Ephrin-A1 (EFNA1), also known as EPLG1, LERK1, TNFAIP4, Immediate early response protein B61, is a member of the A-type ephrin family of cell surface proteins that function as ligands for the A-type Eph receptor tyrosine kinase family. EFNA1 plays an important role in angiogenesis and tumor neovascularization. EFNA1 widely affects tumor growth through enhancing tumor angiogenesis, malignant cell events and invasiveness. Ephrin Type A Receptor 1 (EPHA1) is one of high-affinity ligands for EFNA1, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human EFNA1 and recombinant mouse EPHA1. Briefly, biotin-linked EFNA1 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100 ul were then transferred to EPHA1-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 recombinant human EFNA1 and recombinant mouse EPHA1 was shown in Figure 1, the EC50 for this effect is 0.24 ug/mL.