant differences between the ELISA and MS analysis. Overall, the results of this study may form the basis to improve allergen detection after roasting through improved extraction methods and refined ELISA formats.
Walnut oral immunotherapy for desensitisation of walnut and additional tree nut allergies (Nut CRACKER): a single-centre, prospective cohort study.
BACKGROUND: The safety and efficacy of oral immunotherapy for tree nut allergy has not been demonstrated to date, and its effectiveness is complicated by the high prevalence of co-allergies to several nuts. This study aimed to investigate the use of walnut oral immunotherapy in the desensitisation of walnut and additional tree nuts in patients who are co-allergic to several nuts. METHODS: In a single-centre, prospective cohort study (the Nut Co-Reactivity ACquiring Knowledge for Elimination Recommendations study) at the Institute of Allergy, Immunology, and Paediatric Pulmonology at the Yitzhak Shamir Medical Centre, we recruited patients aged 4 years or older who were allergic to walnut, with or without co-allergy to pecan, hazelnut, and cashew. The diagnosis of each food allergy was based on a positive skin prick test or specific serum IgE (≥0·35 kUA/L) to the corresponding nut together with a positive oral food challenge, unless an immediate (within 2 h of exposure) reaction in the past year had been documented. Patients with uncontrolled asthma or a medical contraindication to receive adrenaline were excluded. Patients were assigned to walnut oral immunotherapy or the control group (observation and strict dietary exclusion) on the basis of the order of presentation to the clinic. Oral immunotherapy began with a 4-day dose-escalation phase to establish the single highest tolerated dose, which was consumed daily at home for 24 days; subsequent monthly dose escalations were repeated until 4000 mg walnut protein was achieved. Patients who were desensitised to walnut continued to consume 1200 mg walnut protein daily for 6 months as maintenance. The primary outcome was walnut desensitisation (passing an oral food challenge with 4000 mg of walnut protein) at the end of the study, analysed by intention to treat. In patients who were co-allergic to pecan, hazelnut, and cashew, the proportion who achieved cross-desensitisation to these nuts in addition to walnut desensitisation was examined. FINDINGS: 73 patients with a walnut allergy were enrolled between May 15, 2016, and Jan 14, 2018. 49 (89%) of 55 patients in the oral immunotherapy group were desensitised to walnut compared with none of 18 patients in the control group (odds ratio 9·2, 95% CI 4·3-19·5; p<0·0001). Following walnut desensitisation, all patients who were co-allergic to pecan (n=46) were also desensitised to pecan. Additionally, 18 (60%) of 30 patients who were co-allergic to hazelnut or cashew, and 14 (93%) of 15 patients who were co-allergic to hazelnut alone, were either fully desensitised or responded to treatment. 47 (85%) of 55 patients had an adverse reaction (mostly grade 1 or 2) during up-dosing in the clinic; eight patients required intramuscular epinephrine in response to a dose at home. Of 45 patients who had follow-up data for the maintenance phase, all maintained walnut desensitisation and one patient required epinephrine during this period. INTERPRETATION: Walnut oral immunotherapy can induce desensitisation to walnut as well as cross-desensitisation to pecan and hazelnut in patients who have tree nut co-allergies, with a reasonable safety profile. A low daily dose of the allergen maintains desensitisation.
Lipophilic allergens, different modes of allergen-lipid interaction and their impact on asthma and allergy.
Molecular allergology research has provided valuable information on the structure and function of single allergenic molecules. There are several allergens in food and inhalant allergen sources that are able to interact with lipid ligands via different structural features: hydrophobic pockets, hydrophobic cavities, or specialized domains. For only a few of these allergens information on their associated ligands is already available. Several of the allergens are clinically relevant, so that it is highly probable that the individual structural features with which they interact with lipids have a direct effect on their allergenic potential, and thus on allergy development. There is some evidence for a protective effect of lipids delaying the enzymatic digestion of the peanut (Arachis hypogaea) allergen Ara h 8 (hydrophobic pocket), probably allowing this molecule to get to the intestinal immune system intact (sensitization). Oleosins from different food allergen sources are part of lipid storage organelles and potential marker allergens for the severity of the allergic reaction. House dust mite (HDM), is more often associated with allergic asthma than other sources of inhalant allergens. In particular, lipid-associated allergens from Dermatophagoides pteronyssinuswhich are Der p 2, Der p 5, Der p 7, Der p 13, Der p 14, and Der p 21 have been reported to be associated with severe allergic reactions and respiratory symptoms such as asthma. The exact mechanism of interaction of these allergens with lipids still has to be elucidated. Apart from single allergens glycolipids have been shown to directly induce allergic inflammation. Several-in parts conflicting-data exist on the lipid (and allergen) and toll-like receptor interactions. For only few single allergens mechanistic studies were performed on their interaction with the air-liquid interface of the lungs, in particular with the surfactant components SP-A and SP-D. The increasing knowledge on protein-lipid-interaction for lipophilic and hydrophobic food and inhalant allergens on the basis of their particular structure, of their capacity to be integral part of membranes (like the oleosins), and their ability to interact with membranes, surfactant components, and transport lipids (like the lipid transfer proteins) are essential to eventually clarify allergy and asthma development.
