Insulin-like Growth Factor 2 (IGF2) is a mitogenic peptide hormone critical for fetal growth and development, predominantly synthesized by the liver and placenta. It mediates signaling through the IGF1 receptor (IGF1R) and insulin receptor isoform A (IR-A) to govern cell proliferation, differentiation, and metabolic processes. Dysregulated IGF2 expression is associated with oncogenesis and overgrowth disorders such as Beckwith-Wiedemann syndrome. IGF2 exhibits high-affinity binding to Insulin-like Growth Factor Binding Protein 5 (IGFBP5), which dynamically modulates IGF2 bioactivity by either enhancing receptor engagement or restricting ligand-receptor interaction, contingent on the cellular microenvironment.Thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human IGF2 and recombinant rat IGFBP5 .Briefly, biotin-linked IGF2 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100μl were then transferred to IGFBP5-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 IGF2 and IGFBP5 was shown in Figure 1, the EC50?for this effect is 0.46ug/mL.