Homing Associated Cell Adhesion Molecule (HCAM), also known as CD44, is a ubiquitous multistructural and multifunctional cells surface adhesion molecule involved in cell-cell and cell-matrix interactions. CD44 is broadly expressed, including in the membranes of B cells, granulocytes, monocytes, and erythrocytes as well as on many thymocytes and mature T cells, besides it is highly expressed in many cancers and regulates metastasis via recruitment of CD44 to the cell surface. This protein is a receptor for hyaluronic acid (HA) and can also interact with other ligands, such as osteopontin, collagens, and matrix metalloproteinases (MMPs). Thus a functional binding ELISA assay was conducted to detect the interaction of recombinant rat HCAM and biotinylated hyaluronan (HA). Briefly, biotin-linked HA were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100 ul were then transferred to HCAM-coated microtiter wells and incubated for 2h at 37℃. Wells were washed with PBST 3 times and incubation with Streptavidin-HRP for 1 hour, 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 450/630 nm immediately. The binding activity of recombinant rat HCAM and biotinylated HA was shown in Figure 1, and this effect was in a dose dependent manner.