Tissue Inhibitors of Metalloproteinase 3 (TIMP3) is a key extracellular matrix protein encoded by the TIMP3 gene, playing pivotal roles in regulating matrix metalloproteinase (MMP) activity. It is widely expressed in various tissues, participating in tissue remodeling, angiogenesis, and inflammation. Dysregulation of TIMP3 is linked to multiple diseases, including cancer, cardiovascular disorders, and degenerative conditions, due to its ability to inhibit MMP-mediated extracellular matrix degradation. TIMP3 binds specifically to MMP9 with high affinity, forming a stable complex that potently suppresses MMP9’s proteolytic activity to maintain tissue homeostasis.Thus a functional ELISA assay was conducted to detect the interaction of recombinant cattle TIMP3 and recombinant human MMP9. Briefly, TIMP3 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to MMP9-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-TIMP3 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 cattle TIMP3 and recombinant human MMP9 was shown in Figure 1, the EC50?for this effect is 0.83911μg/mL.