0.1 μm pvdf filter

The aptamer-STIV binding interaction was measured by enzyme-linked immunosorbent assay (ELISA), as described previously [31 (link)], with some modifications. 5′-Biotinylated ssDNA aptamers were synthesized by Life Technologies. The aptamers were heated at 95 °C for 5 min and cooled on ice for 10 min. STIV (10⁹ TCID₅₀/ml) was incubated with each aptamer (200 nM) in binding buffer and the mixtures were passed through a pre-wetted 0.1-μm PVDF filter (EMD Millipore). After washing with binding buffer, the 5′-biotinylated aptamer-STIV complexes were eluted from the filter and transferred to 96-well plates (Pierce, USA). The bound aptamers were detected using streptavidin-conjugated horseradish peroxidase (HRP) (1:10,000, Pierce). After adding 50 μl 2 M H₂SO₄ to terminate the color reaction, the absorbance of each well was measured at 450 nm using an ELISA plate reader.

The specificity of aptamer-STIV binding was further verified by fluorescent localization, as described previously [30 (link)], with some modifications. STIV was labeled with the aptamers as follows: FITC-aptamers were denatured at 95 °C for 5 min and cooled on ice for 10 min, followed by the addition of 100 μl purified STIV (10⁹ TCID₅₀/ml) and incubation on ice for 1 h. The aptamer-STIV mixtures were passed through a pre-wetted 0.1-μm PVDF filter (EMD Millipore) and eluted from the filter by washing with TN buffer. The chemical dye Hoechst 33,342 was then added to label the virus. After washing three times, the samples (10 μl) were dropped onto a glass coverslip and imaged using a fluorescence microscope (Leica DMRXA, German) at an excitation wavelength of 488 nm (green for FITC) or 350 nm (blue for Hoechst 33,342). The FITC-library pool and SGIV served as controls.