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1. AU2008275917 - Multiplexed analyses of test samples

Note: Text based on automatic Optical Character Recognition processes. Please use the PDF version for legal matters

[ EN ]
CLAIMS
What is claimed is:
1 A method for detecting a target molecule that may be present in a test sample, the method comprising:
(a) preparing a mixture by contacting the test sample with an aptamer comprising a first tag and having a specific affinity for the target molecule, wherein an aptamer affinity complex is formed if said target molecule is present in said test sample;
(b) exposing the mixture to a first solid support comprising a first capture element, and allowing the first tag to associate with the first capture element;
(c) removing any components of the mixture not associated with said first solid support.;
(d) releasing the aptamer affinity complex from said first solid support;
(e) attaching a second tag to said target molecule in the aptamer affinity complex;
(f) exposing the released aptamer affinity complex to a second solid support comprising a second capture element and allowing the second tag to associate with said second capture element;
(g) removing any uncomplexed aptamer from said mixture by partitioning the uncomplexed aptamer from said aptamer affinity complex; and
(h) detecting said target molecule by detecting the aptamer portion of said aptamer affinity complex.
2. The method of claim 1, wherein (h) further comprises dissociating the aptamer from said aptamer affinity complex before detecting the aptamer.
3 The method of claim 1, wherein the first tag is the same as the second tag and the second tag is added to the target molecule at any point after (b) and before (f), and comprising blocking the first capture agent prior to the addition of the second tag.
4 The method of claim 1, wherein the first tag is different than the second tag and the second tag is added to the target molecule at any point before (f).
5 The method of claim 1, further comprising introducing a kinetic challenge at any point after (a) and before (d).
6. The method of claim 5, wherein said kinetic challenge comprises diluting the mixture containing the aptamer affinity complex and incubating the mixture containing the aptamer affinity complex for a time selected from the group consisting of greater than or equal to about 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 30 minutes, and 60 minutes.
7. The method of claim 5, wherein said kinetic challenge comprises diluting the mixture containing the aptamer affinity complex and incubating the mixture containing the aptamer affinity complex for a time such that the ratio of the measured level of aptamer affinity complex to the measured level of the non-specific complex is increased.
8. The method of claim 5, wherein said kinetic challenge comprises adding a competitor to the mixture containing the aptamer affinity complex, and incubating the mixture containing the aptamer affinity complex for a time selected from the group consisting of greater than or equal to about 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 30 minutes, and 60 minutes.
9. The method of claim 5, wherein said kinetic challenge comprises adding a competitor to the mixture containing the aptamer affinity complex and incubating the mixture containing the aptamer affinity complex for a time such that the ratio of the measured level of aptamer affinity complex to the measured level of the non-specific complex is increased. (Check spec!)
10. The method of claim 5, wherein said kinetic challenge comprises diluting the mixture containing the aptamer affinity complex, adding a competitor to the mixture containing the aptamer affinity complex, and incubating the mixture containing the aptamer affinity complex for a time selected from the group consisting of greater than or equal to about 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 30 minutes, and 60 minutes.
11. The method of claim 5, wherein said kinetic challenge comprises diluting the mixture containing the aptamer affinity complex, adding a competitor to the mixture containing the aptamer affinity complex and incubating the mixture containing the aptamer affinity complex for a time such that the ratio of the measured level of aptamer affinity complex to the measured level of the non-specific complex is increased
12. The method of claim 5 wherein the kinetic challenge comprises the introduction of a competitor molecule and said competitor molecule is selected from the group consisting of an oligonucleotide, heparin, herring sperm DNA, salmon sperm DNA, dextran sulfate, polyanion, abasic phosphodiester polymer, dNTP, and pyrophosphate.
13. The method of claim 1 wherein said aptamer affinity complex has a slow rate of dissociation.
14. The method of claim 13 wherein the rate of dissociation of said aptamer affinity complex (t1/2 ) is greater than or equal to about 30 minutes.
