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DNA-linked Inhibitor Antibody Assay (DIANA) for sensitive and selective enzyme detection and inhibitor screening

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    0475093 - ÚOCHB 2018 RIV GB eng J - Journal Article
    Navrátil, Václav - Schimer, Jiří - Tykvart, Jan - Knedlík, Tomáš - Vik, V. - Majer, Pavel - Konvalinka, Jan - Šácha, Pavel
    DNA-linked Inhibitor Antibody Assay (DIANA) for sensitive and selective enzyme detection and inhibitor screening.
    Nucleic Acids Research. Roč. 45, č. 2 (2017), č. článku e10. ISSN 0305-1048. E-ISSN 1362-4962
    R&D Projects: GA MŠMT LO1302
    Institutional support: RVO:61388963
    Keywords : quantitative PCR * enzyme detection * inhibitor screening
    OECD category: Biochemical research methods
    Impact factor: 11.561, year: 2017

    Human diseases are often diagnosed by determining levels of relevant enzymes and treated by enzyme inhibitors. We describe an assay suitable for both ultrasensitive enzyme quantification and quantitative inhibitor screening with unpurified enzymes. In the DNA-linked Inhibitor ANtibody Assay (DIANA), the target enzyme is captured by an immobilized antibody, probed with a small-molecule inhibitor attached to a reporter DNA and detected by quantitative PCR. We validate the approach using the putative cancer markers prostate-specific membrane antigen and carbonic anhydrase IX. We show that DIANA has a linear range of up to six logs and it selectively detects zeptomoles of targets in complex biological samples. DIANA's wide dynamic range permits determination of target enzyme inhibition constants using a single inhibitor concentration. DIANA also enables quantitative screening of small-molecule enzyme inhibitors using microliters of human blood serum containing picograms of target enzyme. DIANA's performance characteristics make it a superior tool for disease detection and drug discovery.
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    0475093.pdf41.8 MBPublisher’s postprintopen-access
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