Tumor Necrosis Factor Alpha (TNFa) is a pleiotropic pro-inflammatory cytokine primarily produced by macrophages and lymphocytes. As a key mediator of systemic inflammation, it binds to TNF receptors (TNFR1/R2) to activate NF-κB and MAPK pathways, regulating immune responses, cell survival, and apoptosis. Clinically, TNFa is implicated in autoimmune diseases and anti-tumor therapies, with its inhibitors widely used clinically. TNFa interacts directly with TNFaIP3, an ubiquitin-editing enzyme that terminates TNFa-induced NF-κB activation through deubiquitination, forming a critical negative feedback loop to control excessive inflammation.Thus a functional binding ELISA assay was conducted to detect the interaction of recombinant mouse TNFa and recombinant human TNFaIP3 .Briefly, biotin-linked TNFa were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100µl were then transferred to TNFaIP3-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST 3 times and incubation with Streptavidin-HRP for 30min, then wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50µl stop solution to the wells and read at 450nm immediately. The binding activity of recombinant mouse TNFa and recombinant human TNFaIP3 was shown in Figure 1, the EC50 for this effect is 0.67 µg/mL.