title: A new enzyme-linked immunosorbent assay (ELISA) for human free and bound kallikrein 9.
abstract: BACKGROUND
  Kallikrein 9 (KLK9) is a member of the human kallikrein-related peptidases family, whose physiological role and implications in disease processes remain unclear. The active form of the enzyme is predicted to have chymotryptic activity. In the present study, we produced for the first time the active recombinant protein and monoclonal antibodies, and developed novel immunoassays for the quantification of free and bound KLK9 in biological samples.
  
  
  METHODS
  The coding sequence of mature KLK9 isoform (mat-KLK9) was expressed in an Expi293F mammalian system and the synthesized polypeptide was purified through a two-step protocol. The purified protein was used as an immunogen for production of monoclonal antibodies in mice. Hybridomas were further expanded and antibodies were purified. Newly-produced monoclonal antibodies were screened for reaction with the KLK9 recombinant protein by a state-of-the-art immunocapture/parallel reaction monitoring mass spectrometry-based methodology.
  
  
  RESULTS
  Anti-KLK9 antibodies were combined in pairs, resulting in the development of a highly sensitive (limit of detection: 15 pg/mL) and specific (no cross-reactivity with other KLKs) sandwich-type ELISA. Highest KLK9 protein levels were found in tonsil and sweat and lower levels in the heart, kidney and liver. Hybrid immunoassays using an anti-KLK9 antibody for antigen capture and various anti-serine protease inhibitor polyclonal antibodies, revealed the presence of an a1-antichymotrypsin-bound KLK9 isoform in biological samples.
  
  
  CONCLUSIONS
  The ELISAs for free and bound forms of KLK9 may be highly useful for the detection of KLK9 in a broad range of biological samples, thus enabling the clarification of KLK9 function and use as a potential disease biomarker.
authors.affiliation: Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada.|Department of Clinical Biochemistry, University Health Network, Toronto, Canada.
authors.forename: Panagiota
authors.initials: P
authors.lastname: Filippou
authors.affiliation: Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada.|Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada.
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Korbakis
authors.affiliation: Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada.|Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Canada.
authors.forename: Sofia
authors.initials: S
authors.lastname: Farkona
authors.affiliation: Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Canada.
authors.forename: Antoninus
authors.initials: A
authors.lastname: Soosaipillai
authors.affiliation: Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada.|Department of Clinical Biochemistry, University Health Network, Toronto, Canada.
authors.forename: Theano
authors.initials: T
authors.lastname: Karakosta
authors.affiliation: Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada.|Department of Clinical Biochemistry, University Health Network, Toronto, Canada.|Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada.|Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Canada.|Mount Sinai Hospital, Joseph & Wolf Lebovic Ctr., 60 Murray St [Box 32], Flr 6 - Rm L6-201, Toronto, ON M5T 3L9 Canada.
authors.forename: Eleftherios P
authors.identifier: 0000-0002-1589-820X
authors.initials: EP
authors.lastname: Diamandis
keywords: ELISA; Hybrid assays; Immunocapture; Kallikrein 9; Kallikreins; PRM; Serine protease inhibitors; a1-Antichymotrypsin