Insulin-like growth factor binding protein 5 (IGFBP5) is a highly conserved secreted protein in the IGFBP family, widely expressed in bone, muscle, and multiple tissues. It modulates cell proliferation, differentiation, and apoptosis through both IGF-dependent and independent pathways, and plays vital roles in skeletal development, tissue repair, and tumor progression. IGFBP5 is involved in extracellular matrix remodeling and can be regulated by proteolytic cleavage and post-translational modifications. It acts as a crucial regulator in physiological and pathological processes, with prominent functions in osteogenesis and tissue homeostasis. IGFBP5 binds IGF1 with high affinity to control its bioavailability and signaling activity.To detect the activity of recombinant human IGFBP5, a functional ELISA assay was performed to evaluate the interaction between recombinant human IGFBP5 and recombinant cattle IGF1. Briefly, biotin-linked IGFBP5 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100μl were then transferred to IGF1-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 IGFBP5 and IGF1 was shown in Figure 1, the EC50?for this effect is 0.381μg/mL.