Complement Component 4b (C4b) is a component of the complement system, which is activated by the recognition of foreign pathogens, such as bacteria and viruses, or altered self-cells. In the classical activation pathway of complement, C4b is produced by the proteolytic cleavage of the precursor protein C4 by the activated enzyme C1s, and it can bind to C2a to form C3 invertase (C4b2a) which is responsible for the cleavage of C3. Besides, the binding of MASP2 to C4b is an important step in the lectin pathway of the complement system, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant mouse C4b and recombinant human MASP2. Briefly, C4b was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to MASP2-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-C4b 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 C4b and recombinant human MASP2 was shown in Figure 1, the EC50 for this effect is 1.39 ug/mL.