Tumor Necrosis Factor Receptor Superfamily, Member 9 (TNFRSF9), also named CD137/4-1BB, acts as a type I transmembrane costimulatory receptor. Expressed mainly on activated T cells and also on B cells, monocytes and transformed cell lines, it mediates T cell clonal expansion, survival and development by activating the NF-κB signaling pathway via TRAF adaptor proteins. It enhances CD8 T cell effector functions, modulates Th1 immune responses and induces monocyte proliferation, playing a pivotal role in anti-tumor and anti-viral immunity. Dysregulated TNFRSF9 expression is linked to autoimmune diseases and tumor progression, making it a promising target for cancer immunotherapy. TNFRSF9 binds trimeric TNFSF9 on APCs via extracellular cysteine-rich domains to trigger bidirectional immune signaling.Briefly, TNFSF9 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μL were then transferred to TNFRSF9-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-TNFSF9 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. Measured by its binding ability in a functional ELISA. When Recombinant human TNFRSF9 is lmmobilized at 2 µg/mL(100 µLwell), the concentration of human TNFSF9 that produces 50% optimal bindingresponse is found to be approximately 0.048 µg/mL.