Ephrin-A5, also known as EFNA5, is a member of the Ephrin family. EFNA5 is localized in cell membranes and was highly expressed in skin, brain, spleen, kidney, heart and fat. EFNA5 can bind to its receptors to play important physiological functions, such as participating in neurogenesis, angiogenesis, promoting cell proliferation and differentiation, balancing glucose homeostasis, maintaining lens development, regulating female mammalian reproduction, and mobilizing fat thermogenesis. EPHA10 is a receptor for members of the ephrin-A family. It binds to EFNA3, EFNA4 and EFNA5. Thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human EFNA5 and recombinant human EPHA10. Briefly, EFNA5 was diluted serially in PBS with 0.01% BSA (pH7.4). Duplicate samples of 100 μl were then transferred to EPHA10-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-EFNA5 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 EFNA5 and recombinant human EPHA10 was shown in Figure 1, the EC50 for this effect is 0.03 ug/mL.