Tumor Necrosis Factor Related Apoptosis Inducing Ligand (TRAIL) is a member of the tumor necrosis factor (TNF) superfamily, mainly functioning to induce selective apoptosis in cancer cells while sparing normal cells. As a type II transmembrane protein, TRAIL binds to death receptors on the cell membrane, initiating the extrinsic apoptotic pathway. This selective cytotoxicity makes TRAIL a promising candidate for tumor immunotherapy. Upon receptor activation, TRAIL triggers a series of intracellular signaling events, including caspase activation and mitochondrial dysfunction, leading to cell death. Dysregulation of TRAIL signaling is associated with tumor progression and drug resistance. TRAIL recruits FADD through death domains to form the death-inducing signaling complex (DISC). Thus a functional ELISA assay was conducted to detect the interaction of recombinant human TRAIL and recombinant human FADD. Briefly, TRAIL was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to FADD-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-TRAIL 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 TRAIL and recombinant human FADD was shown in Figure 1, the EC50?for this effect is 0.00027 μg/mL.