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18?Proteomic and Immunological Characterization of Ragweed Allergens.

ABSTRACT: BackgroundThe prevalence of sensitization to ragweed has risen in North America and across Europe. Although the pectate lyase Amb a 1, the major allergen of ragweed, was identified as long ago as the 1960s, little is known about the allergenicity of the 5 Amb a 1 isoallergens and other allergens present in ragweed pollen. Ragweed extracts and purified Amb a 1 isoallergens have now been characterized for their allergenic potential to determine whether a single Amb a 1 isoallergen, several isoallergens or a combination with other allergens should be included in a recombinant SIT vaccine.MethodsExtracts from North American short ragweed (Ambrosia artemisiifolia) pollen were investigated by mass spectrometry (MS), 2D-PAGE and immunoblotting. Furthermore, Amb a 1 isoallergens were purified and IgE reactivity determined by immunoblotting and IgE inhibition.Results2D-PAGE and MS of ragweed extract proved the presence of all 5 known Amb a 1 isoallergens, of which Amb a 1.01 represents the dominant form. Additionally all other ragweed allergens known by sequence (Amb a 3, Amb a 4, Amb a 5, Amb a 6, Amb a 8, Amb a 9, Amb a 10) were identified. The highest IgE reactivity by immunoblotting was observed for Amb a 1.01 followed by Amb a 1.03; other Amb a 1 isoallergens as well as other detected ragweed allergens showed only weak IgE reactivity. All isoallergens with the exception of Amb a 1.04, which is only of low abundance in ragweed extract, were purified. Similar to the immunoblot analysis with crude extract, the purified isoallergens Amb a 1.02 and Amb a 1.05 showed weak IgE binding, whereas Amb a 1.01 and Amb a 1.03 had high IgE reactivity. First IgE inhibition experiments suggest that Amb a 1.01 contains all relevant IgE epitopes.ConclusionsAmb a 1.01 is the most abundant Amb a 1 isoallergen, and presumably the most important ragweed allergen. However, a larger panel of ragweed-allergic subjects has to be analyzed with regard to IgE and T cell reactivities, to be able to choose a candidate for a recombinant vaccine for specific immunotherapy of ragweed allergy.

SUBMITTER: Augustin S

PROVIDER: S-EPMC3512999 | biostudies-other | 2012 Feb

REPOSITORIES: biostudies-other

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Project description:BACKGROUND:Our knowledge of allergen structure and function continues to rise and new scientific data on the homology and cross-reactivity of allergen sources should be considered to extend the work of Lorenz et al., 2009 (Int Arch Allergy Immunol. 148(1):1-1, 2009) and the concept of homologous groups. In addition to this, sophisticated techniques such as mass spectrometry (MS) are increasingly utilised to better characterise the complex mix and nature of allergen extracts. METHODS:Homology models were used of Fag s 1 (Beech) and Cyn d 1 (Bermuda grass) and compared with template crystal structures of Bet v 1 and Phl p 1 from the 'exemplar' species of Birch and Timothy grass, respectively. ELISA experiments were performed to assess cross-reactivity of Beech (tree) and Bermuda (grass) extracts to rabbit sera raised to either "3-Tree" (Birch, Alder and Hazel) extract or "Grass" (12-grass mix extract), respectively. The comparability of biochemical stability of different allergen sources was assessed through statistical methods for a range of tree and grass species. RESULTS:Allergen cross-reactivity and/or structural homology have been described providing justification for inclusion of Beech within the Birch homologous tree group. Data from Bermuda grass (Cyn d 1) provides further justification for the inclusion of this species into the homologous group of the sweet grasses. However, further characterisation of relevant allergens from Bermuda grass and, in particular, comparison of cross-reactive patterns between subjects specifically in areas with high abundance of both Pooideae and Chloridoideae is sought. CONCLUSION:MS allows the possibility to identify individual proteins or allergens from complex mixes by mass and/or sequence, and this has been extensively applied to the allergen field. New data on the homology, cross-reactivity and biological parameters of allergen sources have been considered to extend the work of Lorenz et al., 2009 in the context of tree and grass species. The concept of homologous groups is certainly dynamic allowing the flexibility and potential in streamlining quality parameters, such as stability profiles, due to extrapolation of exemplar data to a wider range of allergens.

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18?Proteomic and Immunological Characterization of Ragweed Allergens.

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