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Background: An in vitro elicitation test employing human high-affinity IgE receptor-expressing rat mast cell lines appears to be a useful method for measuring mast cell activation using a patient's IgE and an allergen; however, such cell lines are sensitive to human complements in the serum. We have recently developed a new luciferase-reporting mast cell line (RS-ATL8) to detect IgE crosslinking-induced luciferase expression (EXiLE) with relatively low quantities of serum IgE., Methods: A total of 30 patients suspected of having egg white (EW) allergy were subjected to an oral food challenge (OFC) test; then, the performances of EW-specific serum IgE (CAP-FEIA), EW-induced degranulation, and EXiLE responses in RS-ATL8 cells were compared using receiver-operating characteristic (ROC) curve analysis. The patients' sera were diluted to 1 : 100, which causes no cytotoxicity when sensitizing the RS-ATL8 cells for the degranulation and EXiLE tests., Results: The area under the ROC curves was highest in the EXiLE test (0.977), followed by CAP-FEIA (0.926) and degranulation (0.810). At an optimal cutoff range (1.648-1.876) calculated from the ROC curve of the EXiLE test, sensitivity and specificity were 0.944 and 0.917, respectively. A 95% positive predictive value was given at a cutoff level of 2.054 (fold increase in luciferase expression) by logistic regression analysis., Conclusions: In contrast to in vivo tests, the EXiLE test appears to be a useful tool in diagnosing patients suspected of having IgE-dependent EW allergy without the risk of severe systemic reactions.

To produce crops that are more tolerant to stresses such as heat, cold, and salt, transgenic plants have been produced those express stress-associated proteins. In this study, we used immunoproteomic and two-dimensional difference gel electrophoresis (2D-DIGE) methods to investigate the allergenicity of transgenic potatoes expressing Arabidopsis DREB1A (dehydration responsive element-binding protein 1A), driven by the rd29A promoter or the 35S promoter. Immunoproteomic analysis using sera from potato-allergic patients revealed several immunoglobulin E (IgE)-binding protein spots. The patterns of protein binding were almost the same between transgenic and non-transgenic potatoes. The IgE-binding proteins in potato were identified as patatin precursors, a segment of serine protease inhibitor 2, and proteinase inhibitor II by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) MS/MS. 2D-DIGE analysis revealed several differences in protein expression between non-transgenic potato and transgenic potato; those showing increased expression in transgenic potatoes were identified as precursors of patatin, a major potato allergen, and those showing decreased expression in transgenic potatoes were identified as lipoxygenase and glycogen (starch) synthase. These results suggested that transgenic potatoes may express slightly higher levels of allergens, but their IgE-binding patterns were almost the same as those of control potatoes. Further research on changes in protein expressions in response to environmental factors is required to confirm whether the differences observed in this study are due to gene transfection, rather than environmental factors.

Background: The buckwheat 16-kDa protein (BWp16), as reported in our previous study, is a major allergen in buckwheat; however, the IgE-binding epitopes of BWp16 have not as yet been identified., Methods: We screened candidates for IgE-binding epitopes on BWp16 by using arrays of overlapping peptides synthesized on activated cellulose membranes (SPOTs membrane). The mimotope method was also used to analyze IgE-binding epitopes of BWp16. Nine single alanine (Ala) mutants of BWp16 expressed in Escherichia coli were used to confirm the epitopes of BWp16. The IgE-binding activity of single Ala mutants of BWp16 was determined by ELISA with mouse anti-BWp16 polyclonal antiserum or ELISA inhibition with sera from buckwheat allergic patients., Results: The SPOTs assay identified amino acid residues 99-110, i.e. EGVRDLKELPSK, as a candidate for the linear IgE-binding epitope of BWp16. The mimotope method indicated that peptides similar to EGVRDLKE were candidate sequences for epitopes of BWp16. Ala scanning of rBWp16 revealed that all EGVRDLKE peptides containing a single amino acid mutation had weaker IgE-binding activity than rBWp16 WT. An ELISA inhibition assay for rBWp16 WT revealed the inhibitory effect of rBWp16 D103A to be less than that of rBWp16 WT., Conclusions: We identified the peptide EGVRDLKE as a very likely candidate for the IgE-binding epitope of BWp16, and Asp103 as the critical amino acid in BWp16. This is the first report on the identification of IgE-binding epitopes of BWp16. Our findings will contribute to the production of BWp16 hypoallergens, and to allergen-specific immunotherapy for buckwheat allergy., (Copyright © 2010 S. Karger AG, Basel.)

Abstract: Plants may trigger hypersensitivity reactions when individuals with allergies consume foods derived from plant materials or inhale plant pollen. As each plant food or pollen contains multiple allergens, proteomics is a powerful tool to detect the allergens present. Allergen-targeted proteomics, termed allergenomics, has been used for comprehensive identification and/or quantification of plant allergens, because it is a simple and inexpensive tool for rapid detection of proteins that bind to IgE. There are increasing numbers of reports on the applications of allergenomics. In this review, we outline some of the applications of proteomics, including: (i) identification of novel allergens, (ii) allergic diagnoses, (iii) quantification of allergens, and (iv) natural diversity of allergens, and finally discuss (v) the use of allergenomics for safety assessment of genetically modified (GM) plants. Biological significance: Recently, the number of allergic patients is increasing. Therefore, a comprehensive analysis of allergens (allergenomics) in plants is highly important for not only risk assessment of food plants but also diagnosis of allergic symptoms. In this manuscript, we reviewed the recent progress of allergenomics for identification, quantification and profiling of allergens. This article is part of a Special Issue entitled: Translational Plant Proteomics. [Copyright &y& Elsevier]

