Receptor activator of nuclear factor kappa B ligand (RANkL) is a member of the TNF superfamily of proteins. RANkL is expressed by macrophages and osteoblasts, and functions as an activator of RANK and is an important factor in osteoclast differentiation, activation and survival. Cathepsin K (CTSK) plays an essential role in osteoclastic bone resorption and CTSK levels increase with osteoclast differentiation and activation, a process that is controlled by RANkL. Thus a functional binding ELISA assay was conducted to detect the interaction of recombinant mouse RANkL and recombinant human CTSK. Briefly, CTSK was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to RANkL-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-CTSK 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 result was shown in Figure 1. When recombinant mouse RANkL is lmmobilized at 2 ug/mL (100 ul/well), the concentration of recombinant human CTSK that produces 50% optimal binding response is found to be approximately 2.17 ug/mL.