Versican (VCAN) is a large extracellular matrix proteoglycan that plays critical roles in tissue development, inflammation, and cell adhesion by interacting with hyaluronan and other ECM components. It regulates tissue hydration, structural integrity, and immune cell migration, contributing to cardiovascular disease, cancer progression, and fibrosis through modulation of cell proliferation and matrix remodeling. Proteolytic cleavage of VCAN releases bioactive fragments that influence cellular signaling. Importantly, VCAN binds and sequesters TGFβ1 in the ECM, controlling its bioavailability; upon VCAN cleavage, TGFβ1 is released, activating downstream signaling pathways involved in fibrosis, immune regulation, and tissue repair.Thus a functional ELISA assay was conducted to detect the interaction of recombinant mouse VCAN and recombinant dog TGFb1. Briefly, VCAN was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to TGFb1-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-VCAN 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 VCAN and recombinant dog TGFb1 was shown in Figure 1, the EC50?for this effect is 0.005ug/mL.