Tumor Necrosis Factor Alpha (TNFa) is a pro-inflammatory cytokine primarily produced by activated macrophages and immune cells. It plays a pivotal role in regulating inflammation, immune responses, and apoptosis. TNFa binds to its receptors (TNFR1/2), triggering signaling pathways such as NFkB and MAPK, which influence cell survival, proliferation, and cytokine production. Dysregulation of TNFa is associated with autoimmune diseases , cancer, and metabolic disorders.TNFa can interact with TIMP3, which inhibits TNFa-converting enzyme (TACE), thereby reducing TNFa release and modulating inflammation.Thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human TNFa and recombinant human TIMP3 . Briefly, biotin-linked TNFa were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100µl were then transferred to TIMP3-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 TNFa and TIMP3 was shown in Figure 1, the EC50 for this effect is 0.076 µg/mL.