Abstract
Transmissible spongiform encephalopathies are neurodegenerative diseases and are considered to be caused by malformed prion proteins accumulated into fibrillar structures that can then aggregate to form larger deposits or amyloid plaques. The identification of fibril-interfering compounds is of therapeutic and prophylactic interest. A robust and easy-to-perform, high-throughput, in vitro fluorescence assay was developed for the detection of such compounds. The assay was based on staining with the fluorescent probe thioflavin S in polystyrene microtiter plates to determine the amyloid state of synthetic peptides, representing a putative transmembrane domain of human and mouse prion protein. In determining optimal test conditions, it was found that drying peptides from phosphate buffer prior to staining resulted in good reproducibility with an interassay variation coefficient of 8%. Effects of thioflavin S concentration and staining time were established. At optimal thioflavin S concentration of 0.2 mg/ml, the fluorescence signals of thioflavin S with five different prion protein-based fibrillogenic peptides, as well as peptide Aβ (1-42), were found to show a peptide-dependent linear correlation within a peptide concentration range of 10-400 μM. The ability of the assay to identify compounds that interfere with fibril formation and/or dissociate preformed fibrils was demonstrated for tetracyclic compounds by preceding coincubation with human prion protein peptide huPrP106-126.
| Original language | English |
|---|---|
| Pages (from-to) | 372-380 |
| Number of pages | 9 |
| Journal | Analytical Biochemistry |
| Volume | 333 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - Oct 15 2004 |
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Keywords
- Alzheimer's disease
- Amyloid
- BSE
- High-throughput assay
- Prion
- PrP106-126
- Tetracycline
- Thioflavin S
ASJC Scopus subject areas
- Biochemistry
- Biophysics
- Molecular Biology
Cite this
An in vitro screening assay based on synthetic prion protein peptides for identification of fibril-interfering compounds. / Boshuizen, Ronald S.; Langeveld, Jan P M; Salmona, Mario; Williams, Alun; Meloen, Rob H.; Langedijk, Johannes P M.
In: Analytical Biochemistry, Vol. 333, No. 2, 15.10.2004, p. 372-380.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - An in vitro screening assay based on synthetic prion protein peptides for identification of fibril-interfering compounds
AU - Boshuizen, Ronald S.
AU - Langeveld, Jan P M
AU - Salmona, Mario
AU - Williams, Alun
AU - Meloen, Rob H.
AU - Langedijk, Johannes P M
PY - 2004/10/15
Y1 - 2004/10/15
N2 - Transmissible spongiform encephalopathies are neurodegenerative diseases and are considered to be caused by malformed prion proteins accumulated into fibrillar structures that can then aggregate to form larger deposits or amyloid plaques. The identification of fibril-interfering compounds is of therapeutic and prophylactic interest. A robust and easy-to-perform, high-throughput, in vitro fluorescence assay was developed for the detection of such compounds. The assay was based on staining with the fluorescent probe thioflavin S in polystyrene microtiter plates to determine the amyloid state of synthetic peptides, representing a putative transmembrane domain of human and mouse prion protein. In determining optimal test conditions, it was found that drying peptides from phosphate buffer prior to staining resulted in good reproducibility with an interassay variation coefficient of 8%. Effects of thioflavin S concentration and staining time were established. At optimal thioflavin S concentration of 0.2 mg/ml, the fluorescence signals of thioflavin S with five different prion protein-based fibrillogenic peptides, as well as peptide Aβ (1-42), were found to show a peptide-dependent linear correlation within a peptide concentration range of 10-400 μM. The ability of the assay to identify compounds that interfere with fibril formation and/or dissociate preformed fibrils was demonstrated for tetracyclic compounds by preceding coincubation with human prion protein peptide huPrP106-126.
AB - Transmissible spongiform encephalopathies are neurodegenerative diseases and are considered to be caused by malformed prion proteins accumulated into fibrillar structures that can then aggregate to form larger deposits or amyloid plaques. The identification of fibril-interfering compounds is of therapeutic and prophylactic interest. A robust and easy-to-perform, high-throughput, in vitro fluorescence assay was developed for the detection of such compounds. The assay was based on staining with the fluorescent probe thioflavin S in polystyrene microtiter plates to determine the amyloid state of synthetic peptides, representing a putative transmembrane domain of human and mouse prion protein. In determining optimal test conditions, it was found that drying peptides from phosphate buffer prior to staining resulted in good reproducibility with an interassay variation coefficient of 8%. Effects of thioflavin S concentration and staining time were established. At optimal thioflavin S concentration of 0.2 mg/ml, the fluorescence signals of thioflavin S with five different prion protein-based fibrillogenic peptides, as well as peptide Aβ (1-42), were found to show a peptide-dependent linear correlation within a peptide concentration range of 10-400 μM. The ability of the assay to identify compounds that interfere with fibril formation and/or dissociate preformed fibrils was demonstrated for tetracyclic compounds by preceding coincubation with human prion protein peptide huPrP106-126.
KW - Alzheimer's disease
KW - Amyloid
KW - BSE
KW - High-throughput assay
KW - Prion
KW - PrP106-126
KW - Tetracycline
KW - Thioflavin S
UR - http://www.scopus.com/inward/record.url?scp=4644336914&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=4644336914&partnerID=8YFLogxK
U2 - 10.1016/j.ab.2004.06.011
DO - 10.1016/j.ab.2004.06.011
M3 - Article
VL - 333
SP - 372
EP - 380
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 2
ER -