Claims
- 1. A homogeneous method of detecting cleavage of β amyloid precursor protein (βAPP) by gamma-secretase, said method comprising detecting binding of a gamma-cleaved βAPP fragment with a pair of fluorescent adducts, wherein a first fluorescent adduct binds specifically to the carboxy-terminal end of the gamma-cleaved βAPP fragment with substantially no cross-reactivity to uncleaved βAPP or to other types of gamma-cleaved βAPP fragments, and wherein a second fluorescent adduct binds to the gamma-cleaved βAPP fragment within an amino acid sequence which corresponds to amino acid sequence 1-31 of β-amyloid peptide (Aβ); and wherein excitation of one of the fluorescent adducts provides a detectable transfer of energy to the other fluorescent adduct.
- 2. The method according to claim 1, wherein the method is practiced in a fluid sample in the presence of uncleaved βAPP and other types of gamma-cleaved βAPP fragments.
- 3. The method according to claim 2, wherein the sample comprises membrane fractions having endogenous gamma-secretase and Swedish variant βAPP.
- 4. The method according to claim 2, wherein the sample comprises solubilized gamma-secretase complex and βAPP.
- 5. The method according to claim 1, wherein each of the fluorescent adducts separately modifies an antibody.
- 6. The method according to claim 5, wherein the gamma-cleaved βAPP fragment is Aβ-40.
- 7. The method according to claim 6, wherein the first fluorescent adduct modifies a first antibody which binds to Aβ-40 at an epitope comprising amino acid residue 40.
- 8. The method according to claim 7, wherein the second fluorescent adduct modifies a second antibody which binds to Aβ at an epitope comprising amino acid sequence 1-12.
- 9. The method according to claim 1, wherein excitation of the first fluorescent adduct provides a detectable transfer of energy to the second fluorescent adduct.
- 10. The method according to claim 9, wherein the first adduct comprises a molecule selected from the group consisting of lanthanide cryptate or chelate, fluorescein, EDANS, salts of N-[6-amino-9-[2-carboxy-phenyl]-4,5-disulfoxy-3H-xanthen-3-ylidene]aminium ion (2-) and salts of 1-(epsilon-carboxypentyl-1′-ethyl-3,3,3′,3′-tetramethylindocarbocyanine-5,5′-disulfonate ion.
- 11. The method according to claim 10, wherein the first fluorescent adduct comprises a europium cryptate.
- 12. The method according to claim 10, wherein the second fluorescent adduct comprises xl-APC.
- 13. The method according to claim 12, wherein the detectable transfer of energy comprises an amplified signal from the second fluorescent adduct.
- 14. The method according to claim 1, wherein the other fluorescent adduct comprises a fluorescent quencher molecule.
- 15. The method according to claim 14, wherein the fluorescent quencher molecule is selected from the group consisting of dabcyl and salts of 9-[2-[[4-carboxy-piperidin-1-yl]sulfonyl]phenyl]-6-(N-methyl-N-phenyl-amino)-3H-xanthen-3-ylidene]-N-methylbenzenaminium ion.
- 16. The method according to claim 15, wherein each of the fluorescent adducts separately modifies an antibody.
- 17. The method according to claim 16, wherein the detectable transfer of energy comprises a decrease in fluorescent signal from the fluorescent adduct which is excited.
- 18. The method according to claims 13 or 17, wherein excitation is by laser, xenon flash lamp or deuterium-tungsten lamp.
- 19. The method according to claim 18, wherein excitation is by laser.
- 20. A homogeneous method for determining the presence of β-amyloid peptide (Aβ), said method comprising
(1) exposing the sample to a pair of fluorescent adducts, wherein the first fluorescent adduct binds to the carboxy-terminal region of Aβ and the second fluorescent adduct binds to the amino-terminal region of Aβ and at least one fluorescent adduct is substantially free of cross-reactivity to uncleaved βAPP or to other types of gamma-cleaved βAPP fragments; and (2) detecting binding of the pair of fluorescent adducts with Aβ by excitation of one of the fluorescent adducts.
- 21. The method according to claim 20, wherein the first fluorescent adduct binds specifically to the carboxy-terminal end of Aβ with substantially no cross-reactivity to uncleaved βAPP or to other types of gamma-cleaved βAPP fragments.
- 22. The method according to claim 21, wherein Aβ is Aβ-40.
