Hepcidin (Hepc) is a regulator of iron metabolism.Hepcidin inhibits iron transport by binding to the iron export channel ferroportin which is located on the basolateral surface of gut enterocytes and the plasma membrane of reticuloendothelial cells (macrophages). Hepcidin ultimately breaks down the transporter protein in the lysosome. Inhibiting ferroportin prevents iron from being exported and the iron is sequestered in the cells. Besides, Lipocalin-2 (LCN2) has been identified as an interactor of Hepc, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant rat Hepc and recombinant mouse LCN2. Briefly, Hepc was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to LCN2-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-Hepc 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 450/630nm immediately. The binding activity of recombinant rat Hepc and recombinant mouse LCN2 was shown in Figure 1, the EC50 for this effect is 2.267µg/mL.