#6 A highly Sensitive Diagnostic Immunoassay Featuring DNA-tethered Beads
NIH Title: Highly Sensitive Tethered-Bead Immune Sandwich Assay
NIH Reference Number: E-188-2014
Executive Summary:
General Description:
Immune sandwich assays detecting low concentration analytes in body fluid have shown promise in early stage cancer diagnostics. Immune sandwich assay usually involves immobilization of an analyte-recognizing antibody on a solid phase, followed by analyte capture and attachment of a labeled, second antibody. The amount of analyte is quantified in proportion to the label signal. Current methods often require expensive, cumbersome detection equipment to achieve enhanced sensitivity. In order to solve these problems, researchers developed a simple, inexpensive method that has been compared and shown increased sensitivity compared to the standard immune sandwich methods.
Scientific Progress:
Researchers first prepared and attached one end of uniform-length DNA molecules to a glass slide. Next they conjugated a detection antibody to the free end of the DNA via biotin-streptavidin binding. Then they prepared capture beads by conjugation of capture antibody with 2.8 micrometer beads. Capture beads that bind analyte from a test solution then bind to the detection antibody on the DNA ends, forming tethered beads that are easily detected by light microscopy. Beads were deemed tethered if they moved an expected distance based on DNA length in each direction under flow that was maintained in a given direction for a few seconds. Free beads moved much further than the DNA length in each direction under these conditions. These preliminary data indicate that with optimization this assay could be sensitive to very low concentration of analyte and potentially be used in broad biomedical applications.
Future Directions:
Strength:
Weakness:
Patent Status:
US Patent Application No. 62/015,122 filed June 20, 2014
PCT Application No. PCT/US2015/034815 filed June 9, 2015
Publications:
Silver J, et al. PMID 25064819
Inventor Bio: Jonathan Silver, M.D.
Jonathan Silver graduated with a BA in physics from Harvard College in 1968, received a Masters degree in Physics from Stanford University in 1970, and an MD from Harvard Medical School in 1974. After clinical training in hematology and oncology, he came to the NIH in 1980 where he became a tenured principal investigator in 1987. His work focused on retroviruses in cancer and genetics. He is a co- inventor of several extensions of pcr technology including a form of pcr in droplet arrays that was commercially developed by Applied Biosystems, now ThermoFisher.
Inventor Bio: Keir C. Neuman, Ph.D.
Keir Neuman graduated cum laude with a B.A. in physics and applied math from the University of California, Berkeley in 1994 and received his Ph.D. in physics from Princeton University in 2002. He did postdoctoral research with Steven Block at Stanford University from 2002 to 2004, and was a Human Frontiers Fellow with David Bensimon and Vincent Croquette at the Laboratoire de Physique Statistique at the École Normale Supérieure in Paris, France from 2004 to 2007. Dr. Neuman joined the NHLBI as a tenure-track Investigator in 2007. In 2010, he was awarded a Human Frontiers Young Investigator Grant with Mihály Kovács from the Department of Biochemistry at Eötvös University in Hungary. Dr. Neuman is a reviewer for numerous journals and grant organizations. He is also a member of the Biophysical Society, the Optical Society of America, and the American Physical Society.
NIH Reference Number: E-188-2014
Executive Summary:
- Invention Type: Diagnostic
- Patent Status: Patent pending
- Link: https://www.ott.nih.gov/technology/E-188-2014
- NIH Institute or Center: National Heart, Lung and Blood Institute (NHLBI)
- Disease Focus: Cancer and infectious diseases
- Basis of Invention: DNA tethered beads
- How it works: “Tethered bead” technology- optical detection of the motion of micron-sized beads attached via immune sandwiches to the ends of single molecules of DNA on a sensor surface.
- Lead Challenge Inventor: Jonathan Silver (NHLBI)
- Inventors: Jonathan Silver (NHLBI), Zhenyu Li (George Washington Univ.), Keir Neuman (NHLBI)
- Development Stage: Early stage, proof-of-principle prototype available. A prototype design using prostate specific antigen (PSA) shows detection sensitivity of ~.03ng/ml, compared with normal PSA sensitivity of ~< 4ng/ml.
