The development and characterization of an ELISA specifically detecting the active form of cathepsin K

Abstract

ObjectiveCathepsin K plays essential roles in bone resorption and is intensely investigated as a therapeutic target for the treatment of osteoporosis. Hence an assessment of the active form of cathepsin K may provide important biological information in metabolic bone diseases, such as osteoporosis or ankylosing spondylitis. MethodsPresently there are no robust assays for the assessment of active cathepsin K in serum, and therefore an ELISA specifically detecting the N-terminal of the active form of cathepsin K was developed. ResultsThe assay was technically robust, with a lowest limit of detection (LOD) of 0.085ng/mL. The average intra- and inter-assay CV% were 6.60% and 8.56% respectively. The dilution recovery and spike recovery tests in human serum were within 100±20% within the range of the assay.A comparison of latent and active cathepsin K confirmed specificity towards the active form. Quantification of the levels of active cathepsin K in supernatants of purified human osteoclasts compared to corresponding macrophages showed a 30-fold induction (p<0.001).In contrast, in serum samples from osteoporotic women on estrogen or bisphosphonate therapy and from ankylosing spondylitis patients no clinically relevant differences were observed. ConclusionIn summary, we have developed a robust and sensitive assay specifically detecting the active form of cathepsin K; however, while it monitors osteoclasts with high specificity in vitro, it appears that circulating levels of active cathepsin K do not reflect bone changes under these circumstances.