NR ATKP
AU Strammiello,R.; Parchi,P.
TI Effect of different extraction procedures on PrPres detection limits and PrPres "typing" in peripheral tissues
QU International Conference - Prion 2005: Between fundamentals and society's needs - 19.10.-21.10.2005, Congress Center Düsseldorf - Poster Session: Diagnosis DIA-27
PT Konferenz-Poster
AB Although the CNS represents the preferred and ultimate target for prions, it is increasingly recognized that PrPres also accumulates in extra-neural tissue in most, if not all, TSEs. This event usually occurs during the early phases of pathogenesis, making it an ideal target for the development of novel pre-mortem strategies of PrPres detection and strain typing. As a significant drawback, the amount of PrPres in peripheral tissues is from 10^4 to 10^6 times lower than in CNS, making mandatory the selective enrichment of the protein starting from relatively large amount of tissue. In this study, we compared two commonly used extraction procedures, such as sodium phosphotungstic acid (NaPTA) precipitation, and purification in Sarkosyl, to determine their relative efficacy of extraction and application limits in studying PrPres in muscle or lymphoreticular tissues. In particular, we aimed to verify whether these methodologies influence physico-chemical properties of the protein, such as those commonly analysed for strain typing. To this aim, we spiked known amounts of sCJD brain homogenate into homogenates of normal human muscle or lymphoreticular tissue before applying the extraction protocol and compared the results with those obtained applying standard protocols based on total homogenate analyses. Our findings show that: 1) PrPres recovery is largely influenced by the extraction procedure and, to a lesser extent, by the type of tissue. In particular, the highest recovery (~ 80%) rate was obtained with NaPTA precipitation in spleen; 2) PrPres electrophoretic mobility is not significantly influenced by the 2 extraction procedures, provide that band distortion due to gel overloading is avoided; 3) PrPres glycoform ratio is significantly affected. In particular both procedures led to an overestimation of the diglycosylated form; 4) PrP deglycosylation is critical for lowering the detection limit of the protein. Supported by EU contract QLG3-CT-2002-81030.
IN Offenbar erhält man nach Phosphorwolframsäure-Fällung zugunsten der diglykosilierten Form veränderte Verhältnisse zwischen den unterschiedlich glykosilierten Formen des Prionproteins.
AD Rosaria Strammiello, Piero Parchi, Dipartimento Scienze Neurologiche, Università di Bologna, Italy
SP englisch
PO Deutschland