Solid-phase binding assay of biotinylated VEGF to recombinant human VEGFR1–2

The method was performed as described previously [31]. Briefly, 96-well microplate was coated with 500 ng/ml of either VEGFR-1 or -2 in PBS, sealed and incubated overnight at 4°C. After 3 times washes with PBS-Tween 20 (0.05% v/v), the plate was blocked with PBS with 1% (w/v) BSA, and incubated for 2 h at room temperature. After washing, a mixture of bt-VEGF (50ng/ml) and heparin (1 μg/ml) or various concentrations of CaD in PBS were applied overnight at 4°C. After washing, streptavidin-HRP (1:4000) was added for 2 h, washed and substrate solution was added for 30–45 minutes. Stop solution (2N; H₂SO4) was added and fluorescence was measured at 450nm.