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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.
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