A new enzyme-linked immunosorbent assay (ELISA) for human free and bound kallikrein 9

Panagiota Filippou, Dimitrios Korbakis, Sofia Farkona, Antoninus Soosaipillai, Theano Karakosta, Eleftherios P. Diamandis

    Research output: Contribution to journalArticle

    5 Citations (Scopus)

    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.

    Original languageEnglish
    Pages (from-to)1-12
    Number of pages12
    JournalClinical Proteomics
    Volume14
    Issue number1
    DOIs
    Publication statusPublished - 17 Jan 2017

    Fingerprint Dive into the research topics of 'A new enzyme-linked immunosorbent assay (ELISA) for human free and bound kallikrein 9'. Together they form a unique fingerprint.

  • Cite this

    Filippou, P., Korbakis, D., Farkona, S., Soosaipillai, A., Karakosta, T., & Diamandis, E. P. (2017). A new enzyme-linked immunosorbent assay (ELISA) for human free and bound kallikrein 9. Clinical Proteomics, 14(1), 1-12. https://doi.org/10.1186/s12014-017-9140-6