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Permanent link (DOI): https://doi.org/10.7939/R33M34

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Optimization, adaptation and application of protein misfolding cyclic amplification to detection of prions in blood plasma Open Access

Descriptions

Other title
Subject/Keyword
detection
amplification
PMCA
protein misfolding cyclic amplification
plasma
optimization
de novo
prion
blood
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Braithwaite, Shannon Lynn
Supervisor and department
Belosevic, Miodrag (Biological Sciences)
Neumann, Norman (School of Public Health)
Examining committee member and department
Allison, Ted (Biological Sciences)
Aiken, Judd (Agriculture, Food & Nutritional Sciences)
McKenzie, Debbie (Biological Sciences)
Department
Department of Biological Sciences
Specialization

Date accepted
2010-09-14T15:59:52Z
Graduation date
2010-11
Degree
Master of Science
Degree level
Master's
Abstract
The PMCA assay was optimized for adaptation to low level detection of PrPSc in hamster plasma. Evaluation of numerous key variables of the PMCA assay led to an optimized protocol capable of ~3 log10 amplification after 32 cycles (two 16 hour rounds). When commercially purchased normal hamster plasma was added to the PMCA reaction an accentuation in PrPSc amplification was observed (>6.75 log10 after 32 cycles). Only con-specific plasma appeared to enhance the conversion of PrPC to PrPSc, suggesting that a species-specific co-factor may be involved in assembly of protein aggregates. Serial PMCA in the presence of low level (10%) contiguous conspecific plasma resulted in the generation of de novo PrPSc after several rounds of PMCA. Although plasma significantly accentuated PrPSc amplification by PMCA, the formation of de novo PrPSc interfered with the ability of using the PMCA assay to detect prion infections in hamsters experimentally infected with 263K scrapie.
Language
English
DOI
doi:10.7939/R33M34
Rights
License granted by Shannon Braithwaite (shannon.braithwaite@ualberta.ca) on 2010-09-13T22:47:56Z (GMT): Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of the above terms. The author reserves all other publication and other rights in association with the copyright in the thesis, and except as herein provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
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File title: Funding for this thesis work was provided by grants awarded to Dr. Norman Neumann and Dr. Miodrag Belosevic from PrioNet Canada and Alberta Prion Research Institute (APRI) with laboratory space graciously provided by Dr. Jean-Philippe Deslys of th...
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