Fig. 3. Binding of mNGF to purified 9β1 integrin in ELISA assay. 9β1 integrin isolated from 9K562 cells was immobilized in a 96-well plate at a concentration of 0.5 µg/ml by overnight incubation at 4°C in PBS. After blocking with 5% non-fat milk, a range of concentrations of hrNGF were added to the wells, which were incubated for 30 minutes at 37°C. The wells were then washed, and bound ligand was detected by adding the primary anti-NGF polyclonal antibody and incubation for 60 minutes at 37°C. After washing, the goat anti-rabbit alkaline-phosphatase (AP)-conjugated IgG was added and incubation was continued for another 60 minutes at 37°C. The color was developed with AP substrate (4-nitrophenylphosphate) and the plate was read using an ELISA plate reader at a 405 nm single wavelength. The specific binding () was estimated by subtraction of binding of NGF to the blocker () from binding of NGF to immobilized integrin (). The error bars represent the standard deviation from three duplicated independent experiments.