Complement Component 5 (C5), which forms part of the later stages of the complement pathway, is cleaved?into?C5a and C5b. C5a is a chemotactic agent for neutrophils, while?C5b?forms?part?of?the?complement?membrane?attack?complex. Coagulation Factor II (F2) has been identified as an interactor of C5, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human C5 and recombinant mouse F2. Briefly, C5 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to F2-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-C5 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 C5 and recombinant mouse F2 was shown in Figure 1, the EC50?for this effect is 0.22 ug/mL.