Abstract
BackgroundKallikrein 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.MethodsThe 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.ResultsAnti-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.ConclusionsThe 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.
Highlights
Kallikrein 9 (KLK9) is a member of the human kallikrein-related peptidases family, whose physiological role and implications in disease processes remain unclear
We describe the production and characterization of mouse monoclonal antibodies against the mature kallikrein 9 (KLK9) form and the development of a highly sensitive and specific ELISA assay for the free monomer
We developed an ELISA that measures the inhibitor-bound KLK9 form, through a hybrid assay that includes a1-antichymotrypsin antibodies
Summary
Kallikrein 9 (KLK9) is a member of the human kallikrein-related peptidases family, whose physiological role and implications in disease processes remain unclear. Human tissue kallikrein-related peptidases (KLKs) constitute the largest family of secreted serine proteases, with diverse expression patterns and physiological roles [1]. Human tissue kallikrein 9 (KLK9), which was originally identified as the KLK-L3 gene, spans an area of 7.1 kb on chromosome 19, flanked by the KLK8 and KLK10 genes [4]. AF135026) contains five coding exons and the encoded KLK9 protein (UniProt accession: Q9UKQ9 (KLK9_ HUMAN)) is predicted to be synthesized as a pre-proenzyme (1–250 amino acids) which is processed to the mat-KLK9 (lacking the signal peptide and the pro-segment) [4, 5].
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
