Insulin-like growth factor binding protein 4 (IGFBP4) is a key secreted protein belonging to the insulin-like growth factor binding protein family. It is widely distributed in multiple tissues and biological fluids, with high-affinity binding capacity for insulin-like growth factors IGF-I and IGF-II. By sequestering IGFs, IGFBP4 prolongs their circulating half-life, inhibits their interaction with cell surface receptors, and thus negatively regulates IGF-mediated cell proliferation, differentiation, and survival. It participates in diverse physiological and pathological processes including bone development, reproductive function, tumor progression, and angiogenesis. Unlike some other IGFBPs, IGFBP4 often acts as a growth inhibitor independent of IGFs under certain conditions. IGFBP4 serves as a specific proteolytic substrate for PAPPA2, which can cleave IGFBP4 to attenuate its inhibitory effect on IGF signaling.Briefly, PAPPA2 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μL were then transferred to IGFBP4-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-PAPPA2 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 IGFBP4 is lmmobilized at 2 µg/mL(100 µLwell), the concentration of PAPPA2 that produces 50% optimal binding response is found to be approximately 0.051 µg/mL.