Cholesterol-25-Hydroxylase (CH25H) is an endoplasmic reticulum-associated enzyme that catalyzes the hydroxylation of cholesterol at the 25-position to produce 25-hydroxycholesterol (25-HC). This oxysterol plays critical roles in lipid metabolism, immune regulation, and antiviral responses by modulating the activity of sterol-responsive transcription factors such as SREBP and LXR. CH25H is highly induced by inflammatory signals, particularly interferons, linking it to innate immunity and cholesterol homeostasis. Its product, 25-HC, also influences membrane properties and suppresses viral entry. CH25H interacts with Cytochrome P450 7A1 (CYP7A1), the rate-limiting enzyme in bile acid synthesis, by producing 25-HC to inhibit CYP7A1 activity, thereby linking cholesterol oxidation to bile acid regulation. CH25H-derived 25-HC binds and inhibits CYP7A1 to suppress bile acid production. Thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human CH25H and recombinant mouse CYP7A1 . Briefly, biotin-linked CH25H were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100μl were then transferred to CYP7A1-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 CH25H and CYP7A1 was shown in Figure 1, the EC50?for this effect is 0.139 μg/mL.