Urocortin (UCN) is a 40 amino acid peptide hormone that belongs to the corticotropin-releasing factor (CRF) family. It is expressed in specific regions of the brain but is also detectable in other organs notably the heart.UCN is involved in stress response and has diverse physiological functions including modulation of immune and inflammatory responses, as well as effects on the cardiovascular and central nervous systems.Besides,Urocortin 3 (UCN3) has been identified as an interactor of UCN, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human UCN and recombinant mouse UCN3. Briefly, biotin-linked UCN were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100μl were then transferred to UCN3-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST 3 times and incubation with Streptavidin-HRP for 30min, then wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50µl stop solution to the wells and read at 450nm immediately. The binding activity of recombinant human UCN and recombinant mouse UCN3 was shown in Figure 1, the EC50 for this effect is 1.06ug/mL.