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Western Immunoblotting Techniques for the Study of Transmissible Spongiform Encephalopathies

  • Michael J. Stack
Chapter
Part of the Methods and Tools in Biosciences and Medicine book series (MTBM)

Abstract

Initial diagnosis of transmissible spongiform encephalopathies (TSEs) has always been made by the detection of neuronal vacuolation within formaldehyde-fixed brain sections by histopathological examination. Over time, specific antibodies for pathological markers of TSEs have been produced and immunology-based techniques are being increasingly used to aid diagnosis for screening purposes and to provide information at the molecular level for the disease group as a whole. Western immunoblotting is an established technology and, due to its use of specific antibodies, it is a tool that is used across many scientific disciplines to provide diagnosis of particular diseases, or answers to research problems. This chapter outlines some of the Western immunoblotting techniques which are being used to study TSEs, discusses the advantages and disadvantages of each technique, and makes suggestions on the circumstances under which each may be best used.

Keywords

Prion Protein Bovine Spongiform Encephalopathy Western Immunoblotting Transmissible Spongiform Encephalopathy Scrapie Agent 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

  1. 1.
    Bolton DC, McKinley MP, Prusiner SB (1982) Identification of a protein that purifies with the scrapie prion. Science 218: 1309–1311PubMedCrossRefGoogle Scholar
  2. 2.
    Chesebro B, Race R, Wherly K et al. (1985). Identification of scrapie prion protein specific mRNA in scrapie-infected and uninfected brain. Nature 315: 331–333PubMedCrossRefGoogle Scholar
  3. 3.
    Oesch B, Westaway D, Walchli M et al. (1985). A cellular gene encodes scrapie PrP 27–30 protein. Cell 40: 735–746PubMedCrossRefGoogle Scholar
  4. 4.
    Hunter GD, Millson GC, Meek G (1964) The intracellular location of the agent of mouse scrapie. J Gen Microbiol 34: 319PubMedCrossRefGoogle Scholar
  5. 5.
    Millson GC, Hunter GD, Kimberlin RH (1971) An experimental examination of the scrapie agent hi cell membrane mixtures. J Comp Pathol 81: 255–265PubMedCrossRefGoogle Scholar
  6. 6.
    Prusiner SB, Garfm DE, Cochran SP et al. (1980) Experimental scrapie in the mouse: electrophoretic and sedimentation properties of the partially purified agent. J Neurochem 35: 574–582PubMedCrossRefGoogle Scholar
  7. 7.
    Prusiner SB, Groth DF, Cochran SP et al. (1980a) Gel electrophoresis and glass permeation chromatography of the hamster scrapie agent after enzymic digestion and detergent extraction. Biochemistry 19: 4892–4898CrossRefGoogle Scholar
  8. 8.
    Merz PA, Somerville RA, Wisniewski HM, Iqbal K (1981) Abnormal fibrils from scrapie-infected brain. Acta Neuropath 54: 63–74PubMedCrossRefGoogle Scholar
  9. 9.
    Prusiner SB, Bolton DC, Groth DF et al. (1982) Further purification and characterisation of scrapie prions. Biochemistry 21: 6942–6950PubMedCrossRefGoogle Scholar
  10. 10.
    Prusiner SB, Bolton DC, Kent SB, Hood LE (1984) Purification and structural studies of a major scrapie prion protein. Cell 38: 127–134PubMedCrossRefGoogle Scholar
  11. 11.
    Hilmert H, Diringer H (1984) A rapid and efficient method to enrich SAF-protein from scrapie brains of hamsters. Biosci Reps 4: 165–170CrossRefGoogle Scholar
  12. 12.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277: 680–685CrossRefGoogle Scholar
  13. 13.
    Towbin H, Staehlin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Nat Acad Sci USA 76: 4350–4354PubMedCrossRefGoogle Scholar
