Urokinase-type plasminogen activator receptor (uPAR) is a glycosylphosphatidylinositol (GPI)-anchored protein. Besides regulating proteolysis, uPAR could also activate many intracellular signaling pathways that promote cell motility, invasion, proliferation and survival through cooperating with transmembrane receptors. uPAR is overexpressed across a variety of tumors and is associated with cancer invasion and metastasis. ITGb1 has been identified as an interactor of uPAR, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human uPAR and recombinant human ITGb1. Briefly, uPAR was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to ITGb1-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-uPAR 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 450/630 nm immediately. The binding activity of recombinant human uPAR and recombinant human ITGb1 was shown in Figure 1, the EC50?for this effect is 0.165 ug/mL.