WNT2, also known as wingless-type MMTV integration site family member 2, is a ligand for members of the frizzled family of seven transmembrane receptors. Can activate or inhibit canonical Wnt signaling, depending on receptor context. Stimulates cell migration. Decreases proliferation, migration, invasiveness and clonogenicity of carcinoma cells and may act as a tumor suppressor. Besides, Glypican 3 (GPC3) has been identified to bind to Wnt2 and modulate its activity, influencing the Wnt signaling pathway. Thus a binding ELISA assay was conducted to detect the interaction of recombinant human WNT2 and recombinant human GPC3.Briefly, biotin-linked GPC3 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to WNT2-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 50 µL stop solution to the wells and read at 450/630 nm immediately. Measured by its binding ability in a functional ELISA. When Recombinant WNT2 is lmmobilized at 2 ug/mL(100 uLwell), the concentration of GPC3 that produces 50% optimal bindingresponse is found to be approximately0.0534ug/mL.