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 mouse S100A8 and recombinant rat S100A9. Briefly, biotin-linked 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 3 times and incubation with Streptavidin-HRP for 30min, then 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 nm immediately. The binding activity of recombinant mouse S100A8 and recombinant rat S100A9 was shown in Figure 1, the EC50 for this effect is 0.2 ug/mL.