Use of New Extraction Procedure for Prion Protein to Develope a Diagnostic Kit for TSE


PROJ NO: 3625-32000-025-01T AGENCY: ARS 3625
START: 30 JAN 1997 TERM: 30 JAN 2000


AMES, IOWA 50010

OBJECTIVES: Development of an extraction procedure for prion protein that is based on a high performance liquid chromatography method. Development of a diagnostic kit for Transmissible spongiform encephalopathies based on the titration procedure and de tection by a micro analytical method.

APPROACH: Organic solvents will be used to extract prion protein and a high performance liquid chromatographic column that is selective for its binding of hydrophobic compounds will be used to purify and to concentrate the prion protein. Conventional t ests such as Western blot will be used to detect the prion protein. Capillary electrophoresis using fluorescent labelled peptides will be used as a sensitive test to detect minute amounts of the prion protein. CRADA funded by Fort Dodge Laboratories, FY-9 7, $160,816. Agreement Number 58-3K95-7-524; Award date 01/23/97.

PROGRESS: 1999/01 TO 1999/09
1. What major problem or issue is being resolved and how are you resolving it? Transmissible spongiform encephalopathies of present in domestic animals and game animals has caused considerable concern both economic and for human health. The outbreak of bo vine spongiform encephalopathy in the United Kingdom resulted in great economic loss to the farmers of that country as well as public concern for human health after it was shown that this disease can be transmitted to humans. The putative agent that cause s this disease an abnormal protein is an insoluble protein that forms large aggregates. It is present in a rather low concentration in naturally infected animals. Development of a method that would efficiently extract this protein and increase the concent ration would help in the development of a test for this protein. This serious problem has been approached by using analytical methods. Different organic solvents were used to extract this protein. A high performance liquid chromatography method was develo ped using a specialized column matrix to purify the protein. 2. How serious is the problem? Why does it matter? The actual incidence of scrapie in sheep and in game animals ranges from 1-10%. The tests that are presently used are postmortem tests and diag nosis animals that have progressed to clinical disease. This may not help in the control of the disease if animals are infectious in early stages. A pre-clinical test using n accessible tissue or fluid would address this problem. The ability to test blood in animals and humans for the presence of abnormal prion protein would make it possible to eliminate this disease from the animal population and to improve safety for human products. 3. How does it relate to the National Program(s) and National Component (s) to which it has been assigned? National Program 103, Animal Health 100% 4. What were the most significant accomplishments this past year? The ability to extract and concentrate the small amount of abnormal prion protein abnormal prion protein in the b lood of animals infected with a TSE has aided in the development of an assay. Prior to this extraction method, the only way to for abnormal prion protein in blood was by using rodent adapted strains and injecting the material into the brains of these anim als. 5. Describe the major accomplishments over the life of the project including their predicted or actual impact. Over the past few years, a new extraction method was developed that made it possible to develop a test for the abnormal prion protein in th e blood of animals. 6. What do you expect to accomplish, year by year, over the next 3 years? Experiments to improve protocols and methods that are used for extracting the abnormal prion protein. Experiments to study the time at which abnormal prion prote in can be in the blood of TSE exposed animals. 7. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end user (industry, farmer, other scientists)? What are the const raints if known, to the adoption & durability of the technology product? The CRADA partners for the CRADA No. 58-3K95-7-524 Extraction of the Prion Protein and Its Use in a Diagnostic Test have been trained in the techniques using this technology. Thi s included numerous discussions and has included safety training for protocols using TSE infected material. Provisional Patent granted February 2-2-99 Method and Kit for Extracting Prion Protein. 8. List your most important non-peer reviewed publications and presentations to non-scientific organizations, and articles written about your work(NOTE: this does not replace your peer reviewed publications which are listed below).

PUBLICATIONS: 1999/01 TO 1999/09
1. SCHMERR, M.J. and JENNY, A.L. 1998. A diagnostic test for scrapie infected sheep ... electrophoresis immunoassay with fluorescent labeled peptides. Electrophoresis 19:409-414.
2. SCHMERR, M.J. 1998. Diagnostic test kit for transmissible spongiform encephalopathies using a new extraction method for the abnormal prion protein. U.S. Patent Application S/N 0046-98.

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