This grant will develop, validate, and utilize novel protocols for rapid generation of molecular probes. The generation of molecular probes (usually monoclonal antibodies) remains a critical bottleneck in biomedical research, biomarker discovery, and diagnostic test development. Several approaches have been developed to overcome this problem. For example, yeast display libraries express diverse single chain-fragment variable (scFv) antibodies on the surfaces of Saccharomyces cerevisiae cells. By using fluorescent-activated cell sorting (FACS), yeast clones that bind specifically to antigens can be selected from naive libraries in 2 to 3 weeks. However, selected probes that perform well on yeast cell surfaces often perform poorly in solution. In order to achieve a more broadly useful, accelerated pipeline for molecular probes, this grant will leverage the speed and throughput of yeast scFv display, while overcoming its innate limitations. A yeast immunoprecipitation - tandem mass spectroscopy (yeast IP-MS/MS) approach will be used to improve the speed and throughput of biomarker research. Novel nanoparticle scaffolds combined with FACS will be used to improve the functional affinity of scFv binding to antigens. As a model for the development of these approaches, the project will identify novel cyst antigens of Entamoaeba histolytica, an important enteric pathogen and Category B agent. The accelerated molecular probe pipeline (AMPP) will be used first to identify and validate these biomarkers, and then to generate high-activity scFv probes for incorporation into an improved diagnostic test for E. histolytica cysts in stool.

Public Health Relevance

If successful, this project will change the way molecular probes are generated for research, biomarker discovery, and diagnostic testing. It will benefit any research or development effort that utilizes molecular probes, and pave faster routes to new vaccines, drugs, and diagnostics.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project--Cooperative Agreements (U01)
Project #
5U01AI082186-04
Application #
8244366
Study Section
Special Emphasis Panel (ZAI1-MMT-M (J1))
Program Officer
Rao, Malla R
Project Start
2009-04-03
Project End
2012-09-30
Budget Start
2012-04-01
Budget End
2012-09-30
Support Year
4
Fiscal Year
2012
Total Cost
$407,963
Indirect Cost
$134,766
Name
Seattle Biomedical Research Institute
Department
Type
DUNS #
070967955
City
Seattle
State
WA
Country
United States
Zip Code
98109
Grewal, Yadveer S; Shiddiky, Muhammad J A; Gray, Sean A et al. (2013) Label-free electrochemical detection of an Entamoeba histolytica antigen using cell-free yeast-scFv probes. Chem Commun (Camb) 49:1551-3
Gray, Sean A; Weigel, Kris M; Ali, Ibne K M et al. (2012) Toward low-cost affinity reagents: lyophilized yeast-scFv probes specific for pathogen antigens. PLoS One 7:e32042
Kozak, Darby; Anderson, Will; Vogel, Robert et al. (2011) Advances in Resistive Pulse Sensors: Devices bridging the void between molecular and microscopic detection. Nano Today 6:531-545
Kozak, Darby; Kithva, Prakash; Bax, Jacinda et al. (2011) Development of encoded particle-polymer arrays for the accelerated screening of antifouling layers. Chem Commun (Camb) 47:9687-9
Kozak, Darby; Chen, Annie; Bax, Jacinda et al. (2011) Protein resistance of dextran and dextran-poly(ethylene glycol) copolymer films. Biofouling 27:497-503
Gray, Sean A; Weigel, Kris M; Miller, Keith D et al. (2010) Flow cytometry-based methods for assessing soluble scFv activities and detecting antigens in solution. Biotechnol Bioeng 105:973-81