TNFa (Tumor necrosis factor), is mainly secreted by macrophages and can induce cell death of certain tumor cell lines. It has been reported that TNFa can inhibit the proliferation and induce apoptosis of A549 cells, besides, the concentration of IL-1β and IL-8 in cell supernatant will increase after stimulation. Therefore, a stimulation assay of TNFa was conducted using A549 cells. Briefly, A549 cells were incubated in DMEM with different concentrations of TNFa (1ng/mL, 10ng/mL, 100ng/mL, 1000ng/mL) for 8h, after which the concentration of IL-1β and IL-8 in the cell supernatant were detected by ELISA. IL-1β and IL-8 levels in the cell supernatant of A549 cells increased significantly after stimulated with IL-1β, the data was shown in Figure 1 and Figure 2 separately.

Tumor necrosis factor alpha (TNF-a), also known as cachectin and TNFSF2, is mainly secreted by macrophages and is the prototypic ligand of the TNF superfamily. TNF-a plays important roles in processes such as immunomodulation, fever, inflammatory response, inhibition of tumor formation and inhibition of viral replication. TNFa signals through two distinct cell surface receptors, TNFR1 (TNFRSF1A, CD120a, p55) and TNFR2 (TNFRSF1B, CD120b, p75). Thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human TNF-a and recombinant mouse TNFRSF1B. Briefly, TNF-a 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-TNF-a 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 450/630 nm immediately. The binding activity of recombinant human TNF-a and recombinant mouse TNFRSF1B was shown in Figure 1, the EC50 for this effect is 0.11 ug/mL.