Interleukin-9 (IL-9), also known as P40 and MEA (mast cell growth-enhancing activity), is a 30-40 kDa protein which was originally identified in mice as a T cell growth factor and is a member of the common γ-chain-receptor cytokine family, with other members including IL-2, IL-4, IL-7, IL-15 and IL-21. IL-9 is produced predominantly by helper T cells such as Th2 and Th9 cells. It normally functions through the activation of a JAK/STAT pathway and plays a critical role in immunity and the pathogenesis of cancer. S100 Calcium Binding Protein A6 (S100A6) is a high affinity receptor for IL-9. Thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human IL-9 and recombinant mouse S100A6. Briefly, IL-9 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to S100A6-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-IL9 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 IL-9 and recombinant mouse S100A6 was shown in Figure 1, the EC50?for this effect is 0.09 ug/mL.