Netrin 1 (Ntn1) is included in a family of laminin-related secreted proteins. Mouse Ntn1 is a 66-76?kDa glycoprotein that is well known for its involvement in axonal guidance during embryonic development and as an enhancer of cancer cell metastasis. Ntn1 is also involved in outgrowth and migration orientation in the developing CNS and plays a significant role in the morphogenesis of endothelial cells and vascular smooth-muscle cells. It is also involved in the processes of cytoskeleton reorganization, angiogenesis, epithelial cell adhesion, and cell migration in the lungs, mammary gland, and pancreas. Slit Homolog 1 (Slit1) can acts as a potent promoter of both Netrin-1 attractive and repulsive activities on distinct neuronal cell types, thereby opening novel perspectives on the role of combinations of cues in brain wiring. Thus a functional binding ELISA assay was conducted to detect the interaction of recombinant mouse Ntn1 and recombinant human Slit1. Briefly, Ntn1 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to Slit1-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-Ntn1 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 Ntn1 and recombinant human Slit1 was shown in Figure 1, the EC50?for this effect is 0.03 ug/mL.