EphrinB2 (EFNB2), a 40 kDa member of the EphrinB family, is a pivotal bidirectional signaling molecule ubiquitously expressed in mammals and is crucial in angiogenesis during development and disease progression. Besides, EFNB2 is expressed at abnormally high levels in some neoplasms, such as squamous cell carcinoma of the head and neck and colorectal cancer. Its overexpression is associated with the malignant progression of tumors. Ephrin Type B Receptor 3 (EPHB3) has been identified as an interactor of EFNB2, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant mouse EFNB2 and recombinant mouse EPHB3. Briefly, biotin-linked EFNB2 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100 ul were then transferred to EPHB3-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 mouse EFNB2 and recombinant mouse EPHB3 was shown in Figure 1, the EC50?for this effect is 6.3 ug/mL.