Homeobox Protein D9 (HOXD9) is a crucial transcription factor encoded by the HOXD9 gene in the homeobox gene family. It plays essential roles in embryonic development, especially in limb and axial patterning, by binding to specific DNA sequences and regulating the expression of target genes related to cell differentiation and tissue morphogenesis. Mutations or abnormal expression of HOXD9 can lead to developmental abnormalities and certain diseases.Besides,Hedgehog Homolog, Sonic (SHH) has been identified as an interactor of HOXD9, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human HOXD9 and recombinant human SHH. Briefly, biotin-linked HOXD9 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 HOXD9 and SHH was shown in Figure 1, the EC50?for this effect is 0.015ug/mL.