Cathepsin G (CTSG, D993Hu) is a 255-amino-acid serine protease belonging to the peptidase S1 and chymotrypsin families. Encoded by the CTSG gene on human chromosome 14q11.2, it is predominantly stored in the azurophil granules of neutrophils and monocytes. As a key immune protease, CTSG functions intracellularly to degrade engulfed pathogens and extracellularly to break down extracellular matrix (ECM) components at inflammatory sites. It exhibits broad-spectrum antibacterial activity against Gram-positive and Gram-negative bacteria, independent of its proteolytic activity. Beyond pathogen clearance, CTSG regulates inflammation by processing chemokines, cytokines, and cell-surface receptors, and participates in thrombosis and vascular remodeling. Dysregulation of CTSG is linked to chronic inflammation, autoimmune diseases, and cancer metastasis. CTSG directly binds and proteolytically cleaves pro-MMP9 (A553Ca) to convert it into its active, functional matrix-degrading form. Thus a functional ELISA assay was conducted to detect the interaction of recombinant human CTSG and recombinant dog MMP9. Briefly, CTSG 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-CTSG 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 human CTSG and recombinant dog MMP9 was shown in Figure 1, the EC50?for this effect is 0.0158 μg/mL.