Caspase Activated DNase (CAD), corresponding to product code RPB058Hu01, is a key nuclease in eukaryotic apoptosis. Normally, it exists in an inactive state in the cytoplasm, bound to its inhibitor. Upon apoptotic signal activation, effector caspases (e.g., Caspase-3) cleave the inhibitor, releasing active CAD. The activated enzyme translocates to the nucleus, where it cleaves chromosomal DNA into nucleosome-sized fragments, a hallmark of apoptotic cell death. Its strict regulation prevents unintended DNA damage, ensuring genomic stability in healthy cells. CAD interacts with DFFa (product code L426Hu) as its specific inhibitor; DFFa binds CAD to inhibit its nuclease activity in non-apoptotic cells.Thus a functional ELISA assay was conducted to detect the interaction of recombinant human CAD and recombinant human DFFa. Briefly, CAD was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to DFFa-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-CAD pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody for 1h at 37℃, 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 450/630nm immediately. The binding activity of recombinant human CAD and recombinant human DFFa was shown in Figure 1, the EC50?for this effect is 0.01067μg/mL.