15. The method of claim 13 wherein the rate of dissociation of said aptamer affinity complex (t1/2 ) is between about 30 minutes and about 240 minutes.
16. The method of claim 13 wherein the rate of dissociation of said aptamer affinity complex (t1/2 ) is selected from the group consisting of > about 30 minutes, > about 60 minutes, > about 90 minutes,. > about 120 minutes, > about 150 minutes, > about 180 minutes, > about 210 minutes, and > about 240 minutes,
17. The method of claim 1 wherein said aptamer is detected and optionally quantified using a method selected from the group consisting of Q-PCR, MS, and hybridization.
18. The method of claim 17 wherein said Q-PCR is performed using TaqMan* PCR, an intercalating fluorescent dye during the PCR process, or a molecular beacon during the PCR process.
19. The method of claim 1 further comprising adding a detectable moiety to the aptamer.
20. The method of claim 19, wherein said detectable moiety is selected from the group consisting of a dye, a quantum dot, a radiolabel, a electrochemical functional group, an enzyme, and an enzyme substrate.
21. The method of claim 20, wherein said dye is a fluorescent dye.
22. The method of claim 20, wherein said enzyme is alkaline phosphatase or horseradish peroxidase.
23. The method of claim 1, wherein said aptamer is a single-stranded nucleic acid or a double-stranded nucleic acid.
24. The method of claim 1, wherein said aptamer comprises DNA, RNA or both DNA and RNA
25. The method of claim 1, wherein said aptamer comprises at least one chemical modification.
26. The method of claim 25, wherein said at least one chemical modification is a chemical substitution at one or more positions independently selected from a ribose position, a deoxyribose position, a phosphate position, and a base position.
27. The method of claim 25, wherein said at least one chemical modification is independently selected from the group listed in Figure (19).
28. The method of claim 1 wherein said target molecule is selected from the group consisting of a protein, a peptide, a carbohydrate, a polysaccharide, a glycoprotein, a hormone, a receptor, an antigen, an antibody, a virus, a substrate, a metabolite, a transition state analog, a cofactor, an inhibitor, a drug, a dye, a nutrient, a growth factor, a tissue, and a controlled substance.
29. The method of claim 1, wherein said target molecule is a protein or a peptide.
30. The method of claim 1, wherein said test sample is selected from the group consisting of a biological sample, an environmental sample, a chemical sample, a pharmaceutical sample, a food sample, an agricultural sample, and a veterinary sample.
31. The method claim 1, wherein said test sample is a biological sample selected from the group consisting of whole blood, leukocytes, peripheral blood mononuclear cells, plasma, serum, sputum, breath, urine, semen, saliva, meningial fluid, amniotic fluid, glandular fluid, lymph fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, cells, a cellular extract, stool, tissue, a tissue extract, a tissue biopsy, and cerebrospinal fluid.
32. The method of claim 1, wherein said test sample is plasma or serum.
33. The method of claim 1 wherein said first tag and said second tag each comprises at least one component independently selected from the group consisting of a polynucleotide, a polypeptide, a peptide nucleic acid, a locked nucleic acid, an oligosaccharide, a polysaccharide, an antibody, an affybody, an antibody mimic, a cell receptor, a ligand, a lipid, biotin, avidin, strepavidin, Extravidin, neutravidin, a metal, histidine, and any portion of any of these structures.
34. The method of claim 1, wherein said first capture element and said second capture element each comprises at least one component independently selected from a polynucleotide, a polypeptide, a peptide nucleic acid, a locked nucleic acid, an oligosaccharide, a polysaccharide, an antibody, an affybody, an antibody mimic, a cell receptor, a ligand, a lipid, biotin, avidin, strepavidin, Extravidin, neutravidin, a metal, histidine, and any portion of any of these structures.
