Cytochrome P450 3A4 (CYP3A4), a member of the cytochrome P450 superfamily of heme - containing enzymes, is predominantly expressed in the human liver and small intestine, serving as a key player in xenobiotic metabolism and endobiotic biotransformation. It catalyzes the oxidation of over 50% of clinically used drugs, including statins, immunosuppressants, and anticancer agents, by adding oxygen atoms to hydrophobic substrates, thereby facilitating their excretion. CYP3A4 expression and activity are highly variable among individuals due to genetic polymorphisms, environmental inducers (e.g., rifampicin, phenobarbital) and inhibitors (e.g., ketoconazole, grapefruit juice). Beyond drug metabolism, it participates in the breakdown of endogenous compounds such as steroids and bile acids, maintaining physiological homeostasis. Notably, CYP3A4 and CYP1A1, both phase I metabolic enzymes, exhibit potential functional binding to synergistically modulate the metabolism of shared substrates, with their interaction being a critical part of the hepatic metabolic network.To detect the activity of recombinant CYP3A4, a functional ELISA assay was performed to evaluate the interaction between recombinant human CYP3A4 and recombinant human CYP1A1.Briefly, biotin-linked CYP3A4 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100µl were then transferred to CYP1A1-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 CYP3A4 and CYP1A1 was shown in Figure 1, the EC50 for this effect is 0.03849µg/mL..