Complement Component 9 (C9), a component of the Membrane attack Complex (MAC) plays a key role in the innate and adaptive immune response by forming pores in the plasma membrane of target cells. During MAC assembly, multiple copies of C9 are sequentially recruited to membrane associated C5b8 to form a pore. Thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human C9 and recombinant human C5. Briefly, biotin-linked C9 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100 ul were then transferred to C5-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 human C9 and recombinant human C5 was shown in Figure 1, the EC50 for this effect is 0.13 ug/mL.