Matrix Metalloproteinase 14 (MMP14), also known as membrane-type 1 MMP (MT1-MMP), is a pivotal enzyme in the matrix metalloproteinase family. Unlike most MMPs secreted into the extracellular space, MMP14 is a transmembrane protease that anchors to the cell membrane. It plays a crucial role in degrading extracellular matrix (ECM) components, particularly collagen, thereby regulating tissue remodeling, wound healing, and developmental processes. Its ability to activate other MMPs, like MMP2, amplifies its proteolytic cascade. Importantly, MMP14 is a key mediator in cancer progression, facilitating tumor invasion, angiogenesis, and metastasis by breaking down physical barriers in the ECM. Its dysregulation is also linked to inflammatory and vascular diseases.Besides,Tumor Necrosis Factor Alpha (TNFa) has been identified as an interactor of MMP14, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human MMP14 and recombinant human TNFa. Briefly, biotin-linked TNFa were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100µl were then transferred to MMP14-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. Measured by its binding ability in a functional ELISA. When Recombinant MMP14 is lmmobilized at 2 µg/mL(100 uLwell), the concentration of TNFa that produces 50% optimal bindingresponse is found to be approximately 0.178 µg/mL.