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Novelty:
- Highly sensitive single molecule adaptable format
- Automatable for image analysis
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Clinical Applications:
- Cancer biomarker detection such as PSA
- Infectious disease diagnostic
- Therapeutic drug monitoring
- Clinical pharmacokinetic and bioequivalence studies in drug discovery
General Description:
Immune sandwich assays detecting low concentration analytes in body fluid have shown promise in early stage cancer diagnostics. Immune sandwich assay usually involves immobilization of an analyte-recognizing antibody on a solid phase, followed by analyte capture and attachment of a labeled, second antibody. The amount of analyte is quantified in proportion to the label signal. Current methods often require expensive, cumbersome detection equipment to achieve enhanced sensitivity. In order to solve these problems, researchers developed a simple, inexpensive method that has been compared and shown increased sensitivity compared to the standard immune sandwich methods.
Scientific Progress:
Researchers first prepared and attached one end of uniform-length DNA molecules to a glass slide. Next they conjugated a detection antibody to the free end of the DNA via biotin-streptavidin binding. Then they prepared capture beads by conjugation of capture antibody with 2.8 micrometer beads. Capture beads that bind analyte from a test solution then bind to the detection antibody on the DNA ends, forming tethered beads that are easily detected by light microscopy. Beads were deemed tethered if they moved an expected distance based on DNA length in each direction under flow that was maintained in a given direction for a few seconds. Free beads moved much further than the DNA length in each direction under these conditions. These preliminary data indicate that with optimization this assay could be sensitive to very low concentration of analyte and potentially be used in broad biomedical applications.
Future Directions:
- Refinement of the method to further increase sensitivity
- Expand the application to detect infectious disease pathogens
- Automate fluid handling and tethered bead detection
Strength:
- Refinement of the method to further increase sensitivity
- Expand the application to detect infectious disease pathogens
- Automate fluid handling and tethered bead detection
Weakness:
- Sensitivity is limited at present by low efficiency of binding of antibody at the ends of DNA to analyte captured on beads
- The length of DNA used for tether needs to be optimized because long DNA strands may reduce efficiency of binding to analyte on capture beads while short DNA strands make it more difficult to distinguish tethered beads from beads non-specifically stuck to the glass surface
- At present, skills are required for preparing the DNA for efficient tether formation
Patent Status:
US Patent Application No. 62/015,122 filed June 20, 2014
PCT Application No. PCT/US2015/034815 filed June 9, 2015
Publications:
Silver J, et al. PMID 25064819
Inventor Bio: Jonathan Silver, M.D.
Jonathan Silver graduated with a BA in physics from Harvard College in 1968, received a Masters degree in Physics from Stanford University in 1970, and an MD from Harvard Medical School in 1974. After clinical training in hematology and oncology, he came to the NIH in 1980 where he became a tenured principal investigator in 1987. His work focused on retroviruses in cancer and genetics. He is a co- inventor of several extensions of pcr technology including a form of pcr in droplet arrays that was commercially developed by Applied Biosystems, now ThermoFisher.
Inventor Bio: Keir C. Neuman, Ph.D.
Keir Neuman graduated cum laude with a B.A. in physics and applied math from the University of California, Berkeley in 1994 and received his Ph.D. in physics from Princeton University in 2002. He did postdoctoral research with Steven Block at Stanford University from 2002 to 2004, and was a Human Frontiers Fellow with David Bensimon and Vincent Croquette at the Laboratoire de Physique Statistique at the École Normale Supérieure in Paris, France from 2004 to 2007. Dr. Neuman joined the NHLBI as a tenure-track Investigator in 2007. In 2010, he was awarded a Human Frontiers Young Investigator Grant with Mihály Kovács from the Department of Biochemistry at Eötvös University in Hungary. Dr. Neuman is a reviewer for numerous journals and grant organizations. He is also a member of the Biophysical Society, the Optical Society of America, and the American Physical Society.
