NR ATLN
AU Peretz,D.; Michelitisch,M.; Gao,C.; Wang,X.; Connolly,M.; Horn,T.; Shimizu,R.; Gulliver,T.; Hall,J.; Zuckermann,R.; Chien,D.; Yang,Y.; Hu,C.; Phelps,B.
TI Development of binding reagents and Immunoassay for CJD PrPsc in Plasma
QU International Conference - Prion 2005: Between fundamentals and society's needs - 19.10.-21.10.2005, Congress Center Düsseldorf - Poster Session: Diagnosis DIA-51
PT Konferenz-Poster
AB The BSE epidemic and its transmission to humans causing vCJD have heightened public health concerns. Since there are no clinical signs or symptoms of the disease for many years, there is no accurate way to determine how many people could currently be harboring the disease or the magnitude of future cases. Recent reports of two probable cases of human-to-human vCJD transmission via blood transfusion have underscored the presence of infectious prion in the blood and heightened the need for sensitive tests to safeguard donated blood and blood products. Two major hurdles hinder the development of prion blood test; 1) extremely low PrPsc concentration in blood and 2) lack of PrPsc specific high-affinity binding reagents. As direct interaction between PrPc and PrPsc is proposed to drive the accumulation of nascent PrPsc, we reasoned that peptides of PrPc could be utilized to bind PrPsc. A series of prion protein peptides were synthesized and screened against CJD PrPsc spiked into plasma. Selected peptides were used in developing a prototype assay in which the peptide acts as a capture reagent to concentrate PrPsc. PrPsc is then chemically eluted, denatured, and detected by monoclonal antibodies against PrPc. The assay does not require Proteinase K digestion or centrifugation and can be performed in a 96-well plate. The analytical sensitivity was evaluated by testing vCJD and sCJD brain homogenate samples that were serially diluted in negative human plasma. Most of the peptides were found to bind CJD PrPsc and several of them showed a high binding affinity in the presence of plasma. The prototype assay also detected non-human PrPsc samples. In conclusion: We have demonstrated a prototype prion assay with a sensitivity approaching the limit of detection required for blood screening. Our goal is to provide an automated high throughput assay for screening PrPsc in donated human blood and blood products.
IN Mit synthetisierten und an die Plastikoberflächen von Mikrotiterplatten gebundenen Prionproteinpeptiden anstatt mit Antikörpern binden die Autoren das PrPsc und fischen es so ohne Proteasebehandlung und Zentrifugation aus kontaminiertem Blutserum heraus. Sie berichten von einer bereits experimentell erreichten Sensitivität, die für die Entwicklung eines automatisierten TSE-Bluttests ausreichen soll.
AD David Peretz, Melissa Michelitisch, Carol Gao, Xuemei Wang, Michael Connolly, Thomas Horn, Robert Shimizu, Timoteo Gulliver, John Hall, Ronald Zuckermann, David Chien, Yanfeng Yang, Celine Hu, Bruce Phelps, Chiron Corporation Blood Testing Division, USA
SP englisch
PO Deutschland