Anti-Müllerian hormone?(AMH), also named Müllerian inhibiting substance (MIS) belongs to a tissue-specific TGF-beta superfamily growth factor. It can be expressed by male sertoli cells and postnatal testis, and ovarian granulosa cells of females postpartum. AMH expression is critical to sex differentiation at a specific time during fetal development, it appears to be tightly regulated by SF1, GATA factors, DAX1 and FSH. AMH signals through a characteristic receptor consisting of a type I and a type II receptor serine/threonine kinase. Especially the type II receptor is unique and specific receptor for AMH. Besides, Mothers Against Decapentaplegic Homolog 9 (Smad9) has been identified as an interactor of AMH, thus a binding ELISA assay was conducted to detect the interaction of recombinant human AMH and recombinant human (Smad9) Briefly, AMH were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100uL were then transferred to Smad9-coated microtiter wells and incubated for 2h at 37℃. Wells were washed with PBST and incubated for 1h with anti-AMH pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody, wells were aspirated and washed 3 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 of AMH and Smad9 was shown in Figure 1, and this effect was in a dose dependent manner.