The epidermal growth factor receptor (EGFR) is a growth factor receptor that induces cell differentiation and proliferation upon activation through the binding of one of its ligands. The receptor is located at the cell surface, where the binding of a ligand activates a tyrosine kinase in the intracellular region of the receptor. This tyrosine kinase phosphorylates a number of intracellular substrates that activates pathways leading to cell growth, DNA synthesis and the expression of oncogenes such as fos and jun. In addition, the interaction of Heparin Binding Epidermal Growth Factor Like Growth Factor (HBEGF) and EGFR can activate the EGFR signaling pathway, and then affect the biological behaviors of cell proliferation, differentiation and migration. Thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human EGFR and recombinant human HBEGF. Briefly, EGFR 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-EGFR 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 EGFR and recombinant human HBEGF was shown in Figure 1, the EC50 for this effect is 0.16 ug/mL.