Ephrin-A4, also known as EFNA4 and EFL-4, is a member of the ligand of the EPH family. It is mainly expressed in the spleen, lymph nodes, ovary,?small intestine, and colon of adults, as well as in the heart, lungs, liver, and kidneys of the fetus. It is involved in the development of neurons, blood vessels, and epithelium by regulating cell migration, rejection, and adhesion. Ephrin-A4 has been shown to bind EphA1,EphA2, EphA3, EphA4, EphA5, EphA6, EphA7 and EphB1. Thus a functional binding ELISA assay was conducted to detect the interaction of recombinant mouse EFNA4 and recombinant mouse EPHA1. Briefly, EFNA4 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to EphA1-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-EFNA4 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 mouse EFNA4 and recombinant mouse EphA1 was shown in Figure 1, the EC50?for this effect is 0.06 ug/mL.