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University of Southampton Research Repository Eprints Soton University of Southampton Research Repository ePrints Soton Copyright © and Moral Rights for this thesis are retained by the author and/or other copyright owners. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the copyright holder/s. The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the copyright holders. When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given e.g. AUTHOR (year of submission) "Full thesis title", University of Southampton, name of the University School or Department, PhD Thesis, pagination http://eprints.soton.ac.uk UNIVERSITY OF SOUTHAMPTON FACULTY OF MEDICINE, HEALTH & LIFE SCIENCES School of Biological Sciences Proteins as markers of TSE infection in sheep blood Joanne Martin Thesis for the degree of Doctor of Philosophy September 2008 UNIVERSITY OF SOUTHAMPTON ABSTRACT FACULTY OF MEDICINE, HEALTH AND LIFE SCIENCES SCHOOL OF BIOLOGICAL SCIENCES Doctor of Philosophy PROTEINS AS MARKERS OF TSE INFECTION IN SHEEP BLOOD Joanne Martin Transmissible spongiform encephalopathies (TSEs) are a group of fatal infectious neurodegenerative diseases affecting both humans and agricultural animals. TSE transmission via blood transfusion has been demonstrated experimentally in rodent, primate and sheep models. Additionally, in humans, four variant Creutzfeld-Jakob disease (vCJD) cases have been reported which probably resulted from infected blood transfusions. Although TSEs can be transmitted via blood transfusion, little is known about which blood cells are involved in the replication of the TSE agent and how infectivity is spread throughout the body prior to neuroinvasion. There are no currently validated diagnostic tests for TSE infection in blood. Detection of PK-resistant PrPSc has been extensively used as a biochemical marker for TSE infectivity. However, when this project was started it was not known if PK- resistant PrPSc was present in TSE-infected sheep blood in sufficient quantities to explain the infectivity levels shown by bioassay. Following the development of an optimised Western blot method, this project has demonstrated that the pattern of protein detected with novel anti-PrP monoclonal antibodies is very different from the conventional triple banded pattern of PK-resistant PrPSc. High molecular weight bands were apparent in phosphotungstic acid (NaPTA) concentrated scrapie-infected and uninfected blood and may represent a novel form of blood-specific PrP. PK-resistant PrPSc is not therefore a suitable marker for TSE infection in blood. Other proteins in TSE infected blood were also investigated. Using a proteomics approach three protein markers, lactate dehydrogenase, elongation factor 1 and annexin 1 had altered expression patterns in scrapie infected blood. These proteins, in addition to the novel forms of PrP found in blood, may provide new information on the mechanisms of pathogenesis in scrapie-infected sheep and might prove to be useful molecular indicators of diagnostic value. i Table of contents 1. Introduction ………………………………………………………………...... 1 1.1. Transmissible spongiform encephalopathies ……………………………....... 2 1.1.1. The prion protein (PrP) …………………………………………... 3 1.1.2. Animal TSEs …………………………………………………….. 5 1.1.3. Human TSEs …………………………………………………….. 7 1.2. Nature of the infectious agent ……………………………………………….. 10 1.3. The structure and function of the prion protein (PrP) ……………………...... 15 1.3.1. Biosynthesis of PrPC………………………………………………. 17 1.3.2. The function of PrPC……………………………………………… 18 1.3.3. Models of PrPC/ PrPSc conversion ………………………………... 20 1.4. Sheep PrP genetics …………………………………………………………... 22 1.5. Peripheral pathogenesis………………………………………………………. 23 1.5.1. Initial infection routes…………………………………………...... 23 1.5.2. Infectious agent uptake……………………………………………. 24 1.5.3. Follicular dendritic cells, macrophages, T cells, B cells and 27 dendritic cells……………………………………………………... 1.5.4. Neuroinvasion…………………………………………………….. 30 1.5.5. Summary………………………………………………………….. 32 1.6. Role of blood in pathogenesis……………………………………………….. 32 1.6.1. Evidence of infectivity in blood………………………………….. 32 1.6.2. Distribution of infectivity………………………………………… 40 1.6.3. PrPSc detection in blood…………………………………………... 42 1.6.4. Distribution of cellular PrP in blood……………………………… 43 1.6.5. Protein markers of disease other than PrPSc………………………. 45 1.7. Methods of detecting and measuring PrPC/ PrPSc……………………………. 46 1.7.1. Immunohistochemistry……………………………………………. 46 1.7.2. Western blotting…………………………………………………... 46 1.7.3. Sandwich immunoassays…………………………………………. 47 1.7.4. PrPSc specific antibodies………………………………………….. 48 1.7.5. Cell lines …………………………………………………………. 