Aptamer-based methods for detecting environmental biotoxins and allergen potencies.

摘要

Airborne biological allergens and biotoxins are linked to respiratory diseases like allergies and asthma. Climatic factors like temperature could also potentially affect the number of allergenic proteins expressed, thereby influencing the potency of allergens. However, a clear interpretation of the health impact due to allergen and biotoxin exposure is hampered by the high cost and limitations associated with current methods of analysis. The goal of the research is to evaluate the potential of aptamer-based methods for detecting environmental biotoxins and allergen potencies. To achieve this goal, DNA aptamers will be developed for the fungal biotoxin (1→3)-beta-D glucans and the major allergen Asp f1 of the ubiquitous fungus Aspergillus fumigatus. To guide the aptamer work, the impact of sporulation temperature on A. fumigatus spore allergenicity and the transcription of allergenic genes will also be elucidated.;Through in vitro selection, aptamers have been produced that are able to bind with nanomolar affinity to curdlan (KD ∼300 nM), a (1→3)-beta-D glucan, and to an allergenic epitope of Asp f1 (KD ∼90 nM). The aptamers display high selectivity for their respective intended targets over competitor molecules like non-(1 →3)-beta-D polysaccharides and allergenic proteins from other mold species. Inhibition immunoassays targeting human IgE-binding regions on spores demonstrated that the allergenicity per spore of A. fumigatus conidia could increase by up to 300-fold when sporulation temperatures decreased from 32°C to 14°C. Subsequent microarray analysis of A. fumigatus conidiating cultures reveal that a higher number of genes encoding known, major allergens are more highly expressed at lower sporulation temperatures, indicating an underlying transcriptional mechanism driving the observed temperature-allergenicity relationship.;The aptamers developed in the study will enable the production of more cost-effective, less ambiguous assays for the environmental measurement of (1→3)-beta-D glucans and A. fumigatus allergens. In addition, the production of the Asp f1 aptamer provides proof of principle that measurement methods can be developed that indicate allergenicity. Such allergenicity measurements are able to account for substantial variabilities in allergen potency due to factors like sporulation temperature, and therefore offer a more direct indicator of human exposure to help close the gap between environmental measurements and clinical outcomes.