Abstract
(Englisch)
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SUMMARY OF THE FINAL LABORATORY REPORT
Infected animals without protease resistant PrPSc would go undetected by the diagnostic methods presently used. To avoid protease digestion, we developed a quantitative immuno capturing ELISA for both normal (PrPC) and modified PrP (PrPSc), based on the comparison of the affinity of an antiserum to native and denatured samples. The ELISA plates were coated with a PrP-specific monoclonal antibody (6H4, Prionics Ltd., Zürich) and incubated with bovine brain homogenate. For detection of PrP bound to the plates, PrP-specific rabbit antisera were used as second antibody. The rabbit antibody bound to PrP was quantified by a horseradish peroxidase (HRP) reaction. The assay was calibrated with recombinant PrP of known concentration. A PrPC fraction could be detected both in untreated samples from BSE animals and in controls. For the detection of PrPSc, we exploited the effect of GdnSCN and heat on unmasking PrPSc epitopes (Gh-treatment). Gh-treatment exposed both PrPC and PrPSc. The amount of PrPSc was calculated by subtraction of the results of a Gh-treated from an untreated sample. The PrP content of medulla oblongata, thalamus and cerebral cortex of several BSE affected animals and controls was measured. A larger amount of PrPC was found in the cerebral cortex and in the thalamus than in the medulla oblongata. A significant amount of PrPSc was detected solely in the medulla oblongata of BSE affected animals. In few samples (20%) obtained from the thalamus some PrPSc was detected and no PrPSc was found in the cortex. Our results suggest that the epitopes of PrPSc, which are recognized by the antibodies used in our assay, become only accessible following treatment with GdnSCN and heat. This could perhaps be related to complexing of PrPSc with other fractions, or because its conformation differs from PrPC.
Three alleles of different length were detected in BSE positive and BSE negative animals of the Swiss bovine population by partner 5 (Institute of Animal Breeding, Bern), namely allele 5, containing 316 bp, allele 6, containing 340 bp, and allele 7, containing 364 bp. The concentration of PrPC in the medulla oblongata of BSE positiv and negative animals, being homozygous or heterozygous for allele 5 and 6 was determined. There was no significant difference in the results of the groups neither between BSE positive and negative animals, nor between the subgroups with different PrP alleles. The amount of PrPC from all available combinations of alleles, fell well within the PrPC distribution of the most abundant (82%) 6-6 genotype.
The BSE test described above was validated with blinded samples derived from 180 Swiss cattle. From this, 81 had been tested BSE-positive by immunocytochemistry. Two different methods were used for PrPSc detection: PrPSc was quantified by subtraction of PrPC from total PrP obtained after Gh-treatment, or PrPSc was quantified after digestion of PrPC by protease K. The sensitivity of the first method was 69%, and of the second method 81%. The specificity of the first method was 90%, and of the second method 59%. In comparison to other existing tests, both sensitivity and specificity were rather low.
Both the purified normal (protease-sensitive) isoform of the prion portein (PrPC) and recombinant PrP have been found to be in monomeric form, PrPC - PrPC interactions were previously unknown. We confirmed recombinant PrP to be a monomer by analytical ultracentrifugation. However, by three lines of evidence (ELISA, crosslinking experiments and size exclusion chromatography) we could also demonstrate that, under native conditions, at least part of the native bovine PrPC exists as a monomer-dimer equilibrium. In addition, we could deduce the association constant (3.9 x 108 mol-1 at 37 °C), ?G° (- 48.6 kJ mol-1), ?H° (9.5 kJ mol-1) as well as ?S° (0.2 kJ K-1 mol-1). However, our ELISA detected only about 10% of the PrPC present in a homogenate. The nature of the remaining PrPC, was not analyzed further.
We investigated the possibility to use the same assay as described above for PrP-quantification in sheep. Several brain samples from scrapie sheep were obtained from Switzerland, Italy, United Kingdom and Norway. Control animals were from Switzerland and Norway. Samples from the medulla, thalamus, cortex and cerebellum were measured. In contrast to BSE, a significant amount of PrPSc was detected in the thalamus, cerebellum and cerebral cortex of scrapie affected sheep. In addition, the amount of PrPC was significantly different in sheep and bovine when measured in the medulla and cortex, but very similar in the thalamus. In addition, there was a marked reduction of PrPC measured in the thalamus of scrapie affected animals when compared to negativ controls.
As the ELISA validation results were rather disappointing, we investigated into a more sophisticated Gh-treatment. The incubation in a laboratory oven was replaced by heating the samples in an heating block. In a first experiment GdnSCN was added to several samples containing brain homogenate and the samples were incubated at 130 to 150 °C for 2 to 8 minutes. After cooling, the amount of PrP detectable by the ELISA was measured and compared to an unheated control sample. Both with BSE and the negative control, with increasing incubation time a linear decrease of the absorbance was observed. The dynamic nature of the PrP antigen detection was used to develop some kind of a chromatographic method.
Accordingly, brain homogenate from a single animal was mixed with GdnSCN and equally distributed into 30 sample tubes. One of it was incubated at room temperature, all of the others were placed into the heating block set at 120 °C. The temperature was changed to 190 °C and a tube was removed and cooled to room temperature beginning at 132 °C and then at every two degree. All samples were measured in the ELISA. The resulting chromatogram of bovine medulla oblongata, revealed three main peaks located at about 142, 158 and 174 °C. The location of the peak was independent of the BSE status. However, in BSE positive samples only, the two peaks at 158 and 174 °C were significantly larger in comparison to the first one. In addition, a few samples, were measured using sheep brain from scrapie sick and control sheep. In all samples from sheep, additional peaks were revealed, allowing clear distinction of sheep PrP from bovine PrP. However the sample size was too small, to distinguish between differences due to different strains, breeds or the amino acid sequences.
In conclusion, an easy ELISA procedure for BSE and scrapie can be evolved, by applying just an optimized heat and incubation time to each of the sample or a more sophisticated chromatographic procedure can be applied. In contrast to most other procedures described, in our technique the use of proteinase K digestion is optional.
In order to finish the experiments described above, together with other partners we are preparing a new EC proposal.
Part of the work of this report was published in:
Meyer, R. K., B. Oesch, R. Fatzer, A. Zurbriggen, and M. Vandevelde. 1999. Detection of bovine spongiform encephalopathy-specific PrPSc by treatment with heat and guanidine thiocyanate. J. Virol. 73:9386-9392.
Meyer, R.K., A. Lustig, B. Oesch, R. Fatzer, A. Zurbriggen and M. Vandevelde. 2000. A Monomer-Dimer Equilibrium of a Cellular Prion Protein (PrPC) not Observed with Recombinant PrP. J. Biol. Chem. 275: 38081-38087.
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