Glypican-3 (GPC3), a 70 kDa protein, is a member of the glypican family that attaches to the cell surface by a glycosylphosphatidylinositol anchor, is specifically up-regulated in hepatocellular carcinoma (HCC) although rarely or not expressed in normal liver tissues, making it a perfect diagnostic and treatment target for HCC. GPC3 is also a negative transcriptional regulator and tumor suppressor that inhibits the growth of breast, ovary, and lung cancer cells. It is reported that GPC3 can form a complex with insulin-like growth factor 2 (IGF2), and might thereby modulate IGF2 action. Thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human GPC3 and recombinant rat IGF2. Briefly, GPC3 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to IGF2-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-GPC3 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 GPC3 and recombinant rat IGF2 was shown in Figure 1, the EC50?for this effect is 0.08 ug/mL.