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Using the simple purification method of the peanut antigenic proteins which we have developed before, we purified three kinds of allergenic protein from a peanut species and investigated the antigenicity against each protein and cross reactivity with the Balb/c mouse by alum immunization. Ara h1, Ara h2 and Ara h3, which was three kinds of major allergen, were injected in the abdominal cavity of the mouse with aluminum hydroxide gel for immunization. Two weeks later, blood was collected by cardiac puncture, the serum was measured antibody titer by enzyme linked immunosorbent assay. As a result, Ara h1 and h3 antibodies were increased more than Ara h2 antibody in IgG1 and IgG2a subclasses. It is reported that this Ara h2 shows a serious allergic symptom with the allergen of the peanut in human. However, there are increased antibody titer on h1 and h3 in this experiments with using the arum adjuvant. and it seems that it is important for the immunity model of the mouse to establish mouse allergic model of peanut allergens. In addition, it is suggested that the possibility of different antigenicity from human allergy disease to mouse allergic model.

Background Ara h2 sIgE serum levels improve the diagnostic accuracy for predicting peanut allergy, but the use of Ara h2 purified protein as a skin prick test (SPT), has not been substantially evaluated. The fraction of exhaled nitric oxide (FeNO) shows promise as a novel biomarker of peanut allergy. Reproducibility of these measures has not been determined. The aim was to assess the accuracy and reproducibility (over a time-period of at least 12 months) of SPT to Ara h2 in comparison with four predictors of clinical peanut allergy (Peanut SPT, Ara h2 specific Immunoglobulin E (sIgE), Peanut sIgE and FeNO). Methods Twenty-seven children were recruited in a follow-up of a prospective cohort of fifty-six children at least 12 months after an open-labelled peanut food challenge. Their repeat assessment involved a questionnaire, SPT to peanut and Ara h2 purified protein, FeNO and sIgE to peanut and Ara h2 measurements. Results Ara h2 SPT was no worse in accuracy when compared with peanut SPT, FeNO, Ara h2 sIgE and peanut sIgE (AUC 0.908 compared with 0.887, 0.889, 0.935 and 0.804 respectively) for predicting allergic reaction at previous food challenge. SPT for peanut and Ara h2 demonstrated limited reproducibility (ICC = 0.51 and 0.44); while FeNO demonstrated good reproducibility (ICC = 0.73) and sIgE for peanut and Ara h2 were highly reproducible (ICC = 0.81 and 0.85). Conclusions In this population, Ara h2 SPT was no worse in accuracy when compared with current testing for the evaluation of clinical peanut allergy, but had—like peanut SPT—poor reproducibility. FeNO, peanut sIgE and Ara h2 sIgE were consistently reproducible despite an interval of at least 12 months between the repeated measurements. Electronic supplementary material The online version of this article (doi:10.1186/s13223-016-0143-z) contains supplementary material, which is available to authorized users.

This report describes the further development of a peanut allergy model in Brown Norway (BN) rats and in particular the importance of allergen-free breeding of the laboratory animals for the allergen to be used. For this purpose BN rats were bred for 3 generations on soy- and peanut-free feed since it is known that the legumes peanut and soy are cross-reactive. In addition, the effect of cholera toxin (CT), an oral adjuvant often used to increase the sensitivity of food allergy models, was investigated in the BN rat model. BN rats that were bred on both soy- and peanut-free feed could be sensitized orally to peanut (all exposed rats developed peanut-specific IgE, IgG2a and IgG1) and the adjuvant CT could only enhance this sensitization to a limited extent. We also found different protein recognition patterns against purified peanut allergens (Ara h1, Ara h2 and Ara h3) between intraperitoneally (i.p.) and orally sensitized BN rats. Orally sensitized rats recognized all tested allergens whereas i.p. sensitized rats only recognized Ara h1 and Ara h2. Our conclusion is that a model for food allergy should preferably be (A) oral and (B) if possible without the use of adjuvantia. Our model in BN rats unites these preferred characteristics. In addition, we show the importance of dietary control when conducting oral sensitization studies. Special attention must be paid to unscheduled dietary pre-exposure of the animals to the protein under investigation to obtain optimal oral sensitization. © 2006 Elsevier Inc. All rights reserved.

