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Development of a sensitive cell culture system to assess prion infectivity and the efficacy of prion decontamination technologies

Development of a sensitive cell culture system to assess prion infectivity and the efficacy of prion decontamination technologies
Development of a sensitive cell culture system to assess prion infectivity and the efficacy of prion decontamination technologies
Creutzfeldt-Jakob disease (CJD) can be iatrogenically transmitted during transplants, grafts and transfusions from CJD infected donors and also contaminated surgical instruments. A variety of methods to amplify and detect the presence of infectious prions as disease markers are available. However, these techniques do not measure the infectivity potentially associated with these markers. Currently, animal-bioassays are used to detect infectivity; however, they have limitations in detectable prion strains, cost, ethical considerations and assay length. Novel cell-based infectivity assays offer the potential to overcome these limitations, lower the requirements for animal use and the application of different cell lines could detect a wider range of prion strains. This project utilised murine neuroblastoma, N2a #58 cells infected with 22L-murine scrapie to develop a highly sensitive assay for the in situ detection of amyloid-rich prion (PrPSc) accumulation as an indication of prion infectivity. The autofluorescence quenching properties of Sudan black (SB) were incorporated into a novel Thioflavin T (ThT) based protocol for amyloid staining with improved specificity and sensitivity. Cell passages were incorporated into the assay to increase incubation time, improve cell viability and subsequently improve assay sensitivity; thus, demonstrating the detection of infectivity from a final 10-10 dilution of 22L-infected brain homogenate. Introduction of 22L-inoculated, surgical grade stainless steel wires to the N2a #58 cells demonstrated the SB/ThT detection of prion infectivity pre and post decontamination, which was comparable to animal bioassay data. Furthermore, preliminary work on the incorporation of the SB/ThT detection of prion infectivity within neural stem cells (NSC’s), for prion propagation within a cell line that did not require genetic manipulation for increased prion susceptibility, highlighted problems with unspecific fluorescence of dead cells during NSC differentiation. Improvements in culture conditions of the NSC’s regarding atmospheric conditions and trophic support were addressed in preparation for their use in future prion infectivity assays.
Secker, Thomas
16b0a878-984f-4272-bfaa-667c7c63023a
Secker, Thomas
16b0a878-984f-4272-bfaa-667c7c63023a
Keevil, Charles
cb7de0a7-ce33-4cfa-af52-07f99e5650eb
Smyth, Neil
0eba2a40-3b43-4d40-bb64-621bd7e9d505

(2012) Development of a sensitive cell culture system to assess prion infectivity and the efficacy of prion decontamination technologies. University of Southampton, Biological Sciences, Doctoral Thesis, 240pp.

Record type: Thesis (Doctoral)

Abstract

Creutzfeldt-Jakob disease (CJD) can be iatrogenically transmitted during transplants, grafts and transfusions from CJD infected donors and also contaminated surgical instruments. A variety of methods to amplify and detect the presence of infectious prions as disease markers are available. However, these techniques do not measure the infectivity potentially associated with these markers. Currently, animal-bioassays are used to detect infectivity; however, they have limitations in detectable prion strains, cost, ethical considerations and assay length. Novel cell-based infectivity assays offer the potential to overcome these limitations, lower the requirements for animal use and the application of different cell lines could detect a wider range of prion strains. This project utilised murine neuroblastoma, N2a #58 cells infected with 22L-murine scrapie to develop a highly sensitive assay for the in situ detection of amyloid-rich prion (PrPSc) accumulation as an indication of prion infectivity. The autofluorescence quenching properties of Sudan black (SB) were incorporated into a novel Thioflavin T (ThT) based protocol for amyloid staining with improved specificity and sensitivity. Cell passages were incorporated into the assay to increase incubation time, improve cell viability and subsequently improve assay sensitivity; thus, demonstrating the detection of infectivity from a final 10-10 dilution of 22L-infected brain homogenate. Introduction of 22L-inoculated, surgical grade stainless steel wires to the N2a #58 cells demonstrated the SB/ThT detection of prion infectivity pre and post decontamination, which was comparable to animal bioassay data. Furthermore, preliminary work on the incorporation of the SB/ThT detection of prion infectivity within neural stem cells (NSC’s), for prion propagation within a cell line that did not require genetic manipulation for increased prion susceptibility, highlighted problems with unspecific fluorescence of dead cells during NSC differentiation. Improvements in culture conditions of the NSC’s regarding atmospheric conditions and trophic support were addressed in preparation for their use in future prion infectivity assays.

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Published date: 28 September 2012
Organisations: University of Southampton, Centre for Biological Sciences

Identifiers

Local EPrints ID: 354432
URI: http://eprints.soton.ac.uk/id/eprint/354432
PURE UUID: cc53de52-d7ea-4f6a-94a3-6392b896e5f5
ORCID for Charles Keevil: ORCID iD orcid.org/0000-0003-1917-7706

Catalogue record

Date deposited: 21 Oct 2013 13:20
Last modified: 06 Jun 2018 12:49

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Contributors

Author: Thomas Secker
Thesis advisor: Charles Keevil ORCID iD
Thesis advisor: Neil Smyth

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