Tumor necrosis factor receptor superfamily member 1B (TNFRSF1B), also known as tumor necrosis factor receptor 2 (TNFR2) and CD120b, is a membrane receptor that binds tumor necrosis factor-alpha (TNFα). This protein and TNF-receptor 1 form a heter°Complex that mediates the recruitment of two anti-apoptotic proteins, c-IAP1 and c-IAP2, which possess E3 ubiquitin ligase activity. Besides, Tumor Necrosis Factor Alpha (TNFa) has been identified as an interactor of TNFRSF1B, thus a binding ELISA assay was conducted to detect the interaction of recombinant human TNFRSF1B and recombinant human TNFa. Briefly, TNFRSF1B were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100uL were then transferred to TNFa-coated microtiter wells and incubated for 2h at 37°C. Wells were washed with PBST and incubated for 1h with anti-TNFRSF1B pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody, wells were aspirated and washed 3 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37°C. Finally, add 50µL stop solution to the wells and read at 450nm immediately. The binding activity of TNFRSF1B and TNFa was shown in Figure 1, and this effect was in a dose dependent manner.

(A) A549 cells cultured in DMEM contain 1μg/mL TNFα and 1ng/mL TNFRSF1B for 96h; (B) A549 cells cultured in DMEM only contain 1μg/mL TNFα for 96h.