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 b2 globulin synthesized by the liver, whose structure consists of two polypeptide chains, a and b, and is the most abundant complement component in serum. It has reported that P-Selectin (SELP) can interact with C3, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant mouse C3 and recombinant human SELP. Briefly, biotin-linked SELP were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100 ul were then transferred to C3-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. When Recombinant mouse C3 is lmmobilized at 2 ug/mL (100 uL/well), the concentration of human SELP that produces 50% optimal binding response is found to be approximately 3.75 ug/mL.