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Diagnosis of transmissible spongiform encephalopathies using PrPSc/PrPc specific antibodies

Objective



The infectious particle causing bovine spongiform encephalopathy in cattle, Creutzfeldt-Jakob disease in humans and scrapie in sheep contains an altered form (PrPSc) of the cellular prion protein (PrPc). The difference between the two isoforms seems to be in their conformation rather than in a covalent modification or an alteration of the amino acid sequence. It has therefore been attempted by many groups to generate antibodies which would distinguish between the two isoforms. In this proposal we present a monoclonal antibody (named 1 5B3) which exactly does this. Upon immuno-precipitation of PrP from normal or prion-infected animals or humans, only PrP from infected brains is precipitated. The precipitated PrP can then also be shown to be partially protease-resistant i.e. upon proteinase K digestion the prion-specific fragment of 27-30 kDa is observed. PrPSc from cattle, humans and mice has been tested and is recognized. Other species such as sheep have not yet been tested. In the presented project we propose to use the antibody 1 5B3 to further develop diagnostic methods for human and bovine transmissible spongiform encephalopathies such as a capture ELISA. Furthermore, detection of PrPSc by immuno-precipitation with antibody 15B3 will allow the omission of proteinase K digestion for the detection of the disease-specific form. Immunoaffinity purification will therefore allow to analyze PrPSc from diluted samples and thereby increase the sensitivity. We intend to analyze PrPSc from cerebrospinal fluid and peripheral tissues such as blood (white blood cells), Lymph nodes and tonsils. Furthermore, it will be possible to specifically identify tissue culture cells which produce PrPSc using immuno-fluorescence with the 15B3 antibody. This system will yield an in-vitro detection system for the identification of prions. A second antibody (named 6H4) recognizes PrPc or denatured PrPSc from different species on Western blots. This will allow us to detect human, bovine, mouse, or sheep PrPsc on Western blots with the same antibody. Experiments such as a side by side analysis of the PrP glycosylation pattern are possible with this antibody.
The proposed project centers on the use of these novel antibodies available from Prionics. The unique properties of antibody 1 5B3 make it an extremely valuable tool with respect to diagnosis and maybe even therapy. We will therefore also exploit the possibility whether treatment of animals with antibody 1 5B3 will lead to a prolongation of the incubation time. Since the antibody 1 5B3 reacts with bovine, human and mouse PrPSc we will be able to develop the methods in the mouse scrapie system with the prospect of their application to humans with CJD.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

LUDWIG-MAXIMILIANS UNIVERSITY OF MUNICH
Address
Geschwister-scholl-platz 1
80539 Muenchen
Germany

Participants (6)

Consejo Superior de Investigaciones Científicas
Spain
Address
119,Serrano 119
28071 Madrid
CytotestCell Research GmbH. & Co. KG.
Germany
Address
19,In Den Leppsteinswiesen 19
64380 Rossdorf
Prionics AG
Switzerland
Address
Wintherhurestrasse
8057 Z«rich
The Hebrew University of Jerusalem - The Authority for Research and Development
Israel
Address
Hadassah Medical Centre
91120 Jerusalem
The Hebrew University of Jerusalem - The Authority for Research and Development
Israel
Address

91120 Jerusalem
University of Edinburgh
United Kingdom
Address
Crewe Road
EH4 2XU Edinburgh