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In the assessment of human exposure to allergens, however, current measurement techniques neglect the allergenicity or potency of allergens. Allergic diseases stemming from type I hypersensitivity reactions are mediated by IgE GF 109203X in the serum of atopic individuals. The binding of IgE to specific epitopes on allergenic proteins results in the activation of a pharmacological cascade of reactions that ultimately causes the allergic reaction in the sensitized individual. Mass or number measurements of environmental allergen content based on spore counts from culture-based methods [4] and PCR [5], or surrogate approaches that measure fungal-specific polysaccharide masses [6], are unable to account for this IgE-binding. Commercial immunodetection methods [7] are based on anti-allergen capture antibodies that often target the allergen or allergen extract, but might not target human immunogenic epitopes of allergens.
Materials and methods
Cloning and sequencing of aptamers. After in vitro selection, aptamers were cloned using a TOPO TA cloning kit with the pCR?4–TOPO? vector (Invitrogen, Carlsbad, CA) according to manufacturer’s instructions. Colonies were subsequently picked and grown overnight in a culture of 5 ml LB medium under vigorous shaking, and plasmid DNA from mutation rate colonies was harvested with a QIAprep Spin Miniprep kit (Qiagen, Valencia, CA). Cloned inserts from the plasmid DNAs were sequenced at the DNA Analysis Facility at Yale University using an Applied Biosystems 3730 DNA analyzer (Foster City, CA). Sequence analysis and alignments were performed using the ClustalX program. The mfold tool [12] was used to estimate the secondary structures of sequenced aptamers.