Ephrin A3 (EFNA3) , also known as EFL2, EPLG3, LERK3, Ehk1-L, is a member of the Eph/ephrin tyrosine kinase family. EFNA3 is localized in cell membranes and was highly expressed in brain, skin, esophagus, skeletal muscle, spleen, thymus, prostate, testis, ovary, small intestine, peripheral blood leukocytes. EFNA3, like most genes in the ephrin family, plays a central role in embryonic development can associate with multiple signaling pathways involved in cell growth and tumor cell metastasis. Aberrant regulation of EFNA3 is associated with the occurrence and development of various types of cancer. Ephrin Type A Receptor 4 (EPHA4) is a receptor for members of the ephrin-A family. It can bind to EFNA3. Thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human EFNA3 and recombinant human EPHA4. Briefly, EFNA3 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to EPHA4-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-EFNA3 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 EFNA3 and recombinant human EPHA4 was shown in Figure 1, the EC50?for this effect is 0.4 ug/mL.