Hepatocyte Growth Factor (HGF), also known as scatter factor, is a pleiotropic cytokine primarily produced by mesenchymal cells. It plays crucial roles in embryonic development, tissue regeneration, and wound healing by mediating cell proliferation, motility, and morphogenesis. HGF is synthesized as an inactive single-chain precursor (pro-HGF) that is proteolytically cleaved to form the active heterodimer. Its signaling is vital in organogenesis and contributes to pathological processes like cancer metastasis when dysregulated. HGF binds specifically to its receptor, HGFR (c-MET), triggering intracellular pathways that promote cellular responses including proliferation and migration.To detect the activity of recombinant HGF a functional ELISA assay was performed to evaluate the interaction between recombinant mouse HGF and recombinant human HGFR.Briefly, HGF was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to HGFR-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-HGF 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 HGF and recombinant human HGFR was shown in Figure 1, the EC50?for this effect is 0.12μg/mL.