1. Ultra-Sensitive Methods for Pathogen and Cancer Cell Detection Andrew D. Ellington, Ph.D. Department of Chemistry and Biochemistry

2. Opportunities  The sensitive detection of pathogens has broad utility: Homeland securityHomeland security Food safetyFood safety Personal healthcarePersonal healthcare  The early detection of cancer is critical: Increased effectiveness of therapeutic interventionsIncreased effectiveness of therapeutic interventions Reduced health care costs and malpractice exposureReduced health care costs and malpractice exposure  The growth in relevant markets: Homeland security spending at ~$4.7 billion in 2006Homeland security spending at ~$4.7 billion in 2006 Food safety market is $1.6 billion/year and growing at 5.5%.Food safety market is $1.6 billion/year and growing at 5.5%. Molecular diagnostic sales are expected to double by 2010 to >$4 billion/year.Molecular diagnostic sales are expected to double by 2010 to >$4 billion/year. Freedonia; TSG Partners

3. State of the art  DNA detection: polymerase chain reaction (PCR) Highly sensitive detection of nucleic acidHighly sensitive detection of nucleic acid Detection limit ~200-1000 cells or spores/mlDetection limit ~200-1000 cells or spores/ml Requires cell lysis in sample preparationRequires cell lysis in sample preparation Limitations due to sequence driftLimitations due to sequence drift  Surface protein detection: antibody-based assays Highly specific and broadly applicableHighly specific and broadly applicable Detection limit less sensitive than PCR: ~1000 cells or spores/mlDetection limit less sensitive than PCR: ~1000 cells or spores/ml  Clinical uses Both assays presently used in clinical settings.Both assays presently used in clinical settings. Both assays can be adapted to field settings.Both assays can be adapted to field settings.

4. How does PCR work?  Extract DNA from cells (step 1)  Perform repeated PCR cycles (steps 2-4) 1 2 3 4  Product accumulation is exponential  Detect product

5. How do antibody-based assays work? Add antigen solution Well plate Capture antibody (Ab) Antigen bound by Abs Enzyme acts on substrate Add Ab/enzyme 2nd Ab with enzyme Release of signal and detection Detector Add substrate solution Substrate

6. Our solution  Proximity ligation assays (PLA) Links surface binding assays (e.g., antibody-based) with PCR amplificationLinks surface binding assays (e.g., antibody-based) with PCR amplification Limited sample preparation – rapid assayLimited sample preparation – rapid assay Ultra-sensitive:Ultra-sensitive:  Detection of 20 spores/ml, including Bacillus anthracus, B. subtilis, and B. cereus  Detection of 100 cancer cells/ml in mixture with 10 9 healthy cells Low backgroundLow background Common platform – quickly adaptable to new targetsCommon platform – quickly adaptable to new targets

7. 4. PCR amplify Oligonucleotide primer 1 Oligonucleotide primer 2 Antibody or binding aptamer 3. Ligate Splint oligonucleotide 2. Bind spore 1. Mix Proximity Ligation Assay to detect spores

8. Benefits  Improved sensitivity by one or more orders of magnitude  Improved cell typing and detection breath compared with PCR  Direct use on cells or tissue samples without sample preparation  Multiplex PLA will provide quantitative descriptions of cell surfaces and improved detection.  One optimized oligonucleotide primer set can be used with multiple binding agents for rapid assay development.  Adaptable to standard clinical assay equipment  U.S. and PCT patents filed covering technology