Pro-MCH (PMCH), short for Pro-melanin-concentrating hormone, is a precursor protein and it can be cleaved and processed to produce melanin-concentrating hormone, a neuropeptide involved in the regulation of various physiological processes like appetite, sleep-wake cycles and energy balance in organisms. In addition, Pro-MCH and its derivative MCH are also involved in the regulation of mood and stress responses, and have certain effects on the body's adaptation to environmental changes.Neuropeptide Y (NPY) has been identified as an interactor of PMCH, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant rat NPY and recombinant human PMCH . Briefly, NPY was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to PMCH-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-NPY 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 PMCH is lmmobilized at 2 ug/mL(100 uLwell), the concentration of NPY that produces 50% optimal bindingresponse is found to be approximately 0.055ug/mL.