NR AXNR
AU Grigorov,A.; Kamhieh,S.; Todorova,K.; Salama,A.; Kiesewetter,H.
TI Detection of Pathological Prions Following Plasmin Cleavage of the Normal Isoform
QU International Conference - Prion 2007 (26.-28.9.2007) Edinburgh International Conference Centre, Edinburgh, Scotland, UK - Book of Abstracts: Epidemiology, Risk Assessment and Transmission P04.66
IA http://www.prion2007.com/pdf/Prion Book of Abstracts.pdf
PT Konferenz-Poster
AB The transmissible spongiform encephalopathies (TSE) or prion diseases are fatal neurodegenerative diseases which arise due to the conformational conversion of the non-pathological form of the prion protein PrPc, a cellular glycoprotein of unknown function, into the infectious scrapie isoform, PrPsc. A rapid and sensitive method for the detection of the pathological isoform is urgently required and has become a high priority in transfusion medicine, due to the importance of the safety of blood products. To date, most routine methods described rely on the differing sensitivities of both isoforms to protease digestion. Via activation of endogenous plasminogen in human plasma samples, we have demonstrated that native PrPc is cleaved into, at least, two fragments. The primary cleavage site was identified in the binding site of the 3F4 monoclonal antibody (108-110). This epitope is fully accessible in PrPc but becomes inaccessible in misfolded PrPsc. Using the resistance of the misfolded PrP to plasmin cleavage, we have developed a sensitive and simple detection method for PrPsc in body fluids. Following plasmin cleavage of PrPc, samples were measured and quantified in a sandwich ELISA using a variety of monoclonal antibodies (SAF32, SAF61 Spi-Bio and/or 3F4 Covance) in a time-dependent manner. The antibody pairs recognise different epitopes on both sides of the primary cleavage site of plasmin, thus only full-length, non-cleaved PrP-molecules are detected using this described method. Samples with full-length wild-type recombinant human PrP (rhuPrP) and rhuPrP with a deleted lysine cluster 2 were analysed as a model for plasmin cleavage of native PrPc or PrPsc, respectively. Hamster brain homogenates from both normal and scrapieinfected animals (263K), as well as human plasma spiked with scrapie brain homogenate, were used to test the ability of the assay to discriminate between the normal and pathological form of PrP. Further optimisation was undertaken to increase the sensitivity of the assay. Measurement of the concentration prior to and following cleavage was also investigated. The data described here demonstrate the possibility to detect misfolded PrPsc in plasma samples combining plasmin cleavage and conventional ELISA methods.
AD A. Grigorov, S. Kamhieh, K. Todorova, A. Salama, H. Kiesewetter, Charite, Transfusionmedicine, Germany
SP englisch
PO Schottland