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.

Department of Biochemistry Faculty of Science Charles University Prague Prague 128 43 Czech Republic

Department of Urology Thomayer Hospital Prague Prague 140 59 Czech Republic

Gilead Sciences and IOCB Research Centre Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic Prague 166 10 Czech Republic

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Building the SynBio community in the Czech Republic from the bottom up: You get what you give

DNA-linked inhibitor antibody assay (DIANA) as a new method for screening influenza neuraminidase inhibitors