35. The method of claim 1, wherein the first tag comprises a releasable moiety.
36. The method of claim 35 wherein the releasable moiety comprises a photocleavable moiety.
37. The method of claim 1, wherein said first solid support and second solid support each is independently selected from the group consisting of [a polymer bead, an agarose bead, a polystyrene bead, an acrylamide bead, a solid core bead, a porous bead, a paramagnetic bead, glass bead, controlled pore bead...]. a microtitre well, , a cyclo-olefin copolymer substrate, a membrane, a plastic substrate, , nylon, a Langmuir-Bodgett film, glass, a germanium substrate, a silicon substrate, a silicon wafer chip, a flow through chip, a microbead, a polytetrafluoroethylene substrate, a polystyrene substrate, a gallium arsenide substrate, a gold substrate, and a silver substrate.
38. The method of claim 1, further comprising quantifying said target by quantifying said aptamer.
39. The method of claim 1, wherein detection of the aptamer comprises hybridizing the aptamer to a third solid support wherein the third solid support comprises a plurality of addressable features and wherein at least one of said features comprises at least capture element disposed thereon that is complementary to any sequence contained within the aptamer.
40. A method for detecting a target molecule that may be present in a test sample, the method comprising:
(a) preparing a mixture by contacting a test sample with an aptamer having a specific affinity for a target molecule, wherein an aptamer affinity complex is formed if said target molecule is present in said test sample;
(b) at any point prior to (c) adding a tag to said target molecule; (c) exposing the mixture to a solid support comprising a capture element, and allowing the tag on the target molecule to associate with the capture element;
(d) removing any uncomplexed aptamer from said mixture by partitioning the uncomplexed aptamer from the aptamer affinity complex; (e) detecting said target molecule by detecting the aptamer portion of said aptamer affinity complex.
41. The method of claim 40, wherein (e) further comprises dissociating the aptamer from said aptamer affinity complex, before detecting the aptamer.
42. The method of claim 40, further comprising introducing a kinetic challenge at any point after (a) and before (d).
43. The method of claim 42, wherein said kinetic challenge comprises diluting the mixture containing the aptamer affinity complex and incubating the mixture containing the aptamer affinity complex for a time selected from the group consisting of greater than or equal to about 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 30 minutes, and 60 minutes.
44. The method of claim 42, wherein said kinetic challenge comprises diluting the mixture containing the aptamer affinity complex and incubating the mixture containing the aptamer affinity complex for a time such that the ratio of the measured level of aptamer affinity complex to the measured level of the non-specific complex is increased.
45. The method of claim 42, wherein said kinetic challenge comprises adding a competitor to the mixture containing the aptamer affinity complex, and incubating the mixture containing the aptamer affinity complex for a time selected from the group consisting of greater than or equal to about 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 30 minutes, and 60 minutes.
46. The method of claim 42, wherein said kinetic challenge comprises adding a competitor to the mixture containing the aptamer affinity complex and incubating the mixture containing the aptamer affinity complex for a time such that the ratio of the measured level of aptamer affinity complex to the measured level of the non specific complex is increased.
47. The method of claim 42, wherein said kinetic challenge comprises diluting the mixture containing the aptamer affinity complex, adding a competitor to the mixture containing the aptamer affinity complex, and incubating the mixture containing the aptamer affinity complex for a time selected from the group consisting of greater than or equal to about 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 30 minutes, and 60 minutes.
48. The method of claim 42, wherein said kinetic challenge comprises diluting the mixture containing the aptamer affinity complex, adding a competitor to the mixture containing the aptamer affinity complex and incubating the mixture containing the aptamer affinity complex for a time such that the ratio of the measured level of aptamer affinity complex to the measured level of the non-specific complex is increased
49. The method of claim 42 wherein the kinetic challenge comprises the introduction of a competitor molecule and said competitor molecule is selected from the group consisting of an oligonucleotide, heparin, herring sperm DNA, salmon sperm DNA, dextran sulfate, polyanion, abasic phosphodiester polymer, dNTP, and pyrophosphate.
