Complement Component 3 (C3) is a vital component of the complement system that plays a central role in immune response and inflammation. C3 is a β₂ globulin synthesized by the liver, whose structure consists of two polypeptide chains, α and β, and is the most abundant complement component in serum. It's been identified that C3 can interact with Complement Factor B (CFB), thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human C3 and recombinant mouse CFB. Briefly, CFB was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to C3-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 450/630 nm immediately. When Recombinant C3 is immobilized at 2 ug/mL (100 uL/well), the concentration of CFB that produces 50% optimal binding response is found to be approximately 5.96 ug/mL.