HoxD12 (homeobox D12)?is a?275 amino acid protein?that localizes to the nucleus and contains one homeobox DNA-binding domain.?It plays a key role in embryonic limb development,which precisely controls the morphogenesis and pattern formation of bones and soft tissues in different parts of the limb. And it also can regulate the processes of cell differentiation and proliferation, during the differentiation of stem cells in certain tissues, the HOXD12 protein can guide cells to differentiate in a specific direction.It is reported that Hoxd-12?can?bind?to?and?transactivate?the?Shh?promoter?in?vitro.Thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human HOXD12 and recombinant human Shh. Briefly, biotin-linked HOXD12 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100μl were then transferred to Shh-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 HOXD12 and Shh was shown in Figure 1, the EC50?for this effect is 0.05ug/mL.