Chemistry: analytical and immunological testing – Biological cellular material tested
Reexamination Certificate
1999-04-30
2001-06-12
Wallenhorst, Maureen M. (Department: 1743)
Chemistry: analytical and immunological testing
Biological cellular material tested
C436S164000, C436S172000, C435S004000, C435S029000
Reexamination Certificate
active
06245572
ABSTRACT:
TECHNICAL FIELD
The present invention relates to flow cytometry methods for detecting the presence of amyloid fibrils in biological samples for diagnostic, research and therapeutic purposes.
BACKGROUND OF THE INVENTION
Amyloid fibrils are aggregates of normally soluble, innocuous proteins whose deposition is associated with a group of diseases known as amyloidoses. Conditions mediated by the presence of amyloid fibrils include Alzheimer's disease, inflammation-associated amyloid type II diabetes, bovine encephalopathy (BSE), Creutzfeld-Jakob disease (CJD), scrapie and primary amyloidosis. Fibrillogenesis or fibril formation has been monitored in vitro using a combination of turbidity, light scattering and fluorescence measurements yielding both equilibrium and kinetic information. For additional material regarding transmissible spongiform encephalopathies, infectious and noninfectious amyloids, subacute spongiform encephalopathies, and prions, refer to B. Chesebro et al., “Transmissible Spongiform Encephalopathies: A Brief Introduction,” in Fields Virology 2845-2850 (Third ed., B. N. Fields et al., editors; Lippincott-Raven Publ., Philadelphia, Pa. 1996); D. C. Gajdusek, “Infectious Amyloids: Subacute Spongiform Encephalopathies as Transmissible Cerebral Amyloidoses” in Fields Virology 2851-2900; S. B. Prusiner, “Prions” in Fields Virology 2901-2950; L. W. Heck “The Amyloid Diseases” in Cecil Textbook Of Medicine 1504-6 (20th edition, J. C. Bennett et al., editors; W.B. Saunders Co., Philadelphia, Pa., 1996), and the references disclosed therein; these references are herein incorporated by reference in their entirety.
SUMMARY OF THE INVENTION
The present invention provides methods for detecting amyloid fibrils by flow cytometric analysis. These methods analyze biological samples, such as blood, urine, peritoneal fluid, fat samples or aspirates, cerebrospinal fluid (CSF), and stool, and provide a means for detecting amyloid fibrils for purposes of assessing treatment and general therapy efficacy, diagnosis, research, and monitoring of livestock for the presence of transmissible amyloidoses.
In one embodiment, the presence of amyloid fibrils in a biological sample is detected by the following general method. The autofluorescence of a biological sample is optionally determined. A first amyloidophilic dye is added to the sample which is then subjected to flow cytometry analysis and data is collected therefrom. Next, a second amyloidophilic dye is added to the sample which is subjected to flow cytometry analysis and data is collected therefrom. From this data the presence of amyloid fibrils in the biological sample is determined.
In another embodiment, the presence of amyloid fibrils in a biological sample is detected by the following method. A first amyloidophilic dye is added to a biological sample followed by flow cytometry analysis. A second amyloidophilic dye is then added to the sample and flow cytometry analysis is repeated. From the data collected from the flow cytometry analysis, it is determined whether there is a linear dependence between forward scatter and side scatter. The sample is also evaluated to see if it is positive for fluorescence of the first and second amyloidophilic dyes. The positive fluorescence determinations may then be used to calculate an amyloid burden index. Using this index the presence of amyloid fibrils in the biological sample is determined.
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C.L. Shen et al., “Light Scattering Analysis of Fibril Growth from the Amino-Termial Beta(1-28) Fragment of Beta-amyloid Peptide”,Biophysical Journal, vol. 65, No. 6, pp. 2383-2395 (1993).
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M. Palutke et al., “Flow Cytometric Purification of Alzheimer's Disease Amyloid Plaque Core Using Thioflavin T.”,Cytometry, vol. 8, No. 5, pp. 494-499 (1987).
Morgan & Lewis & Bockius, LLP
University of Tennessee Research Corporation
Wallenhorst Maureen M.
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