Immobilization of target for aptamer selection. The target for aptamer selection is a 10 amino
Cyclopamine long peptide having the sequence N-Q-G-D-L-R-L-C-S-H located at the C-terminal end of the Asp f 1 major allergen of A. fumigatus. This peptide region of the allergen has been shown to demonstrate strong IgE-binding reactivity to pooled serum from patients with allergic bronchopulmonary aspergillosis (ABPA), a complication of allergic asthma caused by A. fumigatus [11]. Anhydrous biotinylated Asp f 1 decapeptide (with aminohexanoic acid as a linker) was synthesized (Sigma-Genosys, St. Louis, MO) and solubilized in sterile water to a final concentration of 1 mg/ml. The peptide was subsequently immobilized onto columns (Hydros, Inc., Bourne, MA) – each containing 20 μg of streptavidin covalently coupled to a porous plastic
polymer matrix – by incubating 50 μl of 1 mg/ml peptide solution in the column at room temperature for 1 h. Unbound biotinylated peptide was removed through two washing steps using a wash solution composed of 0.15 M NaCl, 0.001 M MgCl2, and 0.01% SDS.
