Complement factor B (CFB), a 95-kDa protein, is a crucial catalytic element of the alternative pathway (AP) of complement. After binding of CFB to C3b, activation of the AP depends on the proteolytic cleavage of CFB by factor D to generate the C3 convertase (C3bBb). The C3 convertase contains the catalytic subunit of CFB (Bb), the enzymatic site for the cleavage of a new molecule of C3 into C3b. Thus, a binding ELISA assay was conducted to detect the association of recombinant mouse CFB with recombinant mouse CFD. Briefly, biotin-linked recombinant mouse CFB were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to CFD-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 mouse CFB and recombinant mouse CFD was shown in Figure 1, the EC50 for this effect is 1.238 ug/mL.