Insulin Like Growth Factor Binding Protein 2 (IGFBP2) is a crucial member of the IGFBP family, encoded by the IGFBP2 gene and widely expressed in tissues like the liver, brain, and bone. It regulates insulin-like growth factor (IGF) signaling by binding to IGFs, modulating their bioavailability and interactions with receptors. Dysregulated IGFBP2 is associated with cancer progression, metabolic disorders, and developmental abnormalities, as it balances cell proliferation, differentiation, and apoptosis. IGFBP2 binds to IGF1R directly or via IGF bridging, enhancing IGF1R-mediated downstream signaling to promote cellular processes. Briefly, IGF1R was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to IGFBP2-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-IGF1R 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 IGFBP2 is lmmobilized at 2 µg/mL(100 uLwell), the concentration of IGF1R that produces 50% optimal bindingresponse is found to be approximately 0.23754 µg/mL.