Afamin (AFM, also known as Alpha -Albumin), a bioactive protein, is expressed in the liver and secreted into the bloodstream. Human afamin is a 87?kDa protein which is encoded by the fourth member of the human albumin gene family. Mouse AFM shares 66% aa sequence identity with Full-length human AFM. This protein can bind and transport vitamin E across the blood-brain barrier in body fluids under conditions where the lipoprotein system is not sufficient, it also can act as a carrier for hydrophobic molecules in body fluids. Essential for the solubility and activity of lipidated Wnt family members. Thus a functional binding ELISA assay was conducted to detect the interaction of recombinant mouse AFM and recombinant human WNT3A. Briefly, AFM was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to WNT3A-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-AFM 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 mouse AFM and recombinant human WNT3A was shown in Figure 1, the EC50?for this effect is 0.01 ug/mL.