NR AXPH
AU Jones,M.; Peden,A.H.; Prowse,C.V.; Gröner,A.; Manson,J.C.; Turner,M.L.; Ironside,J.W.; MacGregor,I.R.; Head,M.W.
TI In vitro Amplification and Detection of Variant Creutzfeldt-Jakob Disease PrPsc
QU International Conference - Prion 2007 (26.-28.9.2007) Edinburgh International Conference Centre, Edinburgh, Scotland, UK - Book of Abstracts: Oral Abstracts FC1.3
IA http://www.prion2007.com/pdf/Prion Book of Abstracts.pdf
PT Konferenz-Vortrag
AB
Background: Protein misfolding cyclic amplification (PMCA) is an in vitro technique that rapidly replicates the conversion of the host-encoded prion protein (PrPc) to the disease-associated form (PrPsc). Using a scrapie-hamster model, it has been shown that PMCA-generated PrPsc is infectious and that following PMCA, PrPsc can be detected in both brain tissue and blood during the asymptomatic disease stage. However, little has been reported with regard to the amplification of human prion disease associated PrPsc.
Aim(s)/Objectives(s): To evaluate PMCA for the amplification of vCJD PrPsc using non-CJD brain, humanized transgenic mouse brain and human platelets as potential substrate sources. To further investigate the influence of the PRNP codon 129 genotype on the ability of the substrate source to support PMCA and to investigate alternative methods of detecting amplified PrPsc.
Methods: Brain homogenate prepared from vCJD brain tissue was seeded into substrate homogenates prepared from a non-CJD diseased brain, humanized transgenic mouse brain and human platelets. Samples were divided into two equal lots with one lot stored at -80oC (-PMCA) and the other lot subjected to 48 cycles of PMCA (+PMCA). Amplified PrPsc was then detected either by Western blotting following proteinase K digestion or by conformation dependent immunoassay (CDI) in the absence of PK digestion.
Results: Under the PMCA conditions used, vCJD PrPsc was efficiently amplified in substrates prepared from PRNP codon 129 MM homozygous tissue whereas similar substrate sources prepared from PRNP codon 129 MV and VV tissues failed to support PMCA to the same extent. Using human brain tissue as the substrate source the limit of PrPsc detection by Western blotting was increased 1000-fold following PMCA when compared to non-amplified controls and comparable results were obtained using the CDI assay. Both transgenic mouse brain tissue and human platelets were shown to be suitable alternative PMCA substrate sources.
Conclusions: Although originally developed using hamster scrapie models PMCA can be used to amplify PrPsc from vCJD tissues. Efficient PMCA amplification of vCJD PrPsc is dependent on the PRNP codon 129 genotype of the substrate used. Both humanized transgenic mouse brain and human platelets are suitable alternative substrate sources.
AD M. Jones, A.H. Peden, J.W. Ironside, M.W. Head, University of Edinburgh, National CJD Surveillance Unit, UK; C.V. Prowse, I.R. MacGregor, Scottish National Blood Transfusion Service, National Science Laboratory, UK; A. Groner, CSL Behring, Germany; J.C. Manson, Roslin Institute Neuropathogenesis Unit, UK; M.L. Turner, Royal Infirmary of Edinburgh, Edinburgh Blood Transfusion Centre, UK
SP englisch
PO Schottland