Complement Component 4a (C4a) is a component of the complement system, which is a cleavage product of the complement C4 protein. C4a has been implicated in various inflammatory and immune responses. It acts as a chemoattractant, recruiting immune cells such as neutrophils and macrophages to the site of inflammation. Additionally, C4a can stimulate the release of pro-inflammatory cytokines and chemokines, further amplifying the immune response. C4a and C4b are the two subunits of C4, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human C4a and recombinant mouse C4b. Briefly, C4a was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to C4b-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-C4a 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 C4a and recombinant mouse C4b was shown in Figure 1,and this effect was in a dose dependent manner.