Complement Component 3a (C3a) is a component of the complement system, this protein is also known as the C3a anaphylatoxin, modulates inflammation and possesses antimicrobial activity. It's been identified that C3A can interact with Complement Factor I (CFI), thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human C3a and recombinant human CFI. Briefly, C3a was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to CFI-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-C3a 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 C3a and recombinant human CFI was shown in Figure 1, the EC50 for this effect is 0.03 ug/mL.