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. Reelin (RELN) has been identified as an interactor of EFNB2, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human EFNB2 and recombinant human RELN. Briefly, EFNB2 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to RELN-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-EFNB2 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 EFNB2 and recombinant human RELN was shown in Figure 1, the EC50?for this effect is 0.02 ug/mL.