- 23. The method according to claim 22, wherein each of the fluorescent adducts separately binds specifically to either the amino- and carboxy-terminal ends of Aβ with substantially no cross-reactivity to uncleaved βAPP or to other types of gamma-cleaved βAPP fragments.
- 24. The method according to claim 21, wherein excitation is by laser, xenon flash lamp or deuterium-tungsten lamp.
- 25. The method according to claim 24, wherein excitation is by laser.
- 26. A homogeneous method for determining the presence of β-amyloid peptide Aβ-40, said method comprising
(1) exposing the sample to a pair of fluorescent adducts, wherein the first fluorescent adduct binds to the carboxy-terminal end of Aβ-40 and the second fluorescent adduct binds to the amino-terminal region of Aβ-40 and the first fluorescent adduct is substantially free of cross-reactivity to uncleaved βAPP or to other types of gamma-cleaved βAPP fragments; and (2) detecting binding of the pair of fluorescent adducts with Aβ-40 by excitation of the first fluorescent adduct.
- 27. The method according to claim 26, wherein the first fluorescent adduct modifies a first antibody which binds to Aβ-40 at an epitope comprising amino acid residue 40.
- 28. The method according to claim 27, wherein the first fluorescent adduct modifies a europium cryptate.
- 29. The method according to claim 28, wherein the second fluorescent adduct modifies a second antibody which binds to Aβ-40 at an epitope comprising amino acid sequence 1-12.
- 30. The method according to claim 29, wherein the second fluorescent adduct comprises xl-APC.
- 31. The method according to claim 30, wherein the first fluorescent adduct is excited by laser.
- 32. A homogeneous method of detecting cleavage of β-amyloid precursor protein (βAPP) by gamma-secretase, said method comprising detecting binding of a 6 kDa fragment with a pair of fluorescent adducts; wherein a first fluorescent adduct binds to the amino-terminal end of the 6 kDa fragment with substantially no cross-reactivity to uncleaved βAPP or to other types of gamma-cleaved βAPP fragments; and wherein a second fluorescent adduct binds to a portion within the carboxy-terminal region of the 6 kDa fragment; and wherein excitation of one of the fluorescent adducts provides a detectable transfer of energy to the other fluorescent adduct.
- 33. The method according to claim 32, wherein each of the fluorescent adducts separately modifies an antibody.
- 34. The method according to claim 33, wherein one of the fluorescent adducts comprises a molecule selected from the group consisting of lanthanide cryptate or chelate, fluorescein, EDANS, salts of N-[6-amino-9-[2-carboxy-phenyl]-4,5-disulfoxy-3H-xanthen-3-ylidene]aminium ion (2-) and salts of 1-(epsilon-carboxypentyl-1′-ethyl-3,3,3′,3′-tetramethylindocarbocyanine-5,5′-disulfonate ion.
- 35. The method according to claim 34, wherein the other fluorescent adduct comprises a molecule selected from the group consisting of cross-linked allophycocyanins (“xl-APC”), coumarin, rhodamine, tetramethylrhodamine and salts of 1-(epsilon-carboxypentyl)-1′-ethyl-3,3,3′,3′-tetramethylindodicarbocyanine-5,5′-disulfonate ion.
- 36. The method according to claim 34, wherein the other fluorescent adduct comprises a fluorescent quencher molecule selected from the group consisting of dabcyl and salts of 9-[2-[[4-carboxy-piperidin-1-yl]sulfonyl]phenyl]-6-(N-methyl-N-phenyl-amino)-3H-xanthen-3-ylidene]-N-methylbenzenaminium ion.
- 37. A homogeneous method of detecting cleavage of amyloid precursor protein (APP) by gamma-secretase, comprising the steps of
(1) binding a first fluorescent adduct to a 6 kDa fragment and a second fluorescent adduct to either a amyloid peptide (Aβ) or a p3 fragment, wherein at least one of the fluorescent adducts has substantially no cross-reactivity to other portions of uncleaved βAPP, and wherein each fluorescent adduct separately comprises either a donor molecule or an acceptor molecule; and, (2) exciting the donor molecule by laser, xenon flash lamp or deuterium-tungsten lamp; and (3) detecting a substantially decreased transfer of energy to the acceptor molecule.
