NR AXUE
AU Peden,A.H.; Jones,M.; Turner,M.; Gröner,A.; Manson,J.; Ironside,J.W.; MacGregor,I.
TI PrPsc Generated by Protein Misfolding Cyclic Amplification from Variant CJD Patient Specimens is Recognised by Conformation Dependent Immunoassay
QU International Conference - Prion 2007 (26.-28.9.2007) Edinburgh International Conference Centre, Edinburgh, Scotland, UK - Book of Abstracts: Protein Misfolding P01.53
IA http://www.prion2007.com/pdf/Prion Book of Abstracts.pdf
PT Konferenz-Poster
AB
Background: To date there have been four known cases of transmission of variant Creutzfeldt-Jakob disease (vCJD) infection via blood transfusion. In addition, several studies have shown low levels of the disease-associated abnormal form of the prion protein, PrPsc, in a number of peripheral tissues from vCJD patients, including skeletal muscle. These findings highlight an urgent public health need for a blood test capable of identifying people incubating vCJD.
Aim: One approach for the high-sensitivity detection of PrPsc is the Conformation Dependent Immunoassay (CDI) which employs epitope unmasking to distinguish PrPsc from the normal host-encoded prion protein, PrPc, thus avoiding proteinase K resistance as an operational definition of PrPsc. Our aim has been to integrate CDI with Protein Misfolding Cyclic Amplification (PMCA), as a basis for detecting very low levels of PrPsc in vCJD tissues.
Methods: Serial dilutions of vCJD brain (seed) in non-CJD brain (substrate) were prepared and either frozen or subjected to 48 cycles of PMCA, each cycle consisting of a burst of sonication followed by a 30 min. incubation at 37°. These samples were then analysed by CDI and dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA), using Europium-labelled 3F4 as the anti-PrP detection antibody.
Results: Without PMCA, CDI detected vCJD PrPsc to a dilution limit of 10-3 equivalent to 10µg vCJD brain. Treatment of the samples with PMCA increased sensitivity by 100-fold. A similar increase in sensitivity was observed when healthy human platelet homogenate was used as a substrate for PMCA. CDI obviated the problem of high background observed when Western blotting was used to detect PrPsc amplified using platelet homogenates.
Discussion: We have shown that CDI can be combined with PMCA to detect very low levels of PrPsc. As the PMCA-CDI technique is not dependent on proteinase K digestion of the samples, this technique has the potential to detect proteinase sensitive forms of PrPsc. This method can utilize platelets as a readily available source of human PrPc for PMCA and both assays are conducted in a 96 well format. We are currently optimising PMCA-CDI for detecting PrPsc in vCJD peripheral tissues and blood.
AD A. Peden, M. Jones, J. Ironside, National CJD Surveillance Unit, UK; M. Turner, I. MacGregor, Scottish National Blood Transfusion Service, UK; A. Groner, CSL Behring, Germany; J. Manson, Neuropathogenesis Unit, UK
SP englisch
PO Schottland
EA pdf-Datei und Poster (geänderte Autorenliste: zusätzliche Autoren C. Prowse und M.W. Head, gestrichen: J. Manson; Titel: CDI can be combined with PMCA for high-sensitivity detection of vCJD PrPsc)