Patent EA-004953-B1 (EA)
Title: Method for detecting and separating abnormal prion protein and kit therefor
Inventor: ALPERT ANDREW J
IPC: C12P21/00
Filed: 2000-01-07
Abstract: 1. A method for extracting abnormal prion protein from a biological material suspected of containing abnormal protein, said method comprising: (a) providing an isotonic or hypotonic aqueous preparation of biological material; (b) incubating at a temperature ranging from about 20 degree C to about 100 degree C a mixture of an extraction solvent and the isotonic or hypotonic aqueous preparation of biological material, wherein said extraction solvent is a polar organic solvent in which said abnormal prion protein is soluble and which is miscible with a non-lyotropic aqueous solution but immiscible with a lyotropic aqueous solution; and (c) increasing lyotropic activity of the mixture by adding a lyotropic salt to separate said solvent containing abnormal prion protein from the aqueous preparation of the biological material to yield a solvent fraction containing abnormal prion protein. 2. A method according to claim 1, wherein said solvent is hexafluoro-2-propanol. 3. A method according to claim 1, wherein said biological material is a tissue or a biological fluid from a vertebrate. 4. A method according to claim 3, wherein said tissue is brain tissue. 5. A method according to claim 3, wherein said biological fluid is selected from the group consisting of cerebrospinal fluid, blood, plasma, and serum. 6. A method according to claim 3, wherein said biological fluid is human blood. 7. A method according to claim 1, wherein incubation is at about 56 degree C. 8. A method according to claim 1, wherein lyotropic activity is increased by adding about a 1:1 ratio (vol/vol) of 0.5 M sodium sulfate to said mixture. 9. A method according to claim 1, wherein said biological material in said isotonic or hypotonic aqueous preparation of biological material is treated with proteinase K prior to mixing with the extraction solvent. 10. A method according to claim 1, further comprising drying the solvent to obtain a dried abnormal prion protein. 11. A method according to claim 10, further comprising dissolving said dried abnormal prion protein in water and purifying said abnormal prion protein. 12. A method according to claim 11, wherein said purifying is carried out by a method selected from the group consisting of hydrophilic interaction chromatography and capillary electrophoresis. 13. A method for detecting abnormal prion protein in a biological material, said method comprising the steps of: (a) providing an isotonic or hypotonic aqueous preparation of biological material; (b) incubating at a temperature ranging from about 20 degree C to about 100 degree C a mixture of an extraction solvent and the isotonic or hypotonic aqueous preparation of biological material, wherein said extraction solvent is a polar organic solvent in which said abnormal prion protein is soluble and which is miscible with a non-lyotropic aqueous solution but immiscible with a lyotropic aqueous solution; (c) increasing lyotropic activity of the mixture by adding a lyotropic salt to separate said solvent from said aqueous preparation of the biological material to yield a solvent fraction containing abnormal prion protein; and (d) assaying the solvent fraction containing abnormal protein by a method that will detect the presence of said abnormal prion protein. 14. A method according to claim 13, wherein said assay is an immunoassay of abnormal prion protein. 15. A method according to claim 13, wherein said biological material is a tissue or a biological fluid from a vertebrate. 16. A method according to claim 15, wherein said tissue is brain tissue. 17. A method according to claim 15, wherein said biological fluid is selected from the group consisting of cerebrospinal fluid, blood, plasma, and serum. 18. A method according to claim 15, wherein said biological fluid is human blood. 19. A method for extracting abnormal prion protein from a biological material suspected of containing abnormal protein, said method comprising: (a) providing an isotonic or hypotonic aqueous preparation of biological material; (b) incubating at a temperature from about 20 degree C to about 100 degree C a mixture of about equal amounts of hexafluoro-2-propanol and said isotonic or hypotonic aqueous preparation of biological material; and (c) adding about an equal volume of a solution of 0.5M sodium sulfate to said mixture to separate said hexafluoro-2-propanol containing abnormal prion protein from the aqueous preparation of the biological material to yield a solvent fraction containing abnormal prion protein. 20. A kit for extracting abnormal prion protein from a biological material, said kit comprising: (a) an extraction solvent, wherein the extraction solvent is a polar organic solvent in which said abnormal prion protein is soluble and which is miscible with a non-lyotropic aqueous solution of a biological sample but immiscible with a lyotropic aqueous solution; and (b) a lyotropic salt or aqueous lyotropic salt solution to add to an aqueous preparation of a biological material so that said organic solvent becomes immiscible with said aqueous preparation. 21. The kit according to claim 20, further comprising proteinase K for treating said aqueous preparation of a biological material. 22. A kit for detecting abnormal prion protein in a biological material and if required, for isolating abnormal prion protein from biological material, said kit comprising: (1) a means for isolating abnormal prion protein, comprising (a) an extraction solvent, wherein the extraction solvent is a polar organic solvent in which said abnormal prion protein is soluble and which is miscible with a non-lyotropic aqueous solution but immiscible with a lyotropic aqueous solution; and (b) a lyotropic salt or aqueous lyotropic salt solution to add to an aqueous preparation of a biological material so that said extraction solvent becomes immiscible with said aqueous preparation; and (2) an abnormal prion protein detection means. 23. The kit according to claim 22, wherein said means is intended to detect abnormal prion protein by an immunoassay, in particular by an immunochromatographic assay.