Neuropilin 1 (NRP1), as kown as CD304 or VEGF165R, is a 130 - 140 kDa type I transmembrane (TM) glycoprotein. NRP1 is expressed by neurons, blood vessels, immune cells and many other cell types in the mammalian body and binds a range of structurally and functionally diverse extracellular ligands to modulate organ development and function. VEGF165, an angiogenic cytokine, has higher affinity for NRP1. A functional binding ELISA assay was conducted to detect the interaction of recombinant human NRP1 and recombinant human VEGF165. Briefly, NRP1 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to VEGF165-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-NRP1 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 NRP1 and recombinant human VEGF165 was shown in Figure 1, the EC50?for this effect is 2.98 ug/mL.