Homing Associated Cell Adhesion Molecule (HCAM), also known as CD44, is a ubiquitously expressed transmembrane glycoprotein mediating cell responses to the extracellular microenvironment. CD44 is the major surface hyaluronan (HA) receptor, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human CD44 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 CD44-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 human CD44 and biotinylated HA was shown in Figure 1, the EC50?for this effect is 0.08 ug/mL.