S100 calcium-binding protein 9 (S100A9) also known as calgranulin B, is a member of the S100 family of secreted calcium binding 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. Lactoferrin (LTF) is one of targets of S100A9, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant mouse S100A9 and recombinant human LTF. Briefly, S100A9 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to LTF-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-S100A9 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 mouse S100A9 and recombinant human LTF was shown in Figure 1, the EC50 for this effect is 0.44 ug/mL.