Amphiregulin (AREG) is a well-characterized member of the epidermal growth factor (EGF) family and is one of the ligands of the EGF receptor (EGFR). AREG plays a key role in mammalian development and in the control of branching morphogenesis in various organs. Furthermore, AREG participates in a wide range of physiological and pathological processes activating the major intracellular signalling cascades governing cell survival, proliferation and motility. Besides, EGF has been identified as an interactor of AREG, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human AREG and recombinant rat EGF. Briefly, AREG was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to EGF-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-AREG 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 AREG and recombinant rat EGF was shown in Figure 1, the EC50?for this effect is 0.1 ug/mL.