Insulin-like growth factor-binding protein 3, also known as IGFBP3 is one of six IGF binding proteins (IGFBP1 to IGFBP6) that have highly conserved structures and bind the insulin-like growth factors IGF-1 and IGF-2 with high affinity. Within tissues, IGFBP3 can bind IGF1 and IGF2 released by many cell types, and block their access to the IGF-1 receptor (IGF1R), which is activated by both IGFs. IGFBP3 also interacts with cell-surface proteins, affecting cell signaling from outside the cell or after internalization, and also enters the cell nucleus where it binds to nuclear hormone receptors and other ligands. Besides, Insulin (INS) has been identified as an interactor of IGFBP3, thus a binding ELISA assay was conducted to detect the interaction of recombinant human IGFBP3 and recombinant human INS. Briefly, biotin-linked IGFBP3 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100μl were then transferred to INS-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 IGFBP3 and INS was shown in Figure 1, the EC50 for this effect is 0.166 µg/mL.