- 38. The homogeneous method according to claim 37, wherein the donor molecule is selected from the group consisting of lanthanide cryptate or chelate, fluorescein, EDANS, salts of N-[6-amino-9-[2-carboxy-phenyl]-4,5-disulfoxy-3H-xanthen-3-ylidene]aminium ion (2-) and salts of 1-(epsilon-carboxypentyl-1′-ethyl-3,3,3′,3′-tetramethylindocarbocyanine-5,5′-disulfonate ion.
- 39. The method according to claim 38, wherein the acceptor molecule is selected from the group consisting of cross-linked allophycocyanins (“xl-APC”), coumarin, rhodamine, tetramethylrhodamine and salts of 1-(epsilon-carboxypentyl)-1′-ethyl-3,3,3′,3′-tetramethylindodicarbocyanine-5,5′-disulfonate ion.
- 40. The method according to claim 39, wherein the step of detecting a substantially decreased transfer of energy comprises detecting little or no amplified signal from the acceptor molecule.
- 41. The method according to claim 38, wherein the acceptor molecule is a fluorescent quencher molecule selected from the group consisting of dabcyl and salts of 9-[2-[[4-carboxy-piperidin-1-yl]sulfonyl]phenyl]-6-(N-methyl-N-phenyl-amino)-3H-xanthen-3-ylidene]-N-methylbenzenaminium ion.
- 42. The method according to claim 41, wherein the step of detecting a substantially decreased transfer of energy comprises detecting an unchanged fluorescent signal from the donor molecule.
- 43. A homogeneous method of screening for inhibitors of gamma-secretase cleavage in β-amyloid precursor protein (βAPP), said method comprising the steps of
(1) adding a test compound to a sample comprising gamma-secretase and βAPP; (2) then adding a pair of fluorescent adducts to the sample, wherein a first fluorescent adduct has binding specificity to the carboxy-terminal end of a gamma-cleaved βAPP fragment with substantially no cross-reactivity to uncleaved βAPP or to other types of gamma-cleaved βAPP fragments, and a second fluorescent adduct has binding specificity to the gamma-cleaved βAPP within an amino acid sequence corresponding to 1-31 of β-amyloid peptide (Aβ), and wherein each fluorescent adduct separately comprises either a donor molecule or an acceptor molecule; and (3) detecting a substantially decreased transfer of fluorescent energy between the fluorescent adducts after excitation of the donor molecule.
- 44. The method according to claim 43, wherein the donor molecule is selected from the group consisting of lanthanide cryptate or chelate, fluorescein, EDANS, salts of N-[6-amino-9-[2-carboxy-phenyl]-4,5-disulfoxy-3H-xanthen-3-ylidene]aminium ion (2-) and salts of 1-(epsilon-carboxypentyl-1′-ethyl-3,3,3′,3′-tetramethylindocarbocyanine-5,5′-disulfonate ion.
- 45. The method according to claim 44, wherein the acceptor molecule is selected from the group consisting of cross-linked allophycocyanins (“xl-APC”), coumarin, rhodamine, tetramethylrhodamine and salts of 1-(epsilon-carboxypentyl)-1′-ethyl-3,3,3′,3′-tetramethylindodicarbocyanine-5,5′,-disulfonate ion.
- 46. The method according to claim 45, wherein the step of detecting a substantially decreased transfer of energy comprises detecting little or no amplified signal from the acceptor molecule.
- 47. The method according to claim 44, wherein the acceptor molecule is a fluorescent quencher molecule selected from the group consisting of dabcyl and salts of 9-[2-[[4-carboxy-piperidin-1-yl]sulfonyl]phenyl]-6-(N-methyl-N-phenyl-amino)-3H-xanthen-3-ylidenel-N-methylbenzenaminium ion.
- 48. The method according to claim 47, wherein the step of detecting a substantially decreased transfer of energy comprises detecting an unchanged fluorescent signal from the donor molecule.
- 49. A homogeneous method of screening for inhibitors of gamma-secretase cleavage in β-amyloid precursor protein (βAPP), said method comprising the steps of
(1) adding a test compound to a sample comprising gamma-secretase and βAPP; (2) then binding a pair of fluorescent adducts to uncleaved βAPP; wherein a first fluorescent adduct binds to a portion within amino acid sequence 722-770 of uncleaved βAPP, a second fluorescent adduct binds to a portion within amino acid sequence 671-702 of uncleaved βAPP, and at least one of the fluorescent adducts has substantially no cross-reactivity to other portions of uncleaved βAPP, and wherein each fluorescent adduct separately comprises either a donor molecule or an acceptor molecule; and, (3) detecting a transfer of energy between the fluorescent adducts after excitation of the donor molecule.
