Progress 10/01/08 to 09/30/09
Outputs
Progress Report Objectives (from AD-416) The long-term goal of this research is the development of an assay system for viral infectious agents that is highly portable, relatively inexpensive, and easy to use by relatively untrained personnel. Approach (from AD-416) Development of a number of assays for viral targets using surface enhanced Raman scattering (SERS)-based immunoassays and solution RNA hybridization assays. The basic principle of SERS assays is that the Raman spectrum unique for an analyte (eg. dye) is greatly enhanced when the dye is in close proximity to a metal colloid. Standard ELISA and in solution hybridization assays will be modified to utilize this novel detection system. West Nile virus and the related Saint Louis Enchephalitis viruses will be the specific targets for this project. Significant Activities that Support Special Target Populations The goals of this project were to develop assay methods for the clinically similar West Nile Virus (WNV) and St. Louis Encephalitis (SLE) viruses, as well as the instrumentation that allows the assays to be carried out in the field and in laboratory settings. The basis of the assay methods to be investigated is surface enhanced Raman spectroscopy (SERS). It was proposed that the extraordinarily large signal enhancement attendant upon localization of some dyes at silver or gold surfaces would provide the sensitivity necessary for detection of WNV and SLE. The two assay formats chosen for examination were a nucleic acid hybridization (NAH) assay and an immunoassay. The overall project involves a three-way collaboration: organic synthesis (UW), assay development (Animal Borne Diseases Research Lab (ABDRL) and the University of Wyoming (UW) and Raman instrumentation development to complement the assays (DeltaNu). The successful demonstration of an indirect capture SERS-based NAH model assay was confirmed using quartz crystal microbalance-dissipation (QCM-D). QCM-D has allowed us to improve hybridization conditions and identify optimal hybridization target sites in the genome. Two WNV recombinant constructs have also been made expressing truncated regions of WNV envelope glycoprotein. These truncated proteins have been expressed, protein purification protocols developed and antibodies produced. The initial experiment demonstrated "proof-of-concept" for a WNV SERS immunoassay has been achieved, but further optimization by our collaborators will be needed.
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Progress 06/01/05 to 04/30/09
Outputs
Progress Report Objectives (from AD-416) The long-term goal of this research is the development of an assay system for viral infectious agents that is highly portable, relatively inexpensive, and easy to use by relatively untrained personnel. Approach (from AD-416) Develop of a number of assays for viral targets using Raman immunoassays and solution RNA hybridization assays. The basic principle of the Raman assays is that Raman resonance is enhanced when the dye is close proximity to a metal colloid. Standard ELISA and in solution hybridization assays will be modified to take utilize this novel detection system. West Nile virus and the related Saint Louis Enchephalitits viruses will be the specific targets for this project. The goals of this project were to develop assay methods for the clinically similar West Nile Virus (WNV) and St. Louis Encephalitis (SLE) viruses, as well as the instrumentation that allows the assays to be carried out in the field and in laboratory settings. The basis of the assay methods to be investigated is surface enhanced Raman spectroscopy (SERS). It was proposed that the extraordinarily large signal enhancement attendant upon localization of some dyes at silver or gold surfaces would provide the sensitivity necessary for detection of WNV and SLE. The two assay formats chosen for examination were a nucleic acid hybridization (NAH) assay and an immunoassay. The overall project involved a three-way collaboration: organic synthesis (UW), assay development (Animal Borne Diseases Research Lab (ABDRL) and the University of Wyoming (UW) and Raman instrumentation development to complement the assays (DeltaNu). Two manuscripts describing this work have been accepted or published. This research supports NP103 Action Plan Components 1. Biodefense Research, and 3. Prevent and Control Zoonotic Diseases. ADODR is directly involved in performance of the research and also monitors activities to evaluate research progress through site visits, meeting at conferences and through email and phone calls.
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Progress 10/01/07 to 09/30/08
Outputs
Progress Report Objectives (from AD-416) The long-term goal of this research is the development of an assay system for viral infectious agents that is highly portable, relatively inexpensive, and easy to use by relatively untrained personnel. Approach (from AD-416) Develop of a number of assays for viral targets using Raman immunoassays and solution RNA hybridization assays. The basic principle of the Raman assays is that Raman resonance is enhanced when the dye is close proximity to a metal colloid. Standard ELISA and in solution hybridization assays will be modified to take utilize this novel detection system. West Nile virus and the related Saint Louis Enchephalitits viruses will be the specific targets for this project. Significant Activities that Support Special Target Populations The purpose of this Rocky Mountain RCE research project is the development of rapid assays for the presence of West Nile Virus (WNV) and St. Louis Encephalitis (SLE) virus. These two viruses are clinically similar at early stages and may be mistaken for one another by many diagnostic tests. We have developed two different classes of assays that will simultaneously determine the presence or absence of the viruses in a given sample. A positive test for one of the viruses, combined with a negative test for the other in the same sample, will provide an unusually high level of surety of diagnosis. The basis of both of the classes of assays is the use of surface enhanced Raman scattering (SERS) spectroscopy. Advantages of SERS over other spectroscopic methods include the high information content of Raman spectra (allowing multiple reporter dyes to be employed to distinguish between analytes for simultaneous assays), the extraordinarily high sensitivity that results from localizing a reporter dye at a noble metal surface, and the fact that the signal enhancement only occurs at the surface. In the final phase of this project we will fully optimize the assays using physical chemical analysis and further generate biomaterials necessary to expand the signal pathogen assay to a multiple pathogen single assay. The project will involve the further development of instruments capable of carrying out the assays in a rapid fashion in both the laboratory and in the field. These features will make the assay method well suited for the rapid diagnosis of outbreaks of these viruses. The end product resulting from the project will be an assay system that allows two important viral targets to be detected and distinguished from each other, and which will serve as a model platform for the development of rapid assays for other pathogens. Contacts were made via telephone, meetings, email, etc. This work aligns with the Genetic and Biological Determinants of Disease Susceptibility Component of the Animal Health Action Plan.