50. The method of claim 40 wherein said aptamer affinity complex has a slow rate of dissociation.
51. The method of claim 50 wherein the rate of dissociation of said aptamer affinity complex (t12) is greater than or equal to about 30 minutes.
52. The method of claim 50 wherein the rate of dissociation of said aptamer affinity complex (t1 2 ) is between about 30 minutes and about 240 minutes.
53. The method of claim 50 wherein the rate of dissociation of said aptamer affinity complex (t1 2 ) is selected from the group consisting of > about 30 minutes, > about 60 minutes, > about 90 minutes,. > about 120 minutes, > about 150 minutes, > about 180 minutes, > about 210 minutes, and > about 240 minutes,
54. A method for detecting a target molecule that may be present in a test sample, the method comprising:
(a) preparing a mixture by contacting a test sample with a photoaptamer comprising a first tag and having a specific affinity for a target molecule, wherein an aptamer affinity complex is formed if said target molecule is present in said test sample;
(b) converting said aptamer affinity complex into an aptamer covalent complex;
(c) exposing the mixture to a first solid support comprising a first capture element, and allowing the first tag to associate with the first capture element;
(d) removing any components of the mixture not associated with said first solid support.;
(e) releasing the aptamer covalent complex from said first solid support;
(f) attaching a second tag to said target molecule in the aptamer covalent complex;
(g) exposing the released aptamer covalent complex to a second solid support comprising a second capture element and allowing the second tag to associate with said second capture element;
(h) removing any uncomplexed photoaptamer from said mixture by partitioning the uncomplexed aptamer from said aptamer covalent complex; and
(i) detecting said target molecule by detecting the photoaptamer portion of said aptamer covalent complex.
55. The method of claim 54, wherein (i) further comprises release the photoaptamer from said aptamer covalent complex, before detecting the photoaptamer.
56. The method of claim 54, further comprising introducing a kinetic challenge after (a) and before (b).
57. The method of claim 56, wherein said kinetic challenge comprises diluting the mixture containing the aptamer affinity complex and incubating the mixture containing the aptamer affinity complex for a time selected from the group consisting of greater than or equal to about 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 30 minutes, and 60 minutes.
58. The method of claim 56, wherein said kinetic challenge comprises diluting the mixture containing the aptamer affinity complex and incubating the mixture containing the aptamer affinity complex for a time such that the ratio of the measured level of aptamer affinity complex to the measured level of the non-specific complex is increased.
59. The method of claim 56, wherein said kinetic challenge comprises adding a competitor to the mixture containing the aptamer affinity complex, and incubating the mixture containing the aptamer affinity complex for a time selected from the group consisting of greater than or equal to about 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 30 minutes, and 60 minutes.
60. The method of claim 56, wherein said kinetic challenge comprises adding a competitor to the mixture containing the aptamer affinity complex and incubating the mixture containing the aptamer affinity complex for a time such that the ratio of the measured level of aptamer affinity complex to the measured level of the non-specific complex is increased.
61. The method of claim 56, wherein said kinetic challenge comprises diluting the mixture containing the aptamer affinity complex, adding a competitor to the mixture containing the aptamer affinity complex, and incubating the mixture containing the aptamer affinity complex for a time selected from the group consisting of greater than or equal to about 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 30 minutes, and 60 minutes.
62. The method of claim 56, wherein said kinetic challenge comprises diluting the mixture containing the aptamer affinity complex, adding a competitor to the mixture containing the aptamer affinity complex and incubating the mixture containing the aptamer affinity complex for a time such that the ratio of the measured level of aptamer affinity complex to the measured level of the non-specific complex is increased
63. The method of claim 56 wherein the kinetic challenge comprises the introduction of a competitor molecule and said competitor molecule is selected from the group consisting of an oligonucleotide, heparin, herring sperm DNA, salmon sperm DNA, dextran sulfate, polyanion, abasic phosphodiester polymer, dNTP, and pyrophosphate.
