Protein S (PROS) is a vitamin K-dependent plasma protein functions as a cofactor for the anticoagulant protease, activated protein C (APC) to inhibit blood coagulation.It acts as a cofactor for activated protein C, which inactivates factors Va and VIIIa, thus reducing the risk of thrombosis.Coagulation factor VIII (F8) binds to protein S (PROS) to participate in the clotting process, regulating thrombin production and the conversion of fibrinogen to fibrin, thereby maintaining normal blood clotting function.Thus a functional ELISA assay was conducted to detect the interaction of recombinant human PROS and recombinant dog F8.Briefly, PROS was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to F8-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-PROS 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 human PROS and recombinant dog F8 was shown in Figure 1, the EC50 for this effect is 0.058ug/mL.