Cathepsin G (CTSG, D993Mu) is a 26 kDa serine protease of the peptidase S1 (chymotrypsin) family, primarily stored in the azurophil granules of neutrophilic polymorphonuclear leukocytes. Encoded by the CTSG gene on chromosome 14q11.2, it is synthesized as a preproprotein and proteolytically processed to yield the active mature enzyme. As a key immune effector, CTSG participates in phagocytic pathogen killing and digestion, mediates connective tissue remodeling at inflammatory loci, and exerts direct antimicrobial activity against pathogens like Staphylococcus aureus. Beyond innate immunity, it modulates immune signaling, tissue repair, and pathological processes including tumor invasion and chronic inflammation. Functionally, CTSG directly cleaves and activates the latent proenzyme form of MMP9 (A553Ca), converting pro-MMP9 into its catalytically active matrix metalloproteinase.Thus a functional ELISA assay was conducted to detect the interaction of recombinant mouse 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 mouse CTSG and recombinant dog MMP9 was shown in Figure 1, the EC50?for this effect is 0.017 μg/mL.