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.