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) The long-term goal of this research is the development of an assay system for viral infectious agents that is highly portable, relatively inexpensive, and easy to use by relatively untrained personnel. Approach (from AD-416) Develop of a number of assays for viral targets using Raman immunoassays and solution RNA hybridization assays. The basic principle of the Raman assays is that Raman resonance is enhanced when the dye is close proximity to a metal colloid. Standard ELISA and in solution hybridization assays will be modified to take utilize this novel detection system. West Nile virus and the related Saint Louis Enchephalitits viruses will be the specific targets for this project. Significant Activities that Support Special Target Populations This report documents research conducted under a Reimbursable Cooperative Agreement between ARS and Colorado State University. Additional details of research can be found in the report for the in-house associated projects 5410-32000-011-00D & 5410-32000-016-00D (new), Molecular Biology and Pathogenesis of Arboviruses. The goals of this project are to develop assay methods for the clinically similar West Nile Virus (WNV) and St. Louis Encephalitis (SLE) viruses, as well as the instrumentation that allows the assays to be carried out in the field and in laboratory settings. The basis of the assay methods to be investigated is surface enhanced Raman spectroscopy (SERS). It was proposed that the extraordinarily large signal enhancement attendant upon localization of some dyes at silver or gold surfaces would provide the sensitivity necessary for detection of WNV and SLE. The two assay formats chosen for examination were a nucleic acid hybridization (NAH) assay and an immunoassay. The overall project involves a three way collaboration: organic synthesis (UW), assay development (Animal Borne Diseases Research Lab (ABDRL) and the University of Wyoming (UW) and Raman instrumentation development to complement the assays (DeltaNu). We have demonstrated successful capture of a nucleic acid ternary (DNA/DNA/RNA) complex which contained hybridizing sequences identical to those proposed for the SERS-based NAH assay. However, as capture levels of the complex were considerably reduced relative to maximum calculated levels under the conditions employed and we encountered several technical problems endemic to working with Dynal� paramagnetic beads, we saw no reason to pursue the optimization of a model system which differs in several critical features from the actual proposed assay. Therefore we developed a modified RNase Protection assay to evaluate specific oligonucleotides for use in the SERS WNV genome detection. This system has allowed us to improve hybridization conditions and identify optimal hybridization target sites in the genome. High background signals were noted in our initial SERS assays and modifications of the assay are in progress. Two WNV recombinant constructs have also been made expressing truncated regions of WNV envelope glycoprotein. These truncated proteins have been expressed and protein purification protocols developed. The assembled reagents for the WNV SERS immunoassay have been validated using ELISA techniques and "proof-of-concept" experiments are underway.
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Progress 10/01/05 to 09/30/06
Outputs
Progress Report 4d Progress report. This report serves to document research conducted under a reimbursable agreement (No. 5805410-5-430) between ARS and the Colorado State University (Subaward No. G-4728-3). Additional details of research can be found in the report for the parent project 5410-32000-011-00D Molecular Biology and Pathogenesis of Arboviruses. The goals of this project are to develop assay methods for the clinically similar West Nile Virus (WNV) and St. Louis Encephalitis (SLE) viruses, as well as the instrumentation that allows the assays to be carried out in the field and in laboratory settings. The basis of the assay methods to be investigated is surface enhanced Raman spectroscopy (SERS). It was proposed that the extraordinarily large signal enhancement attendant upon localization of some dyes at silver or gold surfaces would provide the sensitivity necessary for detection of WNV and SLE. The two assay formats chosen for examination were a nucleic
acid hybridization (NAH) assay and an immunoassay. The overall project involves a three way collaboration: organic synthesis (UW), assay development (ABADRL and UW) and Raman instrumentation development to complement the assays (DeltaNu). The ABADRL temporary staff for this project have been hired and components for the NAH assay have been synthesized. Preliminary studies have developed the unique hybridization condition necessary for this assay and are being optimized. New Raman instrumentation have been made that afford more flexibility in assay design.
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