Background: We performed an in vitro elicitation test to de-termine the ability of different types of wheat-allergic pa-tients' IgE to induce humanized mast cell activation after the addition of various time-treated acid-hydrolyzed wheat pro-teins (HWPs). Methods: The reactivity of heat- and various time-treated acid-hydrolyzed glutens (acid-HGs) and com-mercial acid-HWP (HWP1), using serum IgE from wheat al-lergy accompanied by skin and rhinoconjunctlval sensitiza-tion to HWP1 in the facial soap, pediatric subjects with food allergy to native wheat, adult wheat-dependent exercise-in-duced anaphylaxis subjects, and nonatopic healthy subjects, was elucidated by dot blot and a luciferase assay-based in vitro elicitation test (EXiLE test). Results: Serum from sub-jects sensitized with HWP1 reacted only to acid-HGs (acid-HGs treated for 0.5-3 or 6 h), but not native gluten, in the results of the dot blot. In contrast, sera from pediatric sub-jects sensitized with native wheat reacted to native gluten more strongly and showed only slight reactions to 0.5- to 1-hour-treated acid-HGs. The results of the in vitro elicitation test showed that acid hydrolyzation of the gluten attenuat-ed antigen-induced luciferase expression in a time-depen-dent manner for sera from native-wheat-sensitized pediatric subjects. On the other hand, in the sera from HWP1-sensi-tized subjects, acid hydrolyzation of the gluten for 0.5 h dra-matically increased luciferase expression. Conclusions: Even after prolonged hydrolyzation, acid-HGs still retained the ability to activate mast cells in the case of HWP1-sensitized subjects. [ABSTRACT FROM AUTHOR]

Abstract: Salt-soluble proteins extracted from non-transgenic and transgenic rice were evaluated for the presence of known and potential allergens by proteomic techniques. The salt-soluble proteins were extracted, separated by 1D and 2D electrophoresis, and analyzed by Western blotting. 1D immunoblot analysis with patients’ sera revealed few qualitative differences between the IgE-binding proteins of the non-transgenic and transgenic rice. 1D immunoblot with antigen-specific-animal sera revealed no qualitative or quantitative differences in two known allergens, RAG2 and glyoxalase I, between non-transgenic and transgenic rice. Multiple spots containing known and novel IgE-binding proteins were detected among the salt-soluble proteins of non-transgenic rice by 2D immunoblotting. Two globulin-like proteins, a 52kDa protein and a 63kDa protein, were identified as novel IgE-binding proteins that are candidates for rice allergens. These globulin-like proteins were homologous to Cupin superfamily allergens. Quantitative analysis of 19, 52, and 63kDa globulins with protein-specific-animal sera showed no significant differences in the expression of these proteins between the transgenic rice and non-transgenic rice. These results indicate that none of the known or novel endogeneous IgE-binding proteins detected in this study appear to be altered by genetic modification. [Copyright &y& Elsevier]

Abstract: Genetically modified (GM) foods must be tested for safety, including by allergenicity tests to ensure that they do not contain new allergens or higher concentrations of known allergens than the same non-GM foods. In this study experimentally developed EGFP-transgenic chickens were used and evaluated the allergenicity of meat from the chicken based on a serological and two-dimensional difference gel electrophoresis (2D-DIGE) analysis. For the serological analysis, a Western blotting with allergen-specific antibodies and a proteomic analysis of chicken meat allergens with patients’ sera, a so-called allergenome analysis, were used. The allergenome analysis allowed us to identify five IgE-binding proteins in chicken meat, including a known allergen, chicken serum albumin, and no qualitative difference in their expressions between the GM and non-GM chicken meat was found. Results of the 2D-DIGE analysis showed that none of the IgE-binding proteins in chicken meat were significantly changed in expression levels between non-GM and GM chicken, and only 3 of the 1500 soluble protein spots including green fluorescence protein were markedly different as a result of gene transfer. These above results showed that the combination of serological and 2D-DIGE analysis is a valid method of evaluating quality and quantity of allergens in GM foods. [Copyright &y& Elsevier]

Abstract: The 2D-DIGE (2-Dimensional Fluorescence Difference Gel Electrophoresis) method was applied to proteomic phenotyping of natural variants in 10 varieties of rice (Nipponbare, Koshihikari, Sasanishiki, Akitakomachi, Hitomebore, Hinohikari, Kasalath, Rexark, Bleiyo, Cho-ko) from the world rice collection (WRC) in the GenBank of the National Institute of Agrobiological Sciences (NIAS), Japan. Salt-soluble protein extracts of Nipponbare brown rice were labeled with Cy2 fluorochrome and used as an internal standard. Protein extracts from nine other rice varieties were labeled with Cy3 or Cy5 fluorochrome and applied to 2D-PAGE (13cm gel length) analysis. Approximately 700 spots of rice proteins were observed. Fluorescence intensities of each of these spots for the nine rice varieties were expressed as relative ratios to that of Nipponbare. Statistical analysis revealed the spot numbers of five Japanese rice varieties above threshold five (relative ratio of each variety to Nipponbare exceeded 5-fold or was less than 1/5) to be less than three, while those of four varieties from other countries were more than five (especially, Kasalath which was 29 and Bleiyo which was 23). The 2D-DIGE method seems to be useful for analyzing natural varieties of different cultivars and also for comparing the expression of allergen proteins. [Copyright &y& Elsevier]