title: The development and characterization of an ELISA specifically detecting the active form of cathepsin K.
abstract: OBJECTIVE
  Cathepsin 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.
  
  
  METHODS
  Presently 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.
  
  
  RESULTS
  The assay was technically robust, with a lowest limit of detection (LOD) of 0.085 ng/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.
  
  
  CONCLUSIONS
  In 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.
authors.affiliation: Nordic Bioscience Biomarkers and Research, Herlev, Denmark. Electronic address: ssu@nordicbioscience.com.
authors.forename: S
authors.initials: S
authors.lastname: Sun
authors.forename: M A
authors.initials: MA
authors.lastname: Karsdal
authors.forename: A C
authors.initials: AC
authors.lastname: Bay-Jensen
authors.forename: M G
authors.initials: MG
authors.lastname: Sørensen
authors.forename: Q
authors.initials: Q
authors.lastname: Zheng
authors.forename: M H
authors.initials: MH
authors.lastname: Dziegiel
authors.forename: W P
authors.initials: WP
authors.lastname: Maksymowych
authors.forename: K
authors.initials: K
authors.lastname: Henriksen
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D050071:Bone Density Conservation Agents / Q000627:therapeutic use; D001842:Bone and Bones / Q000201:enzymology / Q000473:pathology; D056657:Cathepsin K / Q000097:blood*; D004164:Diphosphonates / Q000627:therapeutic use; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D008264:Macrophages / Q000166:cytology / Q000201:enzymology; D051379:Mice; D008875:Middle Aged; D010010:Osteoclasts / Q000166:cytology / Q000201:enzymology*; D010024:Osteoporosis / Q000097:blood* / Q000188:drug therapy / Q000201:enzymology / Q000473:pathology; D012680:Sensitivity and Specificity; D013167:Spondylitis, Ankylosing / Q000097:blood* / Q000201:enzymology / Q000473:pathology
chemical_list: D000911:Antibodies, Monoclonal; D050071:Bone Density Conservation Agents; D004164:Diphosphonates; D056657:Cathepsin K
keywords: AS; Active form; BASDAI; Bone resorption; CV; Cathepsin K; LOD; OP; QC; SEM; Serum; ankylosing spondylitis; bath ankylosing spondylitis disease activity index; coefficient of variation; lowest limit of detection; mSASSS; modified stoke ankylosing spondylitis spinal score; osteoporosis; quality control; standard error of the mean