Tumor Necrosis Factor Alpha (TNFa), also known as TNF-α, is a potent pro-inflammatory cytokine that plays a central role in the body's immune response, cell survival, and apoptosis. It is primarily produced by activated macrophages, but also by other cell types including lymphocytes, neutrophils, and endothelial cells. TNFa exists in two forms: a transmembrane precursor and a soluble cytokine, both of which are biologically active and mediate their effects by binding to its receptors, TNFR1 and TNFR2.Its functions are diverse and crucial. In its beneficial role, TNFa helps orchestrate the inflammatory response to combat infections and can induce the death of tumor cells, which is the origin of its name. It is essential for the proper functioning of the immune system. However, TNFa is a classic "double-edged sword." Dysregulated or excessive production of TNFa is a key driver of pathological inflammation and is heavily implicated in the pathogenesis of a wide range of autoimmune and chronic inflammatory diseases, such as rheumatoid arthritis, inflammatory bowel disease (Crohn's disease), and psoriasis. Consequently, TNFa has become a major therapeutic target, and drugs that inhibit its action (e.g., infliximab, adalimumab) are widely used to treat these conditions, highlighting its profound clinical significance.To detect the activity of recombinant TNFa, a functional ELISA assay was performed to evaluate the interaction between recombinant mouse TNFa and recombinant human TNFR1.Briefly, biotin-linked TNFa were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100µl were then transferred to TNFR1-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 TNFR1 was shown in Figure 1, the EC50 for this effect is 0.26852µg/mL.