48 ii 1.8. Progress of TSE diagnostic tools…………………………………………….. 49 1.8.1. Current status of TSE blood tests……………………………….... 52 1.8.2. Specific ligands…………………………………………………… 52 1.8.3. AS-ELISA………………………………………………………… 53 1.8.4. Conformationally sensitive peptides……………………………… 53 1.8.5. Other approaches………………………………………………….. 53 1.8.6. Summary………………………………………………………….. 54 1.9. Thesis aims…………………………………………………………………… 55 2. Methods……………………………………………………………………….. 57 2.1. Materials……………………………………………………………………… 58 2.1.1. Animal tissues…………………………………………………….. 58 2.1.2. Antibodies………………………………………………………… 59 2.1.3. Chemical reagents………………………………………………… 59 2.1.4. Enzymes…………………………………………………………... 59 2.1.5. Magnetic beads…………………………………………………… 59 2.1.6. Monoclonal antibody purification………………………………… 59 2.1.7. Recombinant protein……………………………………………… 60 2.1.8. Scrapie mouse brain (SMB) cell lines……………………………. 60 2.2. Solutions, buffers and media………………………………………………… 61 2.2.1. Coomassie staining solutions (for mass spectrometry) ………….. 61 2.2.2. Coomassie staining solutions (for 1D SDS-PAGE)……………… 61 2.2.3. Fluorescence activated cell sorting (FACS) solutions and buffers.. 61 2.2.4. Lysis buffers……………………………………………………… 62 2.2.5. Magnetic activated cell sorting (MACS) buffers………………… 62 2.2.6. Monoclonal antibody purification buffers……………………….. 62 2.2.7. Protein digestion and peptide extraction buffers………………… 63 2.2.8. Sodium phosphotungstic acid (NaPTA) buffers…………………. 63 2.2.9. Sodium dodecyl sulphate –polyacrylamide gel electrophoresis 64 Buffers (1D SDS-PAGE)………… … …………………………. 2.2.10. Sodium dodecyl sulphate –polyacrylamide gel electrophoresis 64 buffers (2D SDS-PAGE)……………………………………… 2.2.11. Silver staining solutions (1D PAGE)…………………………… 65 iii 2.2.12. Silver staining solutions (2D PAGE)…………………………… 65 2.2.13. Scrapie mouse brain (SMB) cell culture solutions………………. 66 2.2.14. SYPRO® orange staining solutions……………………………… 66 2.2.15. Western blotting buffers………………………………………… 66 2.3. Methods………………………………………………………………………. 67 2.3.1. Brain tissue methods……………………………………………………….. 67 2.3.1.1. Brain homogenisation………………………………………….. 67 2.3.1.2. Serial dilutions of brain homogenate…………………………… 67 2.3.1.3. Storage conditions of brain homogenate………………………... 68 2.3.2. Preparation of scrapie mouse brain (SMB) cells…………………………... 68 2.3.2.1. SMB cell culture………………………………………………… 68 2.3.2.2. SMB cell thawing and freezing…………………………………. 68 2.3.3. Preparation of leucocytes…………………………………………………... 69 2.3.3.1. Buffy coat isolation from blood………………………………… 69 2.3.3.2. Peripheral blood mononuclear cell (PBMC) isolation from 69 whole Blood……………………………………………………. 2.3.3.3. Cell counting…………………………………………………… 69 2.3.3.4. Leukocyte cell sorting…………………………………………... 70 2.3.3.5. Flow cytometry staining and acquisition……………………….. 71 2.3.3.6. Leukocyte freezing and thawing……………………………….. 71 2.3.4. Sample preparation for protein analysis on Western blots………………… 72 2.3.4.1. Lysis of brain homogenates…………………………………….. 72 2.3.4.2. Lysis of cells……………………………………………………. 72 2.3.4.3. Proteinase K (PK) treatment of brain homogenates……………. 72 2.3.4.4. PK treatment of cell lysates…………………………………….. 73 2.3.4.5. Sodium phosphotungstic acid (NaPTA) precipitation of brain 73 Homogenate: Method 1…………………………………………. 2.3.4.6. NaPTA precipitation of brain homogenate: Method 2 ………… 73 2.3.4.7. NaPTA precipitation of leukocytes and SMB cell lysates 74 Method 1………………………………………………………… 2.3.4.8. NaPTA precipitation of leukocyte cell lysates: Method 2……… 74 2.3.5. Protein analysis by SDS-PAGE and Western blotting…………………….. 74 iv 2.3.5.1. Sodium dodecyl sulphate polyacrylamide gel electrophoresis 74 (SDS-PAGE 1D)………………………………………………… 2.3.5.2. Coomassie staining……………………………………………… 75 2.3.5.3. Silver staining (1D PAGE)……………………………………… 75 2.3.5.4. SYPRO® orange staining……………………………………….. 76 2.3.5.5. Western blotting………………………………………………… 76 2.3.6. Two-dimensional (2D) PAGE protein methods…………………………… 77 2.3.6.1. Sample preparation for 2D PAGE……………………………… 77 2.3.6.2. Sample rehydration and isoelectric focussing (IEF)……………. 78 2.3.6.3. 2D SDS-PAGE gel preparation…………………………………. 78 2.3.6.4. Second dimension separation…………………………………… 78 2.3.6.5. Silver staining (2D PAGE)……………………………………… 79 2.3.6.6. In-gel protein digestion and peptide extraction…………………. 79 2.3.6.7. Mass spectrometry………………………………………………. 80 3. Development and optimisation of a sensitive Western blot immunoassay 81 for PrPSc detection……………………………………………………………. 3.1. Introduction………………………………………………………………….. 82 3.1.1. Antibodies…………………………………………………………………. 84 3.2. Materials and methods……………………………………………………….. 86 3.2.1. Sensitive Western blot method……………………………………. 86 3.3. Results………………………………………………………………………..
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