This report describes the further development of a peanut allergy model in Brown Norway (BN) rats and in particular the importance of allergen-free breeding of the laboratory animals for the allergen to be used. For this purpose BN rats were bred for 3 generations on soy- and peanut-free feed since it is known that the legumes peanut and soy are cross-reactive. In addition, the effect of cholera toxin (CT), an oral adjuvant often used to increase the sensitivity of food allergy models, was investigated in the BN rat model. BN rats that were bred on both soy- and peanut-free feed could be sensitized orally to peanut (all exposed rats developed peanut-specific IgE, IgG2a and IgG1) and the adjuvant CT could only enhance this sensitization to a limited extent. We also found different protein recognition patterns against purified peanut allergens (Ara h1, Ara h2 and Ara h3) between intraperitoneally (i.p.) and orally sensitized BN rats. Orally sensitized rats recognized all tested allergens whereas i.p. sensitized rats only recognized Ara h1 and Ara h2. Our conclusion is that a model for food allergy should preferably be (A) oral and (B) if possible without the use of adjuvantia. Our model in BN rats unites these preferred characteristics. In addition, we show the importance of dietary control when conducting oral sensitization studies. Special attention must be paid to unscheduled dietary pre-exposure of the animals to the protein under investigation to obtain optimal oral sensitization. AD - University Maastricht, Department of Health Risk Analysis and Toxicology, Universiteitssingel 50, Postbus 616, 6200 MD Maastricht, Maastricht, The Netherlands; National Institute of Public Health and the Environment, Laboratory for Toxicology, Pathology and Genetics, Bilthoven, The Netherlands.

Background: A number of allergenic proteins in peanut has been described and the relative importance of these allergens is yet to be determined. Objectives: We have investigated the relevance of previously identified peanut allergens in well-characterized peanut-allergic patients by in vitro, ex vivo and in vivo assays. Methods: Thirty-two adult peanut-allergic patients were included based on careful and standardized patient history and the presence of peanut-specific IgE. The diagnosis peanut allergy was confirmed using double-blind placebo-controlled food challenges in 23 patients. Major peanut allergens Ara h1, Ara h2 and Ara h3 were purified from peanuts using ion-exchange chromatography. IgE immunoblotting was performed and IgE-cross-linking capacity was examined by measuring histamine release (HR) after incubating patient basophils as well as passively sensitized basophils with several dilutions of the allergens. Intracutaneous tests (ICTs) using 10-fold dilution steps of the purified allergens and crude peanut extract were performed. Results: Ara h2 was recognized most frequently (26 out of 32) in all tests and induced both positive skin tests and basophil degranulation at low concentrations, whereas Ara h1 and Ara h3 were recognized less frequently and reacted only at 100-fold higher concentrations as analysed with HR and intracutaneous testing (ICT). Next to the three tested allergens, proteins with molecular weights of somewhat smaller than 15 kDa were identified as a IgE-binding proteins on immunoblot in the majority of the patients (20 out of 32). Conclusion: We conclude that Ara h2 is, for our patient group, the most important peanut allergen, and that previously unidentified peanut proteins with molecular weights of somewhat smaller than 15 kDa may be important allergens as well. ICT in combination with basophil-HR and IgE immunoblotting provides insight in the patient specificity towards the individual peanut allergens.

Background: A number of allergenic proteins in peanut has been described and the relative importance of these allergens is yet to be determined. Objectives: We have investigated the relevance of previously identified peanut allergens in well-characterized peanut-allergic patients by in vitro, ex vivo and in vivo assays. Methods: Thirty-two adult peanut-allergic patients were included based on careful and standardized patient history and the presence of peanut-specific IgE. The diagnosis peanut allergy was confirmed using double-blind placebo-controlled food challenges in 23 patients. Major peanut allergens Ara h1, Ara h2 and Ara h3 were purified from peanuts using ion-exchange chromatography. IgE immunoblotting was performed and IgE-cross-linking capacity was examined by measuring histamine release (HR) after incubating patient basophils as well as passively sensitized basophils with several dilutions of the allergens. Intracutaneous tests (ICTs) using 10-fold dilution steps of the purified allergens and crude peanut extract were performed. Results: Ara h2 was recognized most frequently (26 out of 32) in all tests and induced both positive skin tests and basophil degranulation at low concentrations, whereas Ara h1 and Ara h3 were recognized less frequently and reacted only at 100-fold higher concentrations as analysed with HR and intracutaneous testing (ICT). Next to the three tested allergens, proteins with molecular weights of somewhat smaller than 15 kDa were identified as a IgE-binding proteins on immunoblot in the majority of the patients (20 out of 32). Conclusion: We conclude that Ara h2 is, for our patient group, the most important peanut allergen, and that previously unidentified peanut proteins with molecular weights of somewhat smaller than 15 kDa may be important allergens as well. ICT in combination with basophil-HR and IgE immunoblotting provides insight in the patient specificity towards the individual peanut allergens.

