Tumor Necrosis Factor Related Apoptosis Inducing Ligand (TRAIL), encoded by the TNFSF10 gene, is a type II transmembrane protein belonging to the tumor necrosis factor (TNF) superfamily. It primarily functions to induce selective apoptosis in transformed or cancerous cells while sparing most normal cells, making it a promising candidate for antitumor therapy. TRAIL exerts its pro-apoptotic effects by binding to specific death receptors on the cell surface, triggering the extrinsic apoptotic signaling cascade, including caspase activation and subsequent cellular breakdown. Its selective cytotoxicity offers significant advantages over many conventional chemotherapeutic agents, attracting extensive research in oncology and immunotherapy. TRAIL and FAS are both death receptor ligands in the TNF superfamily and can cooperatively activate extrinsic apoptotic pathways.Thus a functional ELISA assay was conducted to detect the interaction of recombinant mouse TRAIL and recombinant mouse FAS. Briefly, TRAIL was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to FAS-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 mouse TRAIL and recombinant mouse FAS was shown in Figure 1, the EC50?for this effect is 0.0031μg/mL.