S100?calcium-binding protein A8?(S100A8)?also known as calgranulin A, is a member of the S100 family of proteins containing 2 EF-hand calcium-binding motifs. S100 proteins are localized in the cytoplasm and/or nucleus of a wide range of cells, and involved in the regulation of a number of cellular processes such as cell cycle progression and differentiation. Besides, S100 Calcium Binding Protein A9 (S100A9) has been identified as an interactor of S100A8, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant rat S100A8 and recombinant rat S100A9. Briefly, S100A8 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100 ul were then transferred to S100A9-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-NE 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 rat S100A8 and recombinant rat S100A9 was shown in Figure 1, the EC50?for this effect is 0.82 ug/mL.