- 50. The method according to claim 49, wherein the donor molecule is selected from the group consisting of lanthanide cryptate or chelate, fluorescein, EDANS, salts of N-[6-amino-9-[2-carboxy-phenyl]-4,5-disulfoxy-3H-xanthen-3-ylidene]aminium ion (2-) and salts of 1-(epsilon-carboxypentyl-1′-ethyl-3,3,3′,3′-tetramethylindocarbocyanine-5,5′-disulfonate ion.
- 51. The method according to claim 50, wherein the acceptor molecule is selected from the group consisting of cross-linked allophycocyanins (“xl-APC”), coumarin, rhodamine, tetramethylrhodamine and salts of 1-(epsilon-carboxypentyl)-1′-ethyl-3,3,3′,3′-tetramethylindodicarbocyanine-5,5′-disulfonate ion.
- 52. The method according to claim 51, wherein the step of detecting a transfer of energy comprises detecting an amplified signal from the acceptor molecule.
- 53. The method according to claim 52, wherein the acceptor molecule is a fluorescent quencher molecule selected from the group consisting of dabcyl and salts of 9-[2-[[4-carboxy-piperidin-1-yl]sulfonyl]phenyl]-6-(N-methyl-N-phenyl-amino)-3H-xanthen-3-ylidene]-N-methylbenzenaminium ion.
- 54. The method according to claim 53, wherein the step of detecting a transfer of energy comprises detecting a decrease of fluorescent signal from the donor molecule.
- 55. An isolated protein having gamma-secretase activity.
- 56. An isolated protein comprising gamma-secretase.
- 57. The isolated protein of claim 56, wherein the gamma-secretase recognizes and cleaves a substrate having a gamma secretase cleavage site.
- 58. The isolated protein of claim 57, wherein cleavage of the substrate by the gamma secretase at the gamma-secretase cleavage site generates a β-amyloid peptide (Aβ) and a 6 kDa fragment.
- 59. The isolated protein of claim 56 which is a protein complex comprising gamma secretase and PS1.
- 60. A membrane fragment comprising gamma-secretase.
- 61. A method for isolating gamma-secretase from a sample by isolating gamma secretase complexed with PS1.
- 62. The method of claim 61, wherein isolating gamma-secretase complexed with PS1 comprises contacting the sample with an agent that recognizes and binds PS1 so that an agent/PS1/gamma secretase complex forms thereby isolating the molecule having gamma-secretase activity.
- 63. A molecule having gamma-secretase activity isolated by the method of claim 61.
- 64. The method of claim 62, wherein the agent that recognizes and binds PS1 comprises an anti-PS1 antibody.
- 65. A method for isolating a protein complex having gamma-secretase activity from a sample, comprising:
a) contacting the sample with a molecule that recognizes and binds PS1 so that a molecule/PS1 complex forms; and b) removing the molecule/PS1 complex from the sample, thereby isolating the protein complex having gamma secretase activity.
- 66. A protein complex having gamma-secretase activity isolated by the method of claim 65.
- 67. The method of claim 65, wherein the molecule that recognizes and binds PS1 comprises an anti-PS1 antibody.
- 68. The method of claim 65, wherein the protein complex comprises gamma secretase and PS1.
- 69. A protein complex isolated by the method of claim 65.
- 70. A method for isolating a protein complex comprising gamma secretase and PS1, comprising:
a. solubilizing a gamma-secretase positive cell thereby resulting in a mixture of a protein complex comprising gamma-secretase and PS1 and other cell components; and b. contacting the mixture with a molecule that recognizes and binds PS1 so that a molecule/PS1 complex forms; and c. removing the complex from the other cell components thereby isolating a protein complex comprising gamma secretase and PS1.
- 71. A protein complex comprising gamma secretase and PS1 isolated by the method of claim 70.
- 72. The method of claim 70, wherein the molecule that recognizes and binds PS1 is an anti-PS1 antibody.
