Tumor Necrosis Factor Alpha (TNFα, designated A133Hu) is a pivotal pro-inflammatory cytokine involved in immune regulation and host defense. Discovered for its ability to induce tumor cell necrosis, it is primarily secreted by activated macrophages and T lymphocytes. TNFα mediates diverse biological processes, including inflammation, cell proliferation, differentiation, and apoptosis, by binding to specific cell surface receptors. Dysregulated TNFα expression is linked to numerous inflammatory and autoimmune diseases, such as rheumatoid arthritis and Crohn’s disease, making it a key therapeutic target. TNFα (A133Hu) binds directly to mouse-derived TNFRSF1B (B504Mu), initiating downstream signaling pathways that modulate immune and inflammatory responses.Thus a functional ELISA assay was conducted to detect the interaction of recombinant human TNFα and recombinant human TNFRSF1B. Briefly, TNFα was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to TNFRSF1B-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti- TNFα 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 rat TNFα and recombinant human TNFRSF1B was shown in Figure 1, the EC50 for this effect is 0.17655µg/mL.