Glypican 1 (GPC1) is one of the six glycosylphosphatidylinositol-anchored, cell surface heparan sulfate proteoglycans that acts as a growth factor signaling. Cells known to express GPC-1 include neurons, smooth and skeletal muscle cells, keratinocytes, osteoblasts, Schwann cells, immature dendritic cells, and tumor, plus tumor-associated vascular endothelial cells. The function of GPC-1 is complex and varied, it can modulate various signaling pathways including Heparin Binding Epidermal Growth Factor Like Growth Factor (HBEGF), fibroblast growth factors (FGF), vascular endothelial growth factor-A (VEGF-A), transforming growth factor-β (TGF-β), Wnt, Hedgehog (Hh), and bone morphogenic protein (BMP) through specific interactions with pathway ligands and receptors. Thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human GPC1 and recombinant human HBEGF. Briefly, GPC1 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to HBEGF-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-GPC1 pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody for 1h at 37℃, wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50 µL stop solution to the wells and read at 450/630 nm immediately. The binding activity of recombinant human GPC1 and recombinant human HBEGF was shown in Figure 1, the EC50 for this effect is 0.17 ug/mL.