  14. 14.
    Collinge J, Sidle KCL, Meads J et al. (1996) Molecular analysis of prion strain variation and the aetiology of “new” Variant CJD. Nature 383: 685–690PubMedCrossRefGoogle Scholar
  15. 15.
    Hill AF, Desbruslais M, Joiner S et al. (1997) The same prion strain causes vCJD and BSE. Nature 389: 448–450PubMedCrossRefGoogle Scholar
  16. 16.
    Hope J, Reekie LJD, Hunter N et al. (1988) Fibrils from brains of cows with new cattle disease contain scrapie-associated fibrils. Nature 336: 390–392PubMedCrossRefGoogle Scholar
  17. 17.
    Beekes M, Baldauf E, Capens S et al. (1995) Western blot mapping of disease specific amyloid in various animal species and humans with transmissible spongiform encephalopathies using a high-yield purification method. J Gen Virol 76: 2567–2576PubMedCrossRefGoogle Scholar
  18. 18.
    Scott AC, Stack MJ (1994) Protocols II and III. The diagnosis of bovine spongiform encephalopathy (BSE) and scrapie by the detection of BSE fibrils or scrapieassociated fibrils (SAF) by transmission electron microscopy and by Western Immunoblotting. In: European Commision for Agriculture: Transmissible spongiform encephalopathies. Protocols for the laboratory diagnosis and confirmation of bovine spongiform encephalopathy and scrapie. A report from the Scientific Veterinary Committee. European Commission, Directorate General for Agriculture, Unit for Veterinary Legislation and Zootechnics, Brussels, Prot. II: 1–12; Prot. III: 1–18Google Scholar
  19. 19.
    Stack MJ, Keyes P, Scott AC (1996) The Diagnosis of bovine spongiform encephalopathy and scrapie by the detection of fibrils and the abnormal protein isoform. In: HF Baker, RM Ridley (eds): Methods in Molecular Medicine - Prion Diseases. Humana Press, Totowa, N.J., 85–104Google Scholar
  20. 20.
    Benestad SL, Sarradin P, Thu B et al (2003) Cases of scrapie with unusual features in Norway and designation of a new type, Nor98. Vet Rec 153: 202–208PubMedCrossRefGoogle Scholar
  21. 21.
    Polymenidou M, Verrghese-Nikolakaki S, Groschup M et al. (2002) A short purification process for quantitative isolation of PrPse from naturally occurring and experimental transmissible spongiform encephalopathies. BMC Infect Dis 2: 23PubMedCrossRefGoogle Scholar
  22. 22.
    Hadlow W, Kennedy RC, Race RE (1982) Natural infection of Suffolk sheep with scrapie virus. J Infect Dis 146: 657–664PubMedCrossRefGoogle Scholar
  23. 23.
    Wilesmith J, Wells GAH, Cranwell MP, Ryan JBM (1988) Bovine spongiform encephalopathy: Epidemiological studies. Vet Rec 123: 638–644PubMedGoogle Scholar
  24. 24.
    Alpers M (1987) Epidemiology and clinical aspects of kuru. In: SB Prusiner, MP McKinley (eds): Prions - novel infectious pathogens causing scrapie and Creutzfeldt-Jakob disease. Academic Press, Orlando, 451–465Google Scholar
  25. 25.
    Hartsough GR, Burger D (1965) Encephalopathy of mink. 1. Epizootiologic and clinical observations. J Infect Dis 115: 387–392PubMedCrossRefGoogle Scholar
  26. 26.
    Ikegami Y, Ito M, Isomura H et al. (1991) Pre-clinical and clinical diagnosis of scrapie by detection of PrP protein in tissues of sheep. Vet Rec 128: 271–275PubMedCrossRefGoogle Scholar
  27. 27.
    Race RE, Ernst D (1992) Detection of proteinase K-resistant prion protein and infectivity in mouse spleen by 2 weeks after scrapie agent inoculation. J Gen Virol 73: 3319–3323PubMedCrossRefGoogle Scholar
  28. 28.
    Rubenstein R, Merz PA, Kascsak RJ et al. (1991) Scrapie-infected spleens: analysis of infectivity, scrapie-associated and protease-resistant proteins. J Infect Dis 164: 29–35PubMedCrossRefGoogle Scholar
