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

  • Author / Creator
    Braithwaite, Shannon Lynn
  • 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.

  • Subjects / Keywords
  • Graduation date
  • Type of Item
  • Degree
    Master of Science
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
  • Institution
    University of Alberta
  • Degree level
  • Department
    • Department of Biological Sciences
  • Supervisor / co-supervisor and their department(s)
    • Belosevic, Miodrag (Biological Sciences)
    • Neumann, Norman (School of Public Health)
  • Examining committee members and their departments
    • Allison, Ted (Biological Sciences)
    • McKenzie, Debbie (Biological Sciences)
    • Aiken, Judd (Agriculture, Food & Nutritional Sciences)