- 73. The method of claim 70, wherein in step (a) the gamma-secretase positive cell is solubilized in a solution comprising N-[3[(dimethylamino)propyl]3,7,12-trihydroxy(3a,5b,7a,12a)cholan-2-amide].
- 74. An isolated functionally-active substrate which is cleaved by gamma-secretase.
- 75. The functionally-active substrate of claim 74 comprising APP.
- 76. A method for cleaving a functionally-active substrate comprising incubating the functionally-active substrate with a molecule having gamma-secretase activity under conditions so that the molecule having gamma-secretase activity cleaves the functionally-active substrate thereby producing cleavage products.
- 77. A method for detecting gamma-secretase activity in a molecule of interest by determining whether the molecule can cleave a substrate in accordance with the method of claim 76.
- 78. The method of claim 76, wherein the functionally-active substrate comprising βAPP.
- 79. The method of claim 76, wherein the functionally-active substrate and the molecule having gamma-secretase activity are incubated in a solution comprising N-[3[(dimethylamino)propyl]3,7,12-trihydroxy(3a,5b,7a,12a)cholan-2-amide].
- 80. A method for isolating a functionally-active substrate, comprising:
a) generating a substrate comprising a gamma-secretase cleavage sequence; b) inserting the substrate into a microsomal membrane fragment to generate a functionally-active substrate; and c) isolating the microsomal membrane fragment which includes the functionally-active substrate.
- 81. A functionally-active substrate generated by the method of claim 80.
- 82. The method of claim 80, wherein the substrate comprises βAPP.
- 83. The method of claim 80, wherein the substrate comprises the amino acid sequence as described in SEQ ID NO.: 2 or 4.
- 84. The method of claim 80, wherein the functionally-active substrate includes a detectable label.
- 85. The method of claim 80, wherein the functionally-active substrate is solubilized from the microsomal membrane fragment with a solution comprising N-[3[(dimethylamino)propyl]3,7,12-trihydroxy(3a,5b,7a,12a)cholan-2-amide].
- 86. The method of claim 80 further comprising:
a) solubilizing the functionally-active substrate from the microsomal membrane fragment; and b) isolating the functionally-active substrate.
- 87. A method for identifying an agent of interest that inhibits gamma-secretase activity in a sample comprising:
a) contacting the sample and the agent of interest with a functionally-active substrate; and b) detecting whether a cleavage product of the functionally-active substrate is generated in the sample, the lack of the cleavage product in the sample being indicative that the agent inhibits gamma-secretase activity in the sample.
- 88. The method of claim 87, wherein the cleavage product is detected with an antibody that recognizes and binds to the N-terminal end of the cleavage product.
- 89. The method of claim 87, wherein the cleavage product is detected with an antibody that recognizes and binds to the C-terminal end of the cleavage product.
- 90. The method of claim 87, wherein the cleavage product is detected with a pair of fluorescent adducts wherein a first fluorescent adduct binds to the N-terminal end of the cleavage product and a second fluorescent adduct binds to the C-terminal end of the cleavage product, and wherein excitation of one of the fluorescent adducts provides a detectable transfer of energy to the other fluorescent adduct.
- 91. The method according to claim 87 which comprises contacting a plurality of substantially identical samples each separately with a different agent of interest.
- 92. The method of claim 87, wherein the plurality of samples comprises more than about 104 samples.
- 93. The method of claim 87, wherein the plurality of samples comprises more than about 105 samples.
- 94. The method of claim 87, wherein the plurality of samples comprises more than about 106 samples.
- 95. The method of claim 87, wherein the plurality of substantially identical samples are each contacted essentially simultaneously with a different agent of interest.
- 96. A method for isolating an integral membrane protein or protein complex comprising:
a) solubilizing a cell with a solution comprising N-[3[(dimethylamino)propyl]3,7,12-trihydroxy(3a,5b,7a,12a)cholan-2-amide] thereby obtaining a mixture having he integral membrane protein or protein complex and other cell components; and b) isolating the integral membrane protein or protein complex.
- 97. An integral membrane protein or protein complex isolated by the method of claim 96.
Parent Case Info
[0001] This application claims the benefit of U.S. Provisional Patent Application No. 60/194,495, filed Apr. 3, 2000.
Provisional Applications (1)
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Number |
Date |
Country |
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60194495 |
Apr 2000 |
US |