Insulin Like Growth Factor Binding Protein 5 (IGFBP5) is a protein that plays an important role in regulating the activity of Insulin Like Growth Factors (IGFs). It has a high affinity for IGFs and is widely distributed in various tissues. IGFBP5 can bind to Insulin Like Growth Factor 2 (IGF2) in the extracellular space. By binding to IGF2, it can modulate the bioavailability and half-life of IGF2, either enhancing or inhibiting the interaction of IGF2 with its receptors, thus influencing cell growth, proliferation, and differentiation.Thus a functional ELISA assay was conducted to detect the interaction of recombinant rat IGFBP5 and recombinant rabit IGF2. Briefly, IGFBP5 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to IGF2-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-IGFBP5 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. The binding activity of recombinant rat IGFBP5 and recombinant rabit IGF2 was shown in Figure 1, the EC50?for this effect is 7.021ug/mL.