Ephrin A2 (EFNA2) is a cell surface-associated protein belonging to the Ephrin family, which are involved in intercellular communication and cell localization. EFNA2 is mainly expressed in tissues including the brain, heart, lung, kidney and pancreas. And this protein is involved in regulating a variety of biological processes, including cell migration, cell differentiation, and tissue boundary formation, by interacting with Eph receptors. It aslo plays a significant role in the development and maintenance of the cardiovascular system, as well as in the regulation of synaptic plasticity and neuronal circuitry in the brain. A Disintegrin And Metalloprotease 10 (ADAM10), a disintegrin and metalloproteinase, can cleave EFNA2, potentially altering the activity of EFNA2 or its interaction with Eph receptors, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human EFNA2 and recombinant rat ADAM10. Briefly, biotin-linked EFNA2 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100 ul were then transferred to ADAM10-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST 3 times and incubation with Streptavidin-HRP for 30min, then 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 nm immediately. The binding activity of EFNA2 and ADAM10 was shown in Figure 1, the EC50?for this effect is 1.38 ug/mL.