Hydroxysteroid (17β) dehydrogenase 1 (HSD17b1) is a steroidogenic enzyme that especially catalyzes the conversion of low potent 17keto-steroids to highly potent 17β-hydroxysteroids. It has a dual function in estrogen activation and androgen inactivation and plays a major role in establishing the estrogen E2 concentration gradient between serum and peripheral tissues. Cytochrome P450 1B1 is involved in the metabolism of various endogenous substrates, including fatty acids, steroid hormones and vitamins. Thus a binding ELISA assay was conducted to detect the interaction of recombinant human HSD17b1 and recombinant human CYP1B1. Briefly, HSD17b1 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100 ul were then transferred to CYP1B1-coated microtiter wells and incubated for 2h at 37℃. Wells were washed with PBST and incubated for 1h with anti-HSD17B1 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 50 μL stop solution to the wells and read at 450 nm immediately. The binding activity of recombinant human HSD17b1 and recombinant human CYP1B1 was shown in Figure 1, the EC50?for this effect is 0.098 ug/mL.