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Prion detection by an amyloid seeding assay
David W Colby1, Qiang Zhang, Shuyi Wang
1Institute for Neurodegenerative Diseases [corrected], University of California, San Francisco, CA 94143, USA.
Abstract:
Polymerization of recombinant prion protein (recPrP), which was produced in bacteria, into amyloid fibers was accompanied by the acquisition of prion infectivity. We report here that partially purified preparations of prions seed the polymerization of recPrP into amyloid as detected by a fluorescence shift in the dye Thioflavin T. Our amyloid seeding assay (ASA) detected PrP(Sc), the sole component of the prion, in brain samples from humans with sporadic Creutzfeldt-Jakob disease, as well as in rodents with experimental prion disease. The ASA detected a variety of prion strains passaged in both mice and hamsters. The sensitivity of the ASA varied with strain type; for hamster Sc237 prions, the limit of detection was approximately 1 fg. Some prion strains consist largely of protease-sensitive PrP(Sc) (sPrP(Sc)), and these strains were readily detected by ASA. Our studies show that the ASA provides an alternative methodology for detecting both sPrP(Sc) and protease-resistant PrP(Sc) that does not rely on protease digestion or immunodetection.
Insights
A new amyloid seeding assay (ASA) detects prion diseases by observing the polymerization of recombinant prion protein (recPrP) into amyloid fibers. This method identifies PrP(Sc) in human and animal samples, offering a protease-independent detection approach.
Area of Science:
- Neuroscience
- Biochemistry
- Molecular Biology
Background:
- Prion diseases are fatal neurodegenerative disorders.
- Prion infectivity is linked to the polymerization of recombinant prion protein (recPrP) into amyloid fibers.
- The prion protein exists in different forms, including protease-sensitive (sPrP(Sc)) and protease-resistant PrP(Sc).
Purpose of the Study:
- To develop and validate a novel amyloid seeding assay (ASA) for prion detection.
- To assess the sensitivity and strain-typing capability of the ASA.
- To demonstrate the ASA's utility in detecting both protease-sensitive and protease-resistant PrP(Sc) without relying on protease digestion or immunodetection.
Main Methods:
- Utilized Thioflavin T fluorescence to detect recPrP polymerization into amyloid.
- Applied the ASA to brain samples from humans with sporadic Creutzfeldt-Jakob disease and rodents with experimental prion disease.
- Tested the ASA's sensitivity with various prion strains, including hamster Sc237 prions.
Main Results:
- The ASA successfully seeded recPrP polymerization, indicating the presence of prions.
- PrP(Sc) was detected in human and rodent prion disease samples.
- The ASA demonstrated high sensitivity, with a detection limit of approximately 1 fg for hamster Sc237 prions.
- The assay effectively detected various prion strains, including those composed of protease-sensitive PrP(Sc).
Conclusions:
- The amyloid seeding assay (ASA) is a sensitive and versatile method for detecting prion diseases.
- ASA provides an alternative to traditional protease digestion and immunodetection methods for identifying PrP(Sc).
- This assay has significant potential for prion diagnostics and research.