  29. 29.
    Van Keulen LJM, Schreuder BEC, Meloen RH et al. (1996) Immunohistochemical detection of prion protein in lymphoid tissues of sheep with natural scrapie. J Clin Microbiol 34: 1228–1231PubMedGoogle Scholar
  30. 30.
    Schreuder BEC, Van Keulen LJM, Vromans MEW et al. (1996) Preclinical test for prion diseases. Nature 381: 563PubMedCrossRefGoogle Scholar
  31. 31.
    Hill AF, Zeidler M, Collinge J (1997) Diagnosis of new variant Creutzfeldt-Jakob disease by tonsil biopsy. The Lancet 349: 99–100CrossRefGoogle Scholar
  32. 32.
    Kimberlin RH (1986) Scrapie: how much do we really understand? Neuropath App Neurobiol 12: 131–147CrossRefGoogle Scholar
  33. 33.
    Kimberlin RH, Walker CA (1986) Pathogenesis of scrapie (strain 263K) in hamsters infected intracerebrally, intraperitoneally or intraocularly. J Gen Virol 67: 255–263PubMedCrossRefGoogle Scholar
  34. 34.
    Kimberlin RH, Walker CA (1988) Incubation periods in six models of intraperitoneally injected scrapie depend mainly on the dynamics of agent replication within the nervous system and not the lymphoreticular system. J Gen Virol 69: 2953–2960PubMedCrossRefGoogle Scholar
  35. 35.
    Kimberlin RH, Walker CA (1990) Intraperitoneal infection with scrapie is established within minutes of injection and is non-specifically enhanced by a variety of different drugs. Arch Virol 112: 103–114PubMedCrossRefGoogle Scholar
  36. 36.
    Doi S, Ito M, Shinagawa M et al. (1988) Western blot detection of scrapie-associated fibril protein in tissues outside the central nervous system from preclinical scrapie-infected mice. J Gen Virol 69: 955–960PubMedCrossRefGoogle Scholar
  37. 37.
    Mohri S, Farquhar CF, Somerville RA et al. (1992) Immunodection of a disease specific PrP fraction in scrapie-affected sheep and BSE-affected cattle. Vet Rec 131: 537–539PubMedGoogle Scholar
  38. 38.
    Muramatsu Y, Onodera A, Horiuchi M (1994) Detection of PrPse in sheep at the preclinical stage of scrapie and its significance for the diagnosis of insidious infection. Arch ‘Virol 13: 427–432CrossRefGoogle Scholar
  39. 39.
    Race R, Ernst D, Jenny A et al. (1992) Diagnostic implications of detection of proteinase K-resistant protein in spleen, lymph nodes and brain of sheep. Am J Vet Res 53: 883–889PubMedGoogle Scholar
  40. 40.
    Safar J, Wille H, Itri Vet al. (1998) Eight prion strains have PrP(Sc) molecules with different conformations. Nat Med 4: 1157–1165PubMedCrossRefGoogle Scholar
  41. 41.
    Wadsworth JD, Joiner S, Hill AF et al. (2001) Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay. Lancet 358: 171–180PubMedCrossRefGoogle Scholar
  42. 42.
    Kuczius T, Haist I, Groschup MH (1999) Molecular analysis of bovine spongiform encephalopathy and scrapie strain variation. J Infect Dis 178: 693–699CrossRefGoogle Scholar
  43. 43.
    Kuczius T, Groschup MH (1999) Differences in proteinase K resistance and neuronal deposition of abnormal prion proteins characterise bovine spongiform encephalopathy (BSE) and scrapie strains Mol Med 5: 406–418PubMedGoogle Scholar
  44. 44.
    Parchi P, Capellari S, Chen SG et al. (1997) Typing prion isoforms. Nature 386: 232–234PubMedCrossRefGoogle Scholar
  45. 45.
    Somerville RA, Chong A, Mulqueen OU et al. (1997) Biochemical typing of scrapie strains. Nature 386: 564PubMedCrossRefGoogle Scholar
  46. 46.
    Hill AF, Sidle KCL, Joiner S et al. (1998) Molecular screening of sheep for BSE. Neurosci Lett 255: 159–162PubMedCrossRefGoogle Scholar
  47. 47.
    Hope J, Wood SCER, Birkett CR et al. (1999) Molecular analysis of ovine prion protein identifies similarities between BSE and an experimental isolate of natural scrapie, CH1641. J Gen 14rol 80: 1–4Google Scholar
