Inhibitor Of Kappa-Light Polypeptide Gene Enhancer In B-Cells Kinase Beta (IkBKb), a subunit of IKK, plays a crucial role in intracellular signaling, especially in inflammation and immune responses. In addition, the binding of TRAF6 to IkBKB is an important link in the immune response, which is not only involved in the initiation of inflammatory signals, but also in its regulation, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human IkBKb and recombinant mouse TRAF6. Briefly, IkBKb was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to TRAF6-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-IkBKb 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 IkBKb and recombinant mouse TRAF6 was shown in Figure 1, the EC50?for this effect is 0.11 ug/mL.