Purification and immunoglobulin E-binding properties of peanut allergen Ara h 6: evidence for cross-reactivity with Ara h 2 (original) (raw)
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Processing Can Alter the Properties of Peanut Extract Preparations
Journal of Agricultural and Food Chemistry, 2010
As peanut allergy is an increasing public health risk, affecting over 1% of the United States and United Kingdom school children, it is important that methods and reagents for accurate diagnosis of food allergy and detection of allergenic foods are reliable and consistent. Given that most current experimental, diagnostic, and detection tests rely on the presence of soluble allergens in food extracts, we investigated the effects of thermal processing on the solubility and IgE binding of the major peanut allergens, Ara h 1 and Ara h 2. The soluble and insoluble fractions of peanuts that were boiled, fried, and roasted were subjected to electrophoresis and Western blot analysis using anti-Ara h 1 and anti-Ara h 2 antibodies and serum IgE from peanut allergic individuals. Overall protein solubility is reduced with processing and IgE binding increases in the insoluble fractions, due mostly to the increase in the amount of insoluble proteins, with increased time of heating in all processes tested. Therefore, it can be concluded that thermal processing of peanuts alters solubility, and the differences in protein solubility within various extract preparations may contribute to inconsistent skin prick test and immunoassay results, particularly when nonstandardized reagents are used.
Increasing the Solubility and Recovery of Ara h3 Allergen from Raw and Roasted Peanut
Nutrition in Health and Disease - Our Challenges Now and Forthcoming Time, 2019
Ara h3 belongs to the glycinin family of seed storage proteins and is one of the major peanut allergens. It comprises over 20% of the total peanut protein mass, making it a logical target for the detection of trace quantities of undeclared peanut contamination in foods. Both Ara h1 and Ara h3 are detected in lower quantities in cooked foods, either because of the failure to completely resolubilize the denatured proteins or because of the disruption of conformational epitopes required for monoclonal antibody recognition. A new reagent containing a proprietary nondetergent sulfobetaine (NDSB) is described which solubilizes more total protein and yields more Ara h3 protein from both raw and roasted peanut than other commonly used ELISA-compatible reagents.
Sequential Extractions: A New Way for Protein Quantification, Data from Peanut Allergens
Analytical biochemistry, 2015
Quantification of certain protein contents in the matrix is essential in protein analyses. The amount of total protein in the matrix can be determined by Kjeldahl method. However, few methods can quantify certain protein contents in the matrix without extracting all of it in solution. Extracting all of the content is difficult for proteins, especially relatively insoluble ones. A five-step sequential extraction method was developed for the quantification of certain proteins in defatted peanut flour based on the relationship between the extracted protein content and the extraction times. The extracted proteins (i.e., total protein, Ara h 1, and Ara h 2) were quantitatively analyzed in each extraction of the same condition. An exponential equation was obtained between the extraction times and the respective amount of extracted protein, as well as both the total protein and a particular protein. In particular, the amount of protein extracted each time can be a geometric sequence. If al...
Allergen Extraction: Factors Influencing the Yield, Allergenicity and Sensitivity of Immunoassays
Food allergy is an IgE and or IgG immune-mediated reaction to food antigens. Knowledge of the allergenicity properties of proteins, how they react in the body and in diagnostic tests is necessary to adequately assess the allergenic potential of both natural foods and those produced through biotechnological processes. Thus, our aim was analyze the factors those influence the protein extraction of foods in terms of yield, allergenicity and sensitivity in immunoassays. Peanut proteins were extracted using 4 distinct extraction buffers (physiological saline, tris buffer, borate buffer with and without β-mercaptoethanol). The protein concentration of the obtained extracts was determined by the Lowry method. Polyacrylamide electrophoresis (SDS-PAGE) was used to compare the protein profile of each extract. Immunogenicity of each extract was verified by sensitizing two mouse strains (Balb/c and C57/BL6) with 100μg of the proteins, and the immunoreactivity was determined by ELISA. Our result...
