DHCR7 (7-dehydrocholesterol reductase) is a rate-limiting enzyme in cholesterol synthesis, which is mainly involved in the conversion of 7-dehydrocholesterol (7-DHC) to cholesterol. In the cholesterol biosynthetic pathway, this protease can reduce the C7-C8 double bond of cholesta-5,7-dien-3beta-ol (7-dehydrocholesterol/7-DHC) and cholesta-5,7,24-trien-3beta-ol, two intermediates in that pathway. In the absence of functional DHCR7, accumulation of 7DHC and a lack of cholesterol production leads to the devastating developmental disorder, Smith-Lemli-Opitz syndrome. Cholesterol 25-hydroxylase (CH25H) can also repress cholesterol biosynthetic enzymes. A functional ELISA assay was conducted to detect the interaction of recombinant human DHCR7 and recombinant human CH25H. Briefly, biotin-linked DHCR7 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100 ul were then transferred to CH25H-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST 3 times and incubation with Streptavidin-HRP for 30 min, then wells were aspirated and washed 5 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 DHCR7 and recombinant human CH25H was shown in Figure 1, the EC50?for this effect is 0.028 ug/mL.