The Influence of Gamma Radiation Processing on the Allergenicity of Main Pistachio Allergens.
BACKGROUND: Gamma irradiation is a form of processing with an array of applications in medical sciences such as microbial decontamination, viruses inactivation, cervical carcinoma and breast cancer treatment. One of the ways in which gamma irradiation has the potential to be used is in reducing the allergenicity of food allergens. METHODS: In the present study, pistachios were irradiated with either a 1, 10, or 100 kGy dose of gamma irradiation. The binding rate of mice and human antibodies to the allergens of the pistachio extracts were examined via Western blot analysis. RESULTS: Our findings show an inverse dose-response relationship between the binding rate of antibodies to the pistachio allergens and the gamma irradiation dose. Despite these promising findings, the results of our sensory evaluation indicate that gamma irradiation causes undesirable changes to the sensory characteristics of pistachios, especially at the dose of 100 kGy. CONCLUSION: Gamma irradiation appears to be an effective method in reducing the allergenicity of pistachios. Thus, this form of processing has the potential to prevent adverse allergic reactions to the major pistachio allergens in sensitized subjects. However, further research must be dedicated to examining the dose sufficient in reducing allergencity, while maintaining adequate sensory quality for satisfactory consumption.
Preventing peanut allergy: where are we now?
Peanut allergy affects 1% to 3% of the Western world, usually begins in early childhood, is rarely outgrown, and has no currently approved treatment. The identification and application of prevention strategies is therefore essential. In 2015, the Learning Early About Peanuts study findings found that early consumption of peanut protein was effective in preventing peanut allergy in high-risk children as compared with peanut avoidance. These findings resulted in changes to allergy prevention guidelines and policy across the world. There are country-specific variations to guidelines, but, within these variations, feeding peanut to children in infancy is a common theme. There are numerous logistical challenges surrounding the implementation of contemporary guidelines at a population level. In the United States, guidelines advise according to risk level with prescreening recommended for high-risk children (mod/severe eczema, egg allergy). Even though high-risk children represent the minority of the childhood population, there are still significant challenges associated with identifying and screening such infants. The need for conducting allergy testing before first giving peanut protein to high-risk infants is debated; although adopting this approach promotes safety, it is financially and logistically challenging. Clinical trials that explore the real-life application of these guidelines are needed as is an assessment of guidelines (Australia, for example) that do not adopt the approach of screening.
Sensitivity, specificity, and predictive value of oral mucosal brush biopsy for the diagnosis of peanut allergy.
BACKGROUND: Current diagnostic testing methods for peanut allergy, including serum specific immunoglobulin E (sIgE) and skin-prick testing (SPT), have low specificity, whereas oral food challenge (OFC) carries significant risk of provoking adverse events. Mucosal brush biopsy (MBB) from the oral cavity is currently being studied as a new diagnostic test for peanut allergy, but normative data is not yet available with which to begin measuring specificity and predictive value. METHODS: Twenty individuals with no history of adverse reactions from eating peanuts underwent oral MBB and serum testing for peanut sIgE. These data were then compared with previously published data from 10 individuals with a history of clinical peanut allergy, in order to generate receiver operating characteristic (ROC) curves and calculate the sensitivity, specificity, and positive and negative predictive values for both testing modalities. RESULTS: The optimal cutoff levels for oral MBB and sIgE testing were 0.12 kU/L and 1.0 kU/L, respectively. At 0.12 kU/L, the sensitivity of oral MBB testing was 80% and the specificity was 85%, whereas at 1.0 kU/L, the sensitivity of sIgE testing was 50% and the specificity was 100%. From the ROC curves, the areas under the ROC curve (AUC) for oral MBB and sIgE were 0.91 (p < 0.001) and 0.74 (p = 0.007), respectively. Combination testing further increased both sensitivity and accuracy over oral MBB alone. CONCLUSION: In this pilot study, oral MBB demonstrated high sensitivity and specificity for screening individuals with and without oral cavity clinical reactivity to peanuts and may represent a potentially useful testing method for the diagnosis of peanut allergy in the future.
Peanut Allergy in Spanish Children: Comparative Profile of Peanut Allergy versus Tolerance.
