Counting of single prion particles bound to a capture-antibody surface (surface-FIDA)

• Published in: Veterinary microbiology Vol. 123; no. 4; pp. 294 - 304
• Main Authors: Birkmann, Eva, Henke, Franziska, Weinmann, Nicole, Dumpitak, Christian, Groschup, Martin, Funke, Aileen, Willbold, Dieter, Riesner, Detlev
• Format: Journal Article Conference Proceeding
• Language: English
• Published: Amsterdam Elsevier B.V 31-08-2007; Elsevier Science; Elsevier
• Subjects: Animals; antibodies; Biological and medical sciences; biomarkers; Blotting, Western - veterinary; bovine spongiform encephalopathy; BSE; Cattle; cerebrospinal fluid; Cricetinae; CSF; Degenerative and inherited degenerative diseases of the nervous system. Leukodystrophies. Prion diseases; disease detection; Electrophoresis, Polyacrylamide Gel - veterinary; Encephalopathy, Bovine Spongiform - diagnosis; Endopeptidase K - chemistry; Endopeptidase K - metabolism; fluorescent labeling; Fundamental and applied biological sciences. Psychology; Human viral diseases; Infectious diseases; labeling techniques; Medical sciences; Microbiology; Neurology; new methods; new technology; Non conventional transmissible agents; Prion; Prion Diseases - diagnosis; Prion Diseases - veterinary; Prions - isolation & purification; protein aggregates; Proteinase K-free diagnosis; PrPSc proteins; PrPSc Proteins - cerebrospinal fluid; PrPSc Proteins - isolation & purification; Scrapie; Scrapie - diagnosis; Sensitivity and Specificity; sheep; Single particle detection; Spectrometry, Fluorescence - methods; Spectrometry, Fluorescence - veterinary; spectroscopy; Surface-FIDA; surface-fluorescence intensity distribution analysis; TSE diagnosis; Viral diseases; Viral diseases of the nervous system; Scrapie; BSE; Proteinase K-free diagnosis; TSE diagnosis; Surface-FIDA; CSF; Prion; Single particle detection; Degenerative disease; Spongiform encephalopathy; Diagnosis; Ungulata; Prion disease; Nervous system diseases; Farming animal; Enzyme; Antibody; Cerebral disorder; Infection; Peptidases; Vertebrata; Mammalia; Central nervous system disease; Hydrolases; Sheep; Artiodactyla; Veterinary; Detection; Medicine
• ISSN: 0378-1135; 1873-2542
• DOI: 10.1016/j.vetmic.2007.04.001

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.