Early diagnosis of conformational diseases
View Patent ↗A method for the diagnosis or detection of conformational diseases by assaying for a marker (the pathogenic conformer) of such diseases in a sample is described, which method comprises a cyclic amplification system to increase the levels of the pathogenic conformer which causes such diseases. In particular, such transmissible conformational diseases may be prion encephalopathies. Assays, diagnostic kits and apparatus based on such methods are also disclosed.
1. A method for the detection of a conformational disease which is characterized by a conformational transition of Prion Protein between the PrP C non-pathogenic conformer and the PrP SC pathogenic conformer, by assaying a marker of said disease within a sample, which method comprises:
(i) contacting said sample with an amount of the PrP C non-pathogenic conformer;
(ia) incubating said sample with said PrP C non-pathogenic conformer;
(ii) disaggregating any aggregates eventually formed during step (i); and
(iii) determining the presence and/or amount of said PrP SC pathogenic conformer within the sample, the pathogenic conformer being a marker for the presence of said disease, wherein steps (ia) and (ii) form a cycle which is repeated at least twice before carrying out step (iii).
2. The method of claim 1 , wherein the cycle is repeated from 5 to 40 times before carrying out step (iii).
3. The method of claim 1 , wherein step (i) is carried out under physiological conditions.
4. The method of claim 1 , wherein the amount of the PrP C non-pathogenic conformer in step (i) is an excess amount.
5. The method of claim 1 , wherein the conformational disease is a transmissible conformational disease.
6. The method of any one of the preceding claims, wherein the sample to be analysed is subjected to a pre-treatment for selectively concentrating the PrP SC pathogenic conformer in the sample.
7. The method of claim 6 , wherein the pre-treatment is the extraction from the sample of a fraction which is insoluble in mild detergents.
8. An assay for a marker of a conformational disease which is characterized by a conformational transition of Prion Protein between the PrP C non-pathogenic conformer and the PrP SC pathogenic conformer, within a sample, which assay comprises the following steps:
(i) contacting said sample with an amount of the PrP C non-pathogenic conformer;
(ia) incubating said sample with said PrP C non-pathogenic conformer;
(ii) disaggregating any aggregates eventually formed during step (i); and
(iii) determining the presence and/or amount of said PrP SC pathogenic conformer within the sample, the PrP SC pathogenic conformer being a marker for the presence of said disease, wherein steps (ia) and (ii) form a cycle which is repeated at least twice before carrying out step (iii).
9. A kit for use in the assay of claim 8 which comprises a known amount of the non-pathogenic conformer, a multi-well microtitre plate and a multi-well sonicator.
10. A method for identifying a compound which modulates the conformational transition of Prion Protein between the PrP C non-pathogenic conformer and the PrP SC pathogenic conformer, comprising:
(i) contacting an amount of the PrP C non-pathogenic conformer with an amount of the PrP SC pathogenic conformer (a) in the presence of said compound and (b) in the absence of said compound;
(ii) disaggregating any aggregates eventually formed during step (i); and
(iii) determining the amount of the PrP SC pathogenic conformer (a) in the presence of said compound and (b) in the absence of said compound.
11. A method for detecting the presence of a PrP SC pathogenic form of Prion Protein within a sample, comprising:
(i) contacting the sample with an amount of the PrP C non-pathogenic prion protein;
(ia) incubating the sample with the PrP C non-pathogenic prion protein;
(ii) disaggregating any aggregates formed during step (ia); repeating steps (ia)-(ii) two or more times; and then
(iii) determining the presence and/or amount of PrP SC pathogenic prion protein within the sample.
12. Apparatus for use in the method of any one of claims 1 and 2 - 7 or for use in the assay of claim 8 , comprising a microtitre plate, multi-well sonicator and an amount of the PrP C non-pathogenic conformer.