Bone sialoprotein (BSP) is a key non-collagenous protein found primarily in the bone matrix and dentin. Synthesized by osteoblasts, osteocytes, and odontoblasts, it plays a critical role in initiating and regulating bone mineralization by promoting hydroxyapatite crystal formation. BSP contains an RGD sequence that facilitates cell attachment through integrin binding, influencing osteoclast activity and bone remodeling. Its expression is also strongly associated with cancer metastasis to bone. BSP directly interacts with and binds to MMP2, enhancing its proteolytic activity to degrade the extracellular matrix.To detect the activity of recombinant BSP, a functional ELISA assay was performed to evaluate the interaction between recombinant human BSP and recombinant human MMP2.Briefly, biotin-linked BSP were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100µl were then transferred to MMP2-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 BSP and MMP2 was shown in Figure 1, the EC50 for this effect is 0.22 µg/mL.