Background: Peanuts are a major cause of food allergies both in children as in adults which can induce an anaphylactic shock. The identification and characterization of peanut allergens could lead to more insight into the mechanism and contribute to the improvement of diagnostic tests and treatment for peanut allergy. Objectives: In the present study, the peanut protein- specific immunoglobulin concentrations as well as their recognition of the various peanut proteins or protein subunits was determined in the plasma of peanut-allergic (PA) and non-allergic (NA) individuals. Moreover, two peanut allergens were characterized in more detail to confirm them as the earlier described Ara h1 and Ara h2. Methods: The presence of Ig-binding sites in peanut proteins was studied by immunoblotting assays whereas the concentrations of peanut-specific Ig was determined by ELISA. Results: Peanut proteins were found to contain multiple binding sites for immunoglobulins. Of these proteins, six were recognized by peanut-specific IgE present in more than 50% of the plasma samples of the PA group. Their molecular weights were ≃44, 40, 33, 21,20 and 18 kDa. The last three protein bands were recognized by peanut-specific IgE present in more than 70% of the PA plasma samples and were thought to contain Ara h2. This allergen as well as another protein that was thought to be Ara h1, which was not recognized by the majority of the patients' IgE-containing plasma samples, were isolated and the N terminal amino acid sequence was determined. Peanut protein-specific IgA, IgM, IgG and IgG-subclasses showed a more diverse recognition pattern of peanut protein in the PA group compared to the NA group. No differences were found in the plasma concentrations of peanut protein-specific immunoglobulins of the various classes between the PA and NA group. Conclusions: From the present study, we conclude that peanuts contain multiple allergens, of which six can be described as major allergens, Ara h2 included. In our population Ara h1 is not a major allergen. The recognition of peanut proteins by immunoglobulins is more diverse in PA individuals compared with NA individuals which, however, is not substantiated in the concentrations of peanut-specific immunoglobulins in plasma, other than IgE.

Background: Peanuts are a major cause of food allergies both in children as in adults which can induce an anaphylactic shock. The identification and characterization of peanut allergens could lead to more insight into the mechanism and contribute to the improvement of diagnostic tests and treatment for peanut allergy. Objectives: In the present study, the peanut protein- specific immunoglobulin concentrations as well as their recognition of the various peanut proteins or protein subunits was determined in the plasma of peanut-allergic (PA) and non-allergic (NA) individuals. Moreover, two peanut allergens were characterized in more detail to confirm them as the earlier described Ara h1 and Ara h2. Methods: The presence of Ig-binding sites in peanut proteins was studied by immunoblotting assays whereas the concentrations of peanut-specific Ig was determined by ELISA. Results: Peanut proteins were found to contain multiple binding sites for immunoglobulins. Of these proteins, six were recognized by peanut-specific IgE present in more than 50% of the plasma samples of the PA group. Their molecular weights were ≃44, 40, 33, 21,20 and 18 kDa. The last three protein bands were recognized by peanut-specific IgE present in more than 70% of the PA plasma samples and were thought to contain Ara h2. This allergen as well as another protein that was thought to be Ara h1, which was not recognized by the majority of the patients' IgE-containing plasma samples, were isolated and the N terminal amino acid sequence was determined. Peanut protein-specific IgA, IgM, IgG and IgG-subclasses showed a more diverse recognition pattern of peanut protein in the PA group compared to the NA group. No differences were found in the plasma concentrations of peanut protein-specific immunoglobulins of the various classes between the PA and NA group. Conclusions: From the present study, we conclude that peanuts contain multiple allergens, of which six can be described as major allergens, Ara h2 included. In our population Ara h1 is not a major allergen. The recognition of peanut proteins by immunoglobulins is more diverse in PA individuals compared with NA individuals which, however, is not substantiated in the concentrations of peanut-specific immunoglobulins in plasma, other than IgE.