Figure 1. The binding activity of CYP3A4 with HSPA8.
Cytochrome P450 3A4 (CYP3A4) is an important enzyme in the body, mainly found in the liver and in the intestine. It oxidizes small foreign organic molecules (xenobiotics), such as toxins or drugs, so that they can be removed from the body. CYP3A4 is a member of the cytochrome P450 family of oxidizing enzymes and the CYP3A4 is the most common and the most versatile one in drug metabolism. Besides, Heat Shock 70kDa Protein 8 (HSPA8) has been identified as an interactor of CYP3A4, thus a binding ELISA assay was conducted to detect the interaction of recombinant human CYP3A4 and recombinant human HSPA8. Briefly, CYP3A4 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100uL were then transferred to HSPA8-coated microtiter wells and incubated for 2h at 37℃. Wells were washed with PBST and incubated for 1h with anti-CYP3A4 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 CYP3A4 and HSPA8 was shown in Figure 1, and this effect was in a dose dependent manner.