Complement factor B (CFB), a 95-kDa protein, is a crucial catalytic element of the alternative pathway (AP) of complement. Upon cleavage by Complement Factor D (CFD), CFB splits into fragments Ba (inactive) and Bb, the latter forming the active C3 convertase (C3bBb). This enzyme amplifies complement activation by cleaving C3 into C3a/C3b, driving inflammation, opsonization, and membrane attack complex formation. CFB deficiency disrupts immune defense, linking it to infections and autoimmune disorders like atypical hemolytic uremic syndrome (aHUS).Thus a functional ELISA assay was conducted to detect the interaction of recombinant simina CFB and recombinant mouse CFD. Briefly, CFB was 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 and incubated for 1h with anti-CFB 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 450/630nm immediately. The binding activity of recombinant simina CFB and recombinant mouse CFD was shown in Figure 1, the EC50 for this effect is 0.015 ug/mL.