  48. 48.
    Hope J, Wood SCER, Birkett CR et al. (2000) Molecular analysis of ovine prion protein identifies similarities between BSE and an experimental isolate of natural scrapie, CH1641. J Gen Wrol 81 (Pt 4): 1155–1164Google Scholar
  49. 49.
    Baron TGM, Madec J-Y, Calavas D (1999) Similar signature of the prion protein in natural sheep scrapie and bovine encephalopathy-linked diseases. J ain Microbiol 37: 3701–3704Google Scholar
  50. 50.
    Baron TGM, Madec J-Y, Calavas D et al. (2000) Comparison of French natural scrapie isolates with BSE and experimental scrapie infected sheep. Neurosci Lett 284: 175–178PubMedCrossRefGoogle Scholar
  51. 51.
    Sweeney T, Kuczius T, McElroy M et al. (2000) Molecular analysis of Irish sheep scrapie cases. J Gen Nrol 81: 1621–1627Google Scholar
  52. 52.
    Stack MJ, Chaplin MJ, Clark J (2002). Differentiation of prion protein glycoforms from naturally occurring sheep scrapie, sheep passaged scrapie strains (CH1641 and SSBP1), bovine spongiform encephalopathy (BSE) cases and Romney and Cheviot breed sheep experimentally inoculated with BSE using two monoclonal antibodies. Acta Neuropath 104: 279–286PubMedGoogle Scholar
  53. 53.
    Nonno R, Esposito E, Vaccari G et al. (2003) Molecular analysis of cases of Italian sheep scrapie and comparison with cases of bovine spongiform encephaloapthy (BSE) and experimental BSE in sheep. J Clin Microbiol 41: 4127–4133PubMedCrossRefGoogle Scholar
  54. 54.
    Schaller O, Fatzer R, Stack MJ et al. (1999) Validation of a Western immunoblotting procedure for bovine PrPSc detection and its use as a rapid surveillance method for the diagnosis of bovine spongiform encephalopathy (BSE). Acta Neuropathol 98: 437–443PubMedCrossRefGoogle Scholar
  55. 55.
    Moynagh J, Schimmel H (1999) Tests for BSE evaluated. Nature 400: 105PubMedCrossRefGoogle Scholar
  56. 56.
    Foster JD, Dickinson AG (1988) The unusual properties of CH1641, a sheep-passaged isolate of scrapie. Vet Rec 123: 5–8PubMedCrossRefGoogle Scholar
  57. 57.
    Dickinson AG, Fraser H (1979) An assessment of the genetics of scrapie in sheep and mice. In: SB Prusiner, WJ Hadlow (eds): Slow Transmissible Diseases of the Nervous System,Vol 1. Academic Press, New York, 367–386Google Scholar
  58. 58.
    Dickinson AG, Outram GW, Taylor DM, Foster JD (1986) Further evidence that scrapie agent has an independent genome. In: LA Court, D Dormont, P Brown, DT Kingsbury (eds): Unconventional Viruses and Central Nervous System Diseases. Moisdon la Riviere, Abbaye de Mellaray, 446–460Google Scholar
  59. 59.
    Wilson DR, Anderson RD, Smith W (1950) Studies in scrapie. J Comp Pathol 60: 267–275PubMedGoogle Scholar
  60. 60.
    Dickinson AG, Outram GW (1988) Genetic aspects of unconventional virus infections: the basis of the vivino hypothesis. In: G Bock, J Marsh (eds): Novel Infectious Agents and the Central Nervous System. Ciba Foundation Sympo-sium No 135. John Wiley & Sons, Chichester, UK, 63–83Google Scholar
  61. 61.
    Korth C, Stierli B, Streit P et al. (1997) Prion (PrPse) - specific epitope defined by a monoclonal antibody. Nature 390: 74–77PubMedCrossRefGoogle Scholar
  62. 62.
    Harmeyer S, Pfaff E, Groschup MH (1998) Synthetic peptide vaccines yield monoclonal antibodies to cellular and pathological prion proteins of ruminants. J Gen Wol 79: 937–945Google Scholar

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© Springer Basel AG 2004

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  • Michael J. Stack

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