Vitronectin (VTN ) is a glycoprotein of the hemopexin family which is abundantly found in serum, the extracellular matrix and bone. It is a secreted protein and exists in either a single chain form or a clipped, two chain form held together by a disulfide bond. VTN can binds to integrin alpha-V beta-3 and thus promotes cell adhesion and spreading. It also binds to plasminogen activator inhibitor-1 (PAI-1) and serves to regulate proteolysis initiated by plasminogen activation. Besides, Insulin Like Growth Factor Binding Protein 5 (IGFBP5) has been identified as an interactor of VTN, thus a binding ELISA assay was conducted to detect the interaction of recombinant Rat VTN and recombinant Rat IGFBP5. Briefly, VTN were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100ul were then transferred to IGFBP5-coated microtiter wells and incubated for 2h at 37℃. Wells were washed with PBST and incubated for 1h with anti-VTN pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody, wells were aspirated and washed 3 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 VTN and IGFBP5 was shown in Figure 1, and this effect was in a dose dependent manner.