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Nucleic acids research v.45 no.2, 2017년, pp.e10 - e10   SCI SCIE
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DNA-linked Inhibitor Antibody Assay (DIANA) for sensitive and selective enzyme detection and inhibitor screening

Navráátil, Václav (Gilead Sciences and IOCB Research Centre, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, 166 10, Czech Republic ) ; Schimer, Jiáářááří (Gilead Sciences and IOCB Research Centre, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, 166 10, Czech Republic ) ; Tykvart, Jan (Gilead Sciences and IOCB Research Centre, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, 166 10, Czech Republic ) ; Knedlááříášík, Tomááříáááříáš (Gilead Sciences and IOCB Research Centre, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, 166 10, Czech Republic ) ; Vik, Viktor (Department of Urology, Thomayer Hospital in Prague, Prague, 140 59, Czech Republic ) ; Majer, Pavel (Gilead Sciences and IOCB Research Centre, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, 166 10, Czech Republic ) ; Konvalinka, Jan (Gilead Sciences and IOCB Research Centre, Institute of Organic Chemistry and Bio ) ; ááříášíŠááříášíŠácha, Pavel ;
  • 초록  

    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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