Peanut varieties with reduced Ara h 1 content indicating no reduced allergenicity
Molecular Nutrition & Food Research, 2010
Peanut allergy is a major cause of food-induced severe anaphylactic reactions. To date, no medical care is available to prevent and treat peanut allergy and therefore hypoallergenic peanut varieties are of considerable health political and economic interest. Major allergens that induce IgE-responses in peanut-sensitive patients are Ara h 1, Ara h 2 and Ara h 3/4. In order to identify hypoallergenic peanuts, commercially locally available peanut varieties were screened for their allergen content. Ara h 1-deficient peanuts from Southeast Asia were identified by SDS-PAGE, immunoblotting, inhibition assays and ELISA. 2-D PAGE analyses demonstrated the different compositions of the tested extracts and revealed a number of variations of the allergen patterns of peanuts from different varieties. Mediator release experiments of these peanut extracts demonstrated similar allergenicities as compared with standard peanut extract. These results indicate that the allergenicity of peanuts with reduced Ara h 1 content might be compensated by the other allergens, and thus do not necessarily cause a reduction of allergenicity.
Effector activity of peanut allergens: a critical role for Ara h 2, Ara h 6, and their variants
Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology, 2009
Rationale An important property of allergens is their ability to cross-link IgE and activate mast cells and basophils. The effector activity of peanut allergens has not been well characterized.Methods Crude extracts of fresh peanut flour were fractionated by gel filtration. Effector function was assayed by measuring degranulation of RBL SX-38 cells sensitized with IgE from individual sera and from pools of sera of peanut-allergic donors.Results Following gel filtration, 75 ± 7% of the applied protein and 76 ± 16% (n=3) of the applied activity (assayed with a pool of 11 sera) were recovered in the resultant fractions. The majority (85 ± 2%; n=3) of the recovered activity resided in a fraction with a theoretical average molecular weight of ∼20 kDa and a range of 13–25 kDa. When all the individual fractions were recombined, the measured activity was similar to that of the original extract [140 ± 43% when measured with a pool of serum (n=2) and 66 ± 7% when measured with individual sera (n=4)]; when all individual fractions excluding the 20 kDa fraction were recombined, the measured activity was only 8 ± 2% (n=2) of the original extract when assayed with the serum pool and 10 ± 4% (n=3) when assayed with the individual sera. Two-dimensional gel electrophoresis of this biologically active fraction revealed >60 protein spots. Analysis of 50 of the most prominent spots by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry and of the full mixture by automated tandem mass spectrometry coupled to online capillary liquid chromatography revealed that >97% of the protein mass consisted of Ara h 2.0101, Ara h 2.0201, Ara h 6 isoforms, and variants of these proteins.Conclusions Ara h 2 and Ara h 6 account for the majority of the effector activity found in a crude peanut extract.
Food chemistry, 2017
The extraction of Ara h 6 (a peanut allergen) from a complex chocolate-based food matrix was optimized by testing different temperatures, extraction times, and the influence of additives (NaCl and skimmed milk powder) in a total of 36 different conditions. Analyses were carried out using an electrochemical immunosensor. Three conditions were selected since they allowed the extraction of the highest levels of Ara h 6. These extractions were performed using 2g of sample and 20ml of Tris-HNO3 (pH=8) containing: a) 0.1M NaCl and 2g of skimmed milk powder at 21°C for 60min; b) 1M NaCl and 1g of skimmed milk powder at 21°C for 60min; and c) 2g of skimmed milk powder at 60°C for 60min. Recoveries were similar or higher than 94.7%. This work highlights the importance to adjust extraction procedures regarding the target analyte and food matrix components.
Boiling peanut Ara h 1 results in the formation of aggregates with reduced allergenicity
Molecular Nutrition & Food Research, 2011
Roasting rather than boiling and Maillard modifications may modulate peanut allergenicity. We investigated how these factors affect the allergenic properties of a major peanut allergen, Ara h 1. Methods and results: Ara h 1 was purified from either raw (N-Ara h 1) or roasted (R-Ara h 1) peanuts. Boiling (1001C 15 min; H-Ara h 1) resulted in a partial loss of Ara h 1 secondary structure and formation of rod-like branched aggregates with reduced IgE-binding capacity and impaired ability to induce mediator release. Glycated Ara h 1 (G-Ara h 1) formed by boiling in the presence of glucose behaved similarly. However, Hand G-Ara h1 retained the T-cell reactivity of N-Ara h 1. R-Ara h 1 was denatured, comprised compact, globular aggregates, and showed no evidence of glycation but retained the IgE-binding capacity of the native protein. Conclusion: Ara h 1 aggregates formed by boiling were morphologically distinct from those formed by roasting and had lower allergenic activity. Glycation had no additional effect on Ara h 1 allergenicity compared with heating alone. Taken together with published data on the loss of Ara h 2/6 from boiled peanuts, this supports the hypothesis that boiling reduces the allergenicity of peanuts.