Plasminogen (Plg) is a glycoprotein predominantly synthesized in the liver and circulates in plasma as the zymogen (inactive precursor) of plasmin. It serves as a central mediator in fibrinolysis, extracellular matrix (ECM) degradation, and tissue remodeling processes. Upon activation by tPA or uPA, Plg is converted into plasmin—a broad-spectrum serine protease that participates in thrombolysis, cell migration, and inflammatory responses. Notably, emerging evidence suggests that Plg interacts with ECE1; this interaction may modulate Plg’s activation or spatial localization in vascular environments and inflammatory contexts.To detect the activity of recombinant Plg, a functional ELISA assay was performed to evaluate the interaction between recombinant human Plg and recombinant human ECE1. Briefly, biotin-linked Plg were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100µl were then transferred to ECE1-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 Plg and ECE1 was shown in Figure 1, the EC50 for this effect is 0.01µg/mL.