Objective
This proposal aims at improving and controlling techniques in the diagnosis of BSE in humans and animals. For that purpose two techniques have recently been developed, one of which, the paraffin-embedded tissue blot (PET blot) technique for prpSC has proven most sensitive in post mortem brain material and will be used to validate conventional immunohistochemical methods. The other technique uses fluorescence correlation spectroscopy (FCS) to detect seeded multimerization of prion protein molecules in the cerebrospinal fluid (CSF).
1. Currently, transmissible spongiform encephalopathies are most reliably diagnosed using histopathology, immunohistochemistry and Western blotting to detect the pro/ease-resistant prpSC in post-mortem brains. The histoblot technique, which requires fresh unfixed tissue, is superior in sensitivity to immunohistochemistry of formalin-fixed and paraffin-embedded CNS material. This is unfortunate since in the majority of cases, be it in veterinary or human pathology, formalin fixed tissue may be the only tissue available for immunohistochemistry. We have therefore developed a technique which combines the advantage of high sensitivity of tissue blotting (Western or histoblot) and the advantages of paraffin-embedded material, i.e. the high anatomic resolution and ample availability of tissue samples. This technique, which we have termed the paraffin-embedded tissue blot (PET blot), surprisingly is at least as or even more sensitive than the histoblot technique. The technique has been successfully employed for the investigation of human and some animal variants of prion disease. Since it takes at least one week to perform, it will not be used to test meat for human consumption; however, it will be most useful in validating other techniques and in retrospective investigations. It is a present being used to discover covert disease in cattle.
2. We have used fluorescence correlation spectroscopy (FCS) to detect aggregate of prpSC in cerebrospinal fluid from cases of human and animal prion disease. FCS is a recently established, highly sensitive method which allows the unequivocal detection of single fluorescently labeled molecules in solution. The method relies on focussing laser light into a very small, diffraction-limit spot. The focal spot constitutes a volume element of approximately 1 femtoliter which gives a very high signal to noise ratio. Fluorescent molecules present in the focal spot are excited by the laser light and subsequently emit fluorescent light which is detected by con focal microscopy technology. The diffusion of fluorescent particles through the confocal volume causes fluctuations in fluorescence intensity which can be analyzed by auto correlation. In earlier studies (Pitschke et aL, Nature Medicine 1998, see appendix)it has been shown that the multimerization of PrP or b-amyloid protein can be followed with high sensitivity when recorded with FCS. The peptide probe which carries a fluorescent label is added to the sample of interest and is bound to the prion particle in sample volume. This process was called seeded polymerization. The fluorescent label deposited on the prion particle can be detected as a fluorescence peak to to illuminate with the laser beam. Although this completely new diagnostic approach has been shown to work in principle, it will have to be optimized for routine application in everyday diagnosis.
The two techniques, the PET blot and FCS, will be tested in correlation in human sporadic and hereditary prion disease, in an experimental system in the hamster and in primates; the PET blot will also be used to screen a large number (>500 of cattle from Switzerland which will also be investigated by immunohistochemic methods. Thus both techniques will contribute to the validation of existing methodology, to determining the incidence of covert TSE in cattle and to harmonizing and controlling diagnostic methods and early detection of human and animal BSE. In the future, the two techniques may also be used to approach other problems in BSE research diagnosis, validation of other tests etc.
Fields of science (EuroSciVoc)
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CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques.
- natural sciencesbiological scienceszoologymammalogyprimatology
- natural sciencesbiological sciencesbiochemistrybiomoleculesproteins
- natural sciencesphysical sciencesopticsmicroscopy
- natural sciencesphysical sciencesopticslaser physics
- natural sciencesphysical sciencesopticsspectroscopy
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Call for proposal
Data not availableFunding Scheme
CSC - Cost-sharing contractsCoordinator
81377 MUENCHEN
Germany