BACKGROUND: Peanut storage proteins (Ara h 1, Ara h 2, and Ara h 3) have been described as the major peanut allergens in children, although not all peanut-sensitized individuals have clinical reactivity after exposure. OBJECTIVES: We studied the sensitization profile of peanut-allergic and peanut-tolerant children in a pediatric cohort. METHODS: The clinical features and sensitization profile to the peanut storage proteins Ara h 9 and Pru p 3 were compared between peanut-allergic and peanut-tolerant children using component-resolved diagnostics. RESULTS: Thirty-three peanut-sensitized children were included: 22 allergic and 11 tolerant patients. Seventy-two percent of the peanut-allergic children were sensitized to at least one peanut storage protein. The rates of sensitization to Ara h 1, Ara h 2, and Ara h 3 were 63.6, 68.1, and 68.1%, respectively, among the peanut-allergic children and 27.2, 18.1, and 45.4% among the peanut-tolerant children. IgE from the sera of 18% of the peanut-allergic patients recognized Ara h 9, whereas no sensitization to Ara h 9 was detected in the peanut-tolerant children. A total of 59% of the peanut-allergic and 27% of the peanut-tolerant children were sensitized to Pru p 3. Sensitization to Ara h 1 and Ara h 2 was more frequent among the peanut-allergic children (p < 0.05). Although the levels of specific IgE against peanut storage proteins were higher in peanut allergy, there were not statistically significantly different from the levels in peanut tolerance, probably due to the small number of patients included. CONCLUSIONS: In our population, the peanut-allergic children were mainly sensitized to peanut storage proteins, and Ara h 2 sensitization allows a more accurate diagnosis of clinical reactivity to peanuts. More than half of the peanut-allergic patients were sensitized to peach Pru p 3, and 50% of them had fruit allergy at the time of the study.
Nut sensitization profile in Southern Taiwan.
BACKGROUND/PURPOSE: To evaluate the relationship between serum-specific immunoglobulin E (IgE) to peanuts/tree nuts and their clinical manifestations in atopic diseases. METHOD: Serum from people with the classical symptoms of asthma, allergic rhinitis (AR), or atopic dermatitis (AD) was collected for the measurement of serum-specific IgE to peanuts, cashew nuts, Brazil nuts, almonds, and coconuts. Cases with possible sensitization to these nuts (serum specific IgE ≧ 0.35 kU/L) were selected and their clinical relationships with physician-diagnosed asthma, allergic rhinitis, or atopic dermatitis were analyzed. RESULT: Compared with non-sensitization group, people with peanut/tree nut sensitization have higher prevalence of atopic dermatitis, but no such difference noted in the prevalence of allergic rhinitis. In the situation of asthma, people with sensitization to peanuts and Brazil nuts, but not other nuts, have higher prevalence of asthma than people without sensitization to any nut (p < 0.001 and p < 0.05, respectively). Binary logistic regression analysis also showed positive associations between peanut (OR: 1.164, p value = 0.017) and Brazil nut (OR: 1.304, p value = 0.055) sensitization and asthma. The associations between peanut and Brazil nut sensitization and asthma were independent of the prevalence of other atopic diseases. CONCLUSION: People in Asia may have less severe allergic effects as in Western countries, but sensitization to specific food allergens such as peanuts or Brazil nuts may predispose individuals to asthma, which could be helpful in diagnosis and deserves more attention than previously considered.
The functional biology of peanut allergens and possible links to their allergenicity
Peanut is one of the most common food triggers of fatal anaphylaxis worldwide although peanut allergy affects only 1-2% of the general population. Peanuts are the source of highly potent allergenic proteins. It is emerging that the allergenicity of certain proteins is linked to their biological function. Peanut is an unusual crop in that it flowers above ground but produces its seed-containing pods underground. This so called geocarpic fruiting habit exposes pods and seeds during their development to soilborne pathogens and pests. Pest damage can also open routes of entry for opportunistic fungi such as Aspergillus. Although seed proteins have primary functions in nutrient reservoirs, lipid storage bodies, or the cytoskeleton, they have also evolved to act as part of the plant's defense system to enhance fitness and survival of the species. When interacting with pathogens or pests, these proteins modify and damage cells' membranes, interact with immune receptors and modulate signaling pathways. Moreover, following exposure, the immune system of predisposed individuals reacts to these proteins with the production of specific IgE. This review explores the evolutionary biology of peanut and its seed proteins and highlights possible links between the proteins' biological function and their allergenicity.
Rapid Antibody Selection Using Surface Plasmon Resonance for High-Speed and Sensitive Hazelnut Lateral Flow Prototypes.
Lateral Flow Immunoassays (LFIAs) allow for rapid, low-cost, screening of many biomolecules such as food allergens. Despite being classified as rapid tests, many LFIAs take 10⁻20 min to complete. For a really high-speed LFIA, it is necessary to assess antibody association kinetics. By using a label-free optical technique such as Surface Plasmon Resonance (SPR), it is possible to screen crude monoclonal antibody (mAb) preparations for their association rates against a target. Herein, we describe an SPR-based method for screening and selecting crude anti-hazelnut antibodies based on their relative association rates, cross reactivity and sandwich pairing capabilities, for subsequent application in a rapid ligand binding assay. Thanks to the SPR selection process, only the fast mAb (F-50-6B12) and the slow (S-50-5H9) mAb needed purification for labelling with carbon nanoparticles to exploit high-speed LFIA prototypes. The kinetics observed in SPR were reflected in LFIA, with the test line appearing within 30 s, almost two times faster when F-50-6B12 was used, compared with S-50-5H9. Additionally, the LFIAs have demonstrated their future applicability to real life samples by detecting hazelnut in the sub-ppm range in a cookie matrix. Finally, these LFIAs not only provide a qualitative result when read visually, but also generate semi-quantitative data when exploiting freely downloadable smartphone apps.