Homeobox protein Hox - D13 (HOXD13) is a key transcription factor in the homeobox family.HOXD13 is crucial for tissue and organ patterning and differentiation, especially in axial skeleton and limb development. Abnormalities in HOXD13, like mutations or abnormal expressions, can cause developmental disorders such as synpolydactyly, highlighting its importance in normal morphological development.Besides,Hoxd-13 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 HOXD13 and recombinant human SHH .Briefly, biotin-linked HOXD13 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 HOXD13 and SHH was shown in Figure 1, the EC50 for this effect is 0.019ug/mL.