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, UCN3 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to UCN-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-UCN3 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.Measured by its binding ability in a functional ELISA. When Recombinant human UCN is lmmobilized at 2 ug/mL(100 uLwell), the concentration of UCN3 that produces 50% optimal bindingresponse is found to be approximately 0.08ug/mL.