Alpha-Fetoprotein (AFP), as known as fetal alpha globulin or alpha-1-fetoprotein, is a glycoprotein belonging to the albumin family that is produced mainly by the fetal liver and yolk sac during pregnancy. Its primary function in the fetus is to transport calcium and fatty acids across the placenta, supporting fetal growth and development. In adults, elevated AFP levels can indicate certain medical conditions, including liver cancer and other diseases. Besides, Coagulation Factor II (F2) has been identified as an interactor of AFP, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human AFP and recombinant human F2. Briefly, AFP was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to F2-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-AFP 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 human AFP and recombinant human F2 was shown in Figure 1, the EC50?for this effect is 1.06 ug/mL.