64. The method of claim 54 wherein said aptamer affinity complex has a slow rate of dissociation.
65. The method of claim 64 wherein the rate of dissociation of said aptamer affinity complex (t1/2 ) is greater than or equal to about 30 minutes.
66. The method of claim 64 wherein the rate of dissociation of said aptamer affinity complex (t1/2 ) is between about 30 minutes and about 240 minutes.
67. The method of claim 64 wherein the rate of dissociation of said aptamer affinity complex (t1/2 ) is selected from the group consisting of > about 30 minutes, > about 60 minutes, > about 90 minutes,. > about 120 minutes, > about 150 minutes, > about 180 minutes, > about 210 minutes, and > about 240 minutes,
68. The method of claim 54, wherein the first tag comprises a releasable moiety.
69. The method of claim 68 wherein the releasable moiety comprises a nucleic acid sequence that is complementary to a region of the photoaptamer, wherein the complementary sequences can be separated by chemical or thermal conditions that destabilize nucleic acid duplexes.
70. The method of claim 55, wherein the photo-reactive crosslinking group of the photoaptamer is attached to the aptamer via a cleavable moiety.
71. The method of claim 70 wherein the cleavable moiety is a photocleavable moiety.
72. A method of detecting the presence of, or determining the amount of, a target molecule in a sample, the method comprising:
(i) providing a plurality of aptamers to a target molecule, wherein the aptamers have a cleavable capture tag;
(ii) contacting the aptamers with a sample containing target molecules to form a mixture containing aptamer-target molecule complexes;
(iii) providing a solid support having probes adhered to the surface of the support, wherein the probes are capable of binding to the cleavable capture tag;
(iv) contacting the mixture with the solid support such that aptamer-target molecule complexes become bound to the support through binding of the cleavable capture tag and probe;
(v) partitioning aptamer-target molecule complexes bound to the solid support from the remainder of the mixture;
(vi) introducing a second capture tag to the target molecule component of the aptamer target molecule complexes;
(vii) dissociating the aptamer-target molecule complexes from the surface of the solid support by cleaving the cleavable capture tags;
(viii) providing a solid support having probes adhered to the surface of the support, wherein the probes are capable of binding to the second capture tag on target molecules;
(ix) contacting the dissociated aptamer-target molecule complexes with the solid support from (viii) such that the aptamer-target molecule complexes become bound to the support through binding of the second capture tag and probe;
(x) dissociating the aptamer-target molecule complexes to yield free aptamers and target molecules bound to the support;
(xi) detecting the free aptamers.
73. The method of claim 72 wherein after dissociation of the aptamer-target molecule complexes from the surface of the solid support (vii), the dissociated aptamer-target molecule complexes are contacted with an excess of competitor molecule.
74. The method of claim 72 wherein after dissociation of the aptamer-target molecule complexes from the surface of the solid support (vii), the dissociated aptamer-target molecule complexes are diluted.
75. The method of claim 73 wherein after dissociation of the aptamer-target molecule complexes from the surface of the solid support (vii), the dissociated aptamer-target molecule complexes are diluted.
76. The method of any one of claims 73 further comprising the step of measuring the amount of free aptamer detected.
77. The method of any one of claims 74 further comprising the step of measuring the amount of free aptamer detected.
78. The method of any one of claims 75 further comprising the step of measuring the amount of free aptamer detected.
79. A kit comprising:
a) one or more aptamers specific to one or more targets of interest; and b) one or more solid supports; and
c) one or more partitioning reagents; and
d) one or more reagents for the release of an aptamer from the affinity complex.
80. The kit of claim 79 further comprising a reagent for derivatizing the one or more targets of interest.
81. The kits of claims 79 further comprising a reagent to cleave a cleavable moiety in said one or more aptamers.
82. The kits of claims 79 further comprising a reagent for use in a kinetic challenge.