Endoglin (ENG)?is a transmembrane glycoprotein that serves as a co-receptor for transforming growth factor-beta (TGF-b) superfamily ligands, particularly TGFb3 and TGFb3. Primarily expressed on vascular endothelial cells, ENG plays pivotal roles in angiogenesis, vascular remodeling, and maintaining vascular integrity. It modulates TGF-b signaling by forming complexes with type I and II TGF-b receptors, influencing both SMAD-dependent and non-SMAD pathways. ENG exists as two isoforms (long and short) generated by alternative splicing, with differential signaling capabilities. Its critical involvement in cardiovascular development is evidenced by ENG mutations causing hereditary hemorrhagic telangiectasia (HHT), a vascular dysplasia disorder. Beyond vasculature, ENG participates in inflammation, wound healing, and cancer progression, where its expression often correlates with tumor angiogenesis and metastasis. The soluble form (sENG) has emerged as a potential biomarker for preeclampsia and cardiovascular diseases.To detect the activity of recombinant ENG, a functional ELISA assay was performed to evaluate the interaction between recombinant mouse ENG and recombinant human TGFb3.Briefly, biotin-linked ENG were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100μl were then transferred to TGFb3-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST 3 times and incubation with Streptavidin-HRP for 30min, then wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50μl stop solution to the wells and read at 450nm immediately. The binding activity of ENG and TGFb3 was shown in Figure 1, the EC50?for this effect is 0.56 μg/mL.