Tissue Factor Pathway Inhibitor 2 (TFPI2) is a serine protease inhibitor primarily produced by endothelial cells, fibroblasts, and placental trophoblasts. It plays a critical role in regulating coagulation by inhibiting the tissue factor (TF)-dependent extrinsic pathway of blood clotting, specifically targeting factor Xa and the TF-factor VIIa complex. Beyond hemostasis, TFPI2 is involved in extracellular matrix remodeling, wound healing, and angiogenesis. It also exhibits tumor-suppressive properties by inhibiting matrix metalloproteinases (MMPs) and preventing tumor invasion and metastasis. Reduced TFPI2 expression is associated with various cancers, including pancreatic, ovarian, and colorectal cancer, making it a potential biomarker for tumor progression. Additionally, TFPI2 has been implicated in cardiovascular and inflammatory diseases.Besides,Insulin Like Growth Factor 2 (IGF2) has been identified as an interactor of TFPI2, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant rat TFPI2 and recombinant rabbit IGF2. Briefly, TFPI2 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to IGF2-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-TFPI2 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 rat TFPI2 and recombinant rabbit IGF2 was shown in Figure 1, the EC50?for this effect is 0.32μg/mL.