Leptin is a hormone made by adipose cells that helps to regulate energy balance by inhibiting hunger. Many of leptin's effects are mediated through neuropeptide-containing neurons and neuropeptide receptors in the hypothalamus. Although regulation of fat stores is deemed to be the primary function of leptin, it also plays a role in other physiological processes, as evidenced by its multiple sites of synthesis other than fat cells, and the multiple cell types beside hypothalamic cells that have leptin receptors. A functional binding ELISA assay was conducted to detect the interaction of recombinant mouse LEP and recombinant human LEPR. Briefly, LEP were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100 ul were then transferred to LEPR-coated microtiter wells and incubated for 2h at 37℃. Wells were washed with PBST and incubated for 1h with anti-LEP pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody, wells were aspirated and washed 3 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 nm immediately. The binding activity of of LEP and LEPR was shown in Figure 1, the EC50?for this effect is 0.26 ug/mL.