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.