Counting of single prion particles bound to a capture-antibody surface (surface-FIDA)
| Autor: | Eva Birkmann, Franziska Henke, Dieter Willbold, Nicole Weinmann, Martin H. Groschup, Christian Dumpitak, Aileen Funke, Detlev Riesner |
|---|---|
| Rok vydání: | 2007 |
| Předmět: |
PrPSc Proteins
Prions animal diseases Blotting Western Fluorescence correlation spectroscopy Scrapie Biology Protein aggregation Sensitivity and Specificity Microbiology Prion Diseases Cricetinae Animals Prion protein General Veterinary Capture antibody General Medicine Virology nervous system diseases Encephalopathy Bovine Spongiform Electrophoresis Fluorescence intensity Spectrometry Fluorescence Biophysics Cattle Electrophoresis Polyacrylamide Gel Endopeptidase K Spongiform encephalopathy |
| Zdroj: | Veterinary Microbiology. 123:294-304 |
| ISSN: | 0378-1135 |
| DOI: | 10.1016/j.vetmic.2007.04.001 |
| Popis: | Hitherto accredited prion tests use the PK resistance of PrP(Sc), the pathogenic isoform of the prion protein, as a marker for the disease. Because of variations in the amount of disease-related aggregated PrP, which is not PK-resistant, these prion tests offer only limited sensitivity. Therefore, a prion detection method that does not rely on PK digestion would allow for the detection of both PK-resistant as well as PK-sensitive PrP(Sc). Furthermore, single particle counting is more sensitive than methods measuring an integrated signal. Our new test system is based on dual-colour fluorescence correlation spectroscopy (FCS). This method quantifies the number of protein aggregates that have been simultaneously labelled with two different antibodies using dual-colour fluorescence intensity distribution analysis (2D-FIDA). This only counts PrP aggregates, and not PrP monomers. To increase the sensitivity, PrP(Sc) was concentrated in a two-dimensional space by immobilizing it so that the antibodies could be captured on the surface of the slide (surface-FIDA). When the surface was systematically scanned, even single prion particles were detected. Using this new technique, the sensitivity to identify samples from scrapie-infected hamster as well as BSE-infected cattle can be dramatically increased in comparison with identification using FIDA in solution. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect