Reducing sample-to-sample analyte variability in complex biological matrices

Abstract

Described herein are compositions and methods for reducing the variability of sample-to-sample analyte measurements from biological matrices. In some embodiments, the present disclosure relates to methods for reducing sample-to-sample variability in the levels of one or more proteins from biological samples as measured by a proteomic assay.

Description

[Technical field]

[0001] The present disclosure relates generally to reducing the variability of inter-sample analyte measurements from biological samples. In some embodiments, the present disclosure relates to compositions and methods for reducing the variability of inter-sample levels of one or more proteins as measured by a proteomic assay. [Background technology]

[0002] Sample-to-sample variability of analyte measurements in biological samples is problematic in biomarker discovery, metabolic analysis, gene expression analysis, protein pathway analysis, and diagnostic and prognostic tools, especially when the results depend on quantitative biological signals with relatively small differences. Biological sample types that exhibit high sample-to-sample variability are a major challenge in dealing with such sample types. Reducing such variability would result in more consistent and meaningful data sets in experimental and clinical applications.

[0003] Thus, there is a continuing need for alternative compositions and methods for reducing sample-to-sample variability in analyte measurements in biological matrices. The present disclosure meets such a need by providing novel compositions and methods for normalizing biological signals in complex matrices, which reduce, minimize, or eliminate such variability. Summary of the Invention

[0004] Provided herein are buffer exchanged biological samples and methods of preparing biological samples for detecting proteins that include buffer exchange. In some embodiments, the sample-to-sample variation of analyte levels measured in test samples that include or are generated from a buffer exchanged biological sample is reduced compared to samples that include or are generated from a biological sample that is not buffer exchanged. In some embodiments, the sample-to-sample variation of analyte levels in test samples generated using a method that includes buffer exchange is reduced compared to a method that does not include buffer exchange. In some embodiments, the linear range of measurements for the analyte in a test sample that includes or is generated from a buffer exchanged biological sample is greater than the linear range of measurements for the analyte in a test sample that includes or is generated from a non-buffer exchanged biological sample. In some embodiments, the analyte is a protein (e.g., a target protein). In some embodiments, the biological sample is a urine sample. In some embodiments, the total protein concentration in the test sample is between 2 μg / mL and 100 μg / mL. In some embodiments, the total protein concentration in the test sample is between 2 μg / mL and 60 μg / mL. In some such embodiments, the total protein concentration of the test sample or biological sample is adjusted to a range of about 2 μg / mL to about 60 μg / mL after it is determined that the total protein concentration is not within that range. In some embodiments, the total protein concentration of the test sample is in the range of about 2 μg / mL to about 60 μg / mL without adjustment of the total protein concentration. In some such embodiments, the total protein concentration is measured using a fluorescent readout assay. In some embodiments, the total protein concentration in the test sample is 70 μg / mL to 100 μg / mL. In some such embodiments, the total protein concentration of the test sample is adjusted to a range of about 70 μg / mL to about 100 μg / mL after it is determined that the total protein concentration is not within that range. In some embodiments, the total protein concentration of the test sample is in the range of about 70 μg / mL to about 100 μg / mL without adjustment of the total protein concentration.In some such embodiments, the total protein concentration is measured using a BCA assay. In some embodiments, the total protein concentration of each of the multiple test samples or multiple biological samples is adjusted to be the same.

[0005] Certain specific embodiments provided herein are listed below.

[0006] Embodiment 1. A method for preparing a biological sample for detecting a protein, comprising: a) performing buffer exchange on the biological sample with a formulation comprising a buffer, one or more salts, a chelating agent, and a non-ionic surfactant to generate a test sample; b) adjusting the total protein concentration of the test sample to produce an adjusted test sample, wherein the total protein concentration in the adjusted test sample is from about 2 μg / mL to about 60 μg / mL.

[0007] Embodiment 2. The method of embodiment 1, comprising quantifying the protein concentration of the test sample.

[0008] Embodiment 3. A method for preparing a biological sample for detecting a protein, comprising: a) generating a test sample by performing buffer exchange on the biological sample with a formulation comprising a buffer, one or more salts, a chelating agent, and a non-ionic surfactant; b) quantifying the total protein concentration of the test sample; and c) adjusting the total protein concentration of the test sample to produce an adjusted test sample, wherein the total protein concentration in the adjusted test sample is from about 2 μg / mL to about 60 μg / mL.

[0009] Embodiment 4. The method of any one of embodiments 1 to 3, comprising performing buffer exchange on a plurality of biological samples to generate a plurality of test samples, wherein the total protein concentration of each adjusted test sample is approximately the same.

[0010] Embodiment 5. The method of any one of embodiments 1 to 3, comprising performing buffer exchange on a plurality of biological samples to generate a plurality of test samples, wherein each adjusted test sample has the same total protein concentration.

[0011] Embodiment 6. A method for preparing a biological sample for detecting a protein, comprising: a) generating a test sample by performing buffer exchange on the biological sample with a formulation comprising a buffer, one or more salts, a chelating agent, and a non-ionic surfactant; b) quantifying the protein concentration of the test sample; c) if the total protein concentration of the test sample is not within the range of about 2 μg / mL to about 60 μg / mL, adjusting the total protein concentration of the test sample to produce an adjusted test sample, wherein the total protein concentration in the adjusted test sample is about 2 μg / mL to about 60 μg / mL.

[0012] Embodiment 7. The method of embodiment 6, wherein if the total protein concentration of the test sample is not within the range of about 2 μg / mL to about 50 μg / mL, or about 2 μg / mL to about 45 μg / mL, or about 4 μg / mL to about 40 μg / mL, or about 8 μg / mL to about 40 μg / mL, or about 10 μg / mL to about 40 μg / mL, or about 10 μg / mL to about 30 μg / mL, or less than 40 μg / mL, or less than 30 μg / mL, or less than 20 μg / mL, or about 15 μg / mL, or about 10 μg / mL to about 20 μg / mL, the total protein concentration of the biological sample is adjusted to said range to produce an adjusted test sample.

[0013] Embodiment 8. The method of any one of embodiments 1-7, wherein the one or more salts are each independently selected from a sodium salt, a potassium salt, and a magnesium salt.

[0014] Embodiment 9. The method of any one of embodiments 1-7, wherein the one or more salts include a sodium salt, a potassium salt, and a magnesium salt.

[0015] Embodiment 10. The method of any one of embodiments 8 or 9, wherein the sodium salt is NaCl, the potassium salt is KCl, and the magnesium salt is MgCl2.

[0016] Embodiment 11. The method of embodiment 10, wherein the NaCl in the formulation is at a concentration of about 10 mM to about 500 mM, or about 50 mM to about 250 mM, or about 100 mM to about 200 mM, or about 102 mM.

[0017] Embodiment 12. The method of embodiment 10 or 11, wherein the KCl in the formulation is at a concentration of about 0.5 mM to about 30 mM, or about 1 mM to about 20 mM, or about 2 mM to about 15 mM, or about 4 mM to about 10 mM, or about 5 mM.

[0018] Embodiment 13. The method of any one of embodiments 10-12, wherein the MgCl2 in the formulation is at a concentration of about 0.5 mM to about 30 mM, or about 1 mM to about 20 mM, or about 2 mM to about 15 mM, or about 4 mM to about 10 mM, or about 5 mM.

[0019] Embodiment 14. The method of any one of embodiments 1 to 13, wherein the buffer is selected from HEPES, MES, Bis-Trismethane, ADA, ACES, Bis-Trispropane, PIPES, MOPSO, cholamine chloride, MOPS, BES, TES, DIPSO, MOB, acetamidoglycine, TAPSO, TEA, POPSO, HEPPSO, EPS, HEPPS, Tricine, Tris, glycinamide, glycylglycine, HEPBS, bicine, TAPS, AMPB, CHES, AMP, AMPSO, CAPSO, CAPS, and CABS.

[0020] Embodiment 15. The method of embodiment 14, wherein the buffer in the formulation is at a concentration of about 4 mM to about 400 mM, or about 10 mM to about 300 mM, or about 20 mM to about 200 mM, or about 30 mM to about 100 mM, or 35 mM to about 50 mM, or about 40 mM.

[0021] Embodiment 16. The method of any one of embodiments 1 to 15, wherein the chelating agent is selected from EDTA, EGTA, DTPA, BAPTA, DMPS, and ALA.

[0022] Embodiment 17. The method of embodiment 16, wherein the chelating agent in the formulation is at a concentration of about 0.1 mM to about 10 mM, or about 0.5 mM to about 5 mM, or about 1 mM.

[0023] Embodiment 18. The method of any one of embodiments 1 to 17, wherein the nonionic surfactant is selected from polyoxyethylene (20) sorbitan monolaurate (Tween-20), polyoxyethylene (40) sorbitan monolaurate (Tween-40), and polyoxyethylene (80) sorbitan monolaurate (Tween-80).

[0024] Embodiment 19. The method of embodiment 18, wherein the nonionic surfactant is about 0.01% to about 1% of the formulation, or about 0.02% to about 0.5% of the formulation, or about 0.03% to about 0.1% of the formulation, or about 0.04% to about 0.08% of the formulation, or about 0.05% of the formulation, or about 0.1% of the formulation (volume to volume).

[0025] Embodiment 20. The method according to any one of embodiments 1 to 19, wherein the total protein concentration of the adjusted test sample is about 2 μg / mL to about 50 μg / mL, or about 2 μg / mL to about 45 μg / mL, or about 4 μg / mL to about 40 μg / mL, or about 8 μg / mL to about 40 μg / mL, or about 10 μg / mL to about 40 μg / mL, or about 10 μg / mL to about 30 μg / mL, or less than 40 μg / mL, or less than 30 μg / mL, or less than 20 μg / mL, or about 15 μg / mL, or about 10 μg / mL to about 20 μg / mL.

[0026] Embodiment 21. The method of any one of embodiments 2 to 20, wherein the total protein concentration of the test sample or the adjusted test sample is quantified by an assay selected from the group consisting of a fluorescent readout assay, a Bradford assay, a bicinchoninic acid (BCA) assay, a Lowry assay, and an ELISA assay.

[0027] Embodiment 22. The method of embodiment 21, wherein the fluorescent readout assay comprises a reagent selected from a merocyanine dye and epicocconone.

[0028] Embodiment 23. The method of any one of embodiments 1 to 22, wherein the formulation comprises 40 mM HEPES, 102 mM NaCl, 5 mM KCl, 5 mM MgCl2, 1 mM EDTA, and 0.05% Tween-20.

[0029] Embodiment 24. The method of any one of embodiments 1 to 23, wherein the pH of the formulation is from about pH 5 to about pH 9, or from about pH 6 to about pH 8, or from about pH 7 to about pH 7.9, or about pH 7.5.

[0030] Embodiment 25. The method of any one of embodiments 1 to 24, wherein the formulation consists of 40 mM HEPES, 102 mM NaCl, 5 mM KCl, 5 mM MgCl2, 1 mM EDTA, and 0.05% Tween-20, and has a pH of 7.5.

[0031] Embodiment 26. The method of any one of embodiments 1 to 25, wherein the buffer exchange is not performed by ultrafiltration.

[0032] Embodiment 27. The method of any one of embodiments 1 to 26, wherein the buffer exchange is performed by gel filtration chromatography.

[0033] Embodiment 28. The method of any one of embodiments 1 to 27, wherein the biological sample is a urine sample.

[0034] Embodiment 29. The method of any one of embodiments 1 to 27, wherein the biological sample is a serum sample.

[0035] Embodiment 30. The method of any one of embodiments 1 to 29, wherein the method does not include concentrating the biological sample.

[0036] Embodiment 31. A test sample comprising a buffer-exchanged biological sample, wherein the test sample has a total protein concentration of about 2 μg / mL to about 60 μg / mL, and the buffer-exchanged biological sample comprises a buffering agent, one or more salts, a chelating agent, and a non-ionic surfactant.

[0037] Embodiment 32. The test sample of embodiment 31, further comprising one or more protein capture reagents.

[0038] Embodiment 33 The test sample of embodiment 32, wherein each of the one or more protein capture reagents is an aptamer or an antibody.

[0039] Embodiment 34. The test sample according to any one of embodiments 31 to 33, wherein the total protein concentration of the test sample is about 2 μg / mL to 50 μg / mL, or about 2 μg / mL to 45 μg / mL, or about 4 μg / mL to about 40 μg / mL, or about 8 μg / mL to 40 μg / mL, or about 10 μg / mL to 40 μg / mL, or about 10 μg / mL to 30 μg / mL, or less than 50 μg / mL, or less than 40 μg / mL, or less than 30 μg / mL, or less than 20 μg / mL, or about 15 μg / mL, or about 10 μg / mL to about 20 μg / mL.

[0040] Embodiment 35. The test sample of any one of embodiments 31-34, wherein the one or more salts are each independently selected from a sodium salt, a potassium salt, and a magnesium salt.

[0041] Embodiment 36. The test sample of any one of embodiments 31 to 34, wherein the one or more salts include a sodium salt, a potassium salt, and a magnesium salt.

[0042] Embodiment 37. The test sample of embodiment 35 or 36, wherein the sodium salt is NaCl, the potassium salt is KCl, and the magnesium salt is MgCl2.

[0043] Embodiment 38. The test sample of embodiment 37, wherein the NaCl in the test sample is at a concentration of about 10 mM to about 500 mM, or about 50 mM to about 250 mM, or about 100 mM to about 200 mM, or about 102 mM.

[0044] Embodiment 39. The test sample of embodiment 37 or 38, wherein the KCl in the test sample is at a concentration of about 0.5 mM to about 30 mM, or about 1 mM to about 20 mM, or about 2 mM to about 15 mM, or about 4 mM to about 10 mM, or about 5 mM.

[0045] Embodiment 40. The test sample of any one of embodiments 37 to 39, wherein the MgCl2 in the test sample is at a concentration of about 0.5 mM to about 30 mM, or about 1 mM to about 20 mM, or about 2 mM to about 15 mM, or about 4 mM to about 10 mM, or about 5 mM.

[0046] Embodiment 41. The test sample of any one of embodiments 31 to 40, wherein the buffer is selected from HEPES, MES, Bis-Trismethane, ADA, ACES, Bis-Trispropane, PIPES, MOPSO, cholamine chloride, MOPS, BES, TES, DIPSO, MOB, acetamidoglycine, TAPSO, TEA, POPSO, HEPPSO, EPS, HEPPS, Tricine, Tris, glycinamide, glycylglycine, HEPBS, bicine, TAPS, AMPB, CHES, AMP, AMPSO, CAPSO, CAPS, and CABS.

[0047] Embodiment 42. A test sample according to any one of embodiments 31 to 41, wherein the buffer in the test sample is at a concentration of about 4 mM to about 400 mM, or about 10 mM to about 300 mM, or about 20 mM to about 200 mM, or about 30 mM to about 100 mM, or 35 mM to about 50 mM, or about 40 mM.

[0048] Embodiment 43. The test sample of any one of embodiments 31 to 42, wherein the chelating agent is selected from EDTA, EGTA, DTPA, BAPTA, DMPS, and ALA.

[0049] Embodiment 44. A test sample according to any one of embodiments 31 to 43, wherein the chelating agent in the test sample is at a concentration of about 0.1 mM to about 10 mM, or about 0.5 mM to about 5 mM, or about 1 mM.

[0050] Embodiment 45. The test sample of any one of embodiments 31 to 44, wherein the nonionic surfactant is selected from polyoxyethylene (20) sorbitan monolaurate (Tween-20), polyoxyethylene (40) sorbitan monolaurate (Tween-40), and polyoxyethylene (80) sorbitan monolaurate (Tween-80).

[0051] Embodiment 46. The method of any one of embodiments 31 to 45, wherein the nonionic surfactant is about 0.01% to about 1% of the test sample, or about 0.02% to about 0.5% of the test sample, or about 0.03% to about 0.1% of the test sample, or about 0.04% to about 0.08% of the test sample, or about 0.05% of the test sample (volume to volume).

[0052] Embodiment 47. The test sample described in any one of embodiments 31 to 46, wherein the total protein concentration of the test sample is quantified by an assay selected from the group consisting of a fluorescent readout assay, a Bradford assay, a bicinchoninic acid (BCA) assay, a Lowry assay, and an ELISA assay.

[0053] Embodiment 48. The test sample of embodiment 47, wherein the fluorescent readout assay comprises a reagent selected from a merocyanine dye and epicocconone.

[0054] Embodiment 49. The test sample of any one of embodiments 31 to 48, wherein the test sample comprises 40 mM HEPES, 102 mM NaCl, 5 mM KCl, 5 mM MgCl2, 1 mM EDTA, and 0.05% Tween-20.

[0055] Embodiment 50. A test sample described in any one of embodiments 31 to 49, wherein the pH of the test sample is about pH 5 to about pH 9, or about pH 6 to about pH 8, or about pH 7 to about pH 7.9, or about pH 7.5.

[0056] Embodiment 51. The test sample of any one of embodiments 31 to 50, wherein at least one capture reagent is an aptamer, and said at least one aptamer comprises a 5-position modified pyrimidine.

[0057] Embodiment 52 The test sample of embodiment 51, wherein the 5-modified pyrimidine comprises a hydrophobic moiety at the 5-position of the pyrimidine.

[0058] Embodiment 53. The test sample of embodiment 52, wherein the hydrophobic moiety is selected from a naphthyl moiety, a phenyl moiety, a tyrosyl moiety, an indole moiety, and a morpholino moiety.

[0059] Embodiment 54. The test sample of embodiment 52 or 53, wherein the hydrophobic moiety is covalently attached to the 5-position of the base via a linker comprising a group selected from an amide linker, a carbonyl linker, a propynyl linker, an alkyne linker, an ester linker, a urea linker, a carbamate linker, a guanidine linker, an amidine linker, a sulfoxide linker, and a sulfone linker.

[0060] Embodiment 55. The test sample of any one of embodiments 31 to 54, wherein the biological sample is a urine sample.

[0061] Embodiment 56. The test sample of any one of embodiments 31 to 54, wherein the biological sample is a serum sample.

[0062] Embodiment 57. A method for detecting a target protein in a test sample, comprising: a) contacting the test sample with at least one capture reagent, the at least one capture reagent capable of binding to the target protein to form a complex; b) incubating the test sample with the at least one capture reagent under conditions that allow the formation of the complex; c) quantifying the level of the target protein in the test sample by measuring the level of the at least one capture reagent, the complex, or the protein, wherein the level of the at least one capture reagent or the complex is a surrogate for the level of the target protein; the test sample is generated by performing buffer exchange on the biological sample with a formulation comprising a buffer, a salt, a chelating agent, and a non-ionic surfactant; The method, wherein the test sample has a total protein concentration of about 2 μg / mL or more and less than about 60 μg / mL.

[0063] Embodiment 58 The method of embodiment 57, wherein the at least one protein capture reagent is an aptamer or an antibody.

[0064] Embodiment 59 The method of embodiment 57 or 58, comprising a plurality of capture reagents, each capture reagent being an aptamer.

[0065] Embodiment 60. The method of any one of embodiments 57 to 59, wherein the total protein concentration of the test sample is about 2 μg / mL to 50 μg / mL, or about 2 μg / mL to 45 μg / mL, or about 4 μg / mL to about 40 μg / mL, or about 8 μg / mL to 40 μg / mL, or about 10 μg / mL to 40 μg / mL, or about 10 μg / mL to 30 μg / mL, or less than 50 μg / mL, or less than 40 μg / mL, or less than 30 μg / mL, or less than 20 μg / mL, or about 15 μg / mL, or about 10 μg / mL to about 20 μg / mL.

[0066] Embodiment 61. The method of any one of embodiments 57-60, wherein the one or more salts are selected from sodium salts, potassium salts, and magnesium salts.

[0067] Embodiment 62. The method of any one of embodiments 57-60, wherein the one or more salts include sodium salts, potassium salts, and magnesium salts.

[0068] Embodiment 63 The method of embodiment 61 or 62, wherein the sodium salt is NaCl, the potassium salt is KCl, and the magnesium salt is MgCl2.

[0069] Embodiment 64. The method of embodiment 63, wherein the NaCl in the formulation is at a concentration of about 10 mM to about 500 mM, or about 50 mM to about 250 mM, or about 100 mM to about 200 mM, or about 102 mM.

[0070] Embodiment 65. The method of embodiment 63 or 64, wherein the KCl in the formulation is at a concentration of about 0.5 mM to about 30 mM, or about 1 mM to about 20 mM, or about 2 mM to about 15 mM, or about 4 mM to about 10 mM, or about 5 mM.

[0071] Embodiment 66. The method of any one of embodiments 63 to 65, wherein the MgCl2 in the formulation is at a concentration of about 0.5 mM to about 30 mM, or about 1 mM to about 20 mM, or about 2 mM to about 15 mM, or about 4 mM to about 10 mM, or about 5 mM.

[0072] Embodiment 67. The method of any one of embodiments 57 to 66, wherein the buffer is selected from HEPES, MES, bis-trismethane, ADA, ACES, bis-trispropane, PIPES, MOPSO, cholamine chloride, MOPS, BES, TES, DIPSO, MOB, acetamidoglycine, TAPSO, TEA, POPSO, HEPPSO, EPS, HEPPS, tricine, tris, glycinamide, glycylglycine, HEPBS, bicine, TAPS, AMPB, CHES, AMP, AMPSO, CAPSO, CAPS, and CABS.

[0073] Embodiment 68. The method of any one of embodiments 57 to 67, wherein the buffer in the formulation is at a concentration of from 4 mM to about 400 mM, or from about 10 mM to about 300 mM, or from about 20 mM to about 200 mM, or from about 30 mM to about 100 mM, or from 35 mM to about 50 mM, or about 40 mM.

[0074] Embodiment 69. The method of any one of embodiments 57 to 68, wherein the chelating agent is selected from EDTA, EGTA, DTPA, BAPTA, DMPS, and ALA.

[0075] Embodiment 70. The method of any one of embodiments 57 to 69, wherein the chelating agent in the formulation is at a concentration of about 0.1 mM to about 10 mM, or about 0.5 mM to about 5 mM, or about 1 mM.

[0076] Embodiment 71. The method of any one of embodiments 57 to 70, wherein the nonionic surfactant is selected from polyoxyethylene (20) sorbitan monolaurate (Tween-20), polyoxyethylene (40) sorbitan monolaurate (Tween-40), and polyoxyethylene (80) sorbitan monolaurate (Tween-80).

[0077] Embodiment 72. The method of any one of embodiments 57-71, wherein the nonionic surfactant is from about 0.01% to about 1% of the formulation, or from about 0.02% to about 0.5% of the formulation, or from about 0.03% to about 0.1% of the formulation, or from about 0.04% to about 0.08% of the formulation, or about 0.05% of the formulation.

[0078] Embodiment 73. The method of any one of embodiments 57 to 72, wherein the total protein concentration of the method is measured by an assay selected from the group consisting of a fluorescent readout assay, a Bradford assay, a bicinchoninic acid (BCA) assay, a Lowry assay, and an ELISA assay.

[0079] Embodiment 74 The method of embodiment 73, wherein the fluorescent readout assay comprises a reagent selected from a merocyanine dye and epicocconone.

[0080] Embodiment 75. The method of any one of embodiments 57 to 74, wherein the formulation comprises 40 mM HEPES, 102 mM NaCl, 5 mM KCl, 5 mM MgCl2, 1 mM EDTA, and 0.05% Tween 20.

[0081] Embodiment 76. The method of any one of embodiments 57 to 74, wherein the formulation consists of 40 mM HEPES, 102 mM NaCl, 5 mM KCl, 5 mM MgCl2, 1 mM EDTA, and 0.05% Tween-20 (pH 7.5).

[0082] Embodiment 77. The method of any one of embodiments 57 to 75, wherein the pH of the formulation is from about pH 5 to about pH 9, or from about pH 6 to about pH 8, or from about pH 7 to about pH 7.9, or about pH 7.5.

[0083] Embodiment 78. The method of any one of embodiments 58 to 77, wherein the aptamer comprises a 5-position modified pyrimidine.

[0084] Embodiment 79. The method of embodiment 78, wherein the 5-modified pyrimidine comprises a hydrophobic moiety at the 5-position of the pyrimidine.

[0085] Embodiment 80. The method of embodiment 79, wherein the hydrophobic moiety is selected from a naphthyl moiety, a phenyl moiety, a tyrosyl moiety, an indole moiety, and a morpholino moiety.

[0086] Embodiment 81. The method of embodiment 79 or 80, wherein the hydrophobic moiety is covalently attached to the 5-position of the base via a linker comprising a group selected from an amide linker, a carbonyl linker, a propynyl linker, an alkyne linker, an ester linker, a urea linker, a carbamate linker, a guanidine linker, an amidine linker, a sulfoxide linker, and a sulfone linker.

[0087] Embodiment 82. The method of any one of embodiments 57 to 81, wherein the complex is a non-covalent complex.

[0088] Embodiment 83. The method of any one of embodiments 57 to 82, wherein the at least one capture reagent is bound to a first solid support prior to contacting with the test sample, or the capture reagent is bound to a first solid support after contacting with the test sample.

[0089] Embodiment 84 The method of any one of embodiments 57 to 83, further comprising binding the complex to a first solid support via the capture reagent.

[0090] Embodiment 85 The method of embodiment 84, further comprising releasing the complex from the first solid support and binding the complex to a second solid support.

[0091] Embodiment 86 The method of embodiment 85, wherein the complex is bound to the second solid support via the protein.

[0092] Embodiment 87. The method of any one of embodiments 57 to 86, further comprising adding a competitor molecule to the test sample and / or adding a competitor molecule and diluting the test sample.

[0093] Embodiment 88 The method of embodiment 87, wherein the competitor molecule is a polyanion competitor.

[0094] Embodiment 89. The method of embodiment 88, wherein the polyanion competitor is selected from an oligonucleotide, a polydextran, DNA, heparin, and dNTPs.

[0095] Embodiment 90 The method of embodiment 89, wherein the polyanionic competitor is polydextran, and the polydextran is dextran sulfate.

[0096] Embodiment 91 The method of embodiment 89, wherein the polyanion competitor is an oligonucleotide, and the oligonucleotide contains one or more 5-position modified pyrimidines.

[0097] Embodiment 92. The method of any one of embodiments 57 to 91, wherein the biological sample is urine.

[0098] Embodiment 93. The method of any one of embodiments 57 to 91, wherein the biological sample is serum.

[0099] Embodiment 94. The method of any one of embodiments 1 to 30 or 57 to 93, wherein the buffer exchange is performed using gel filtration chromatography.

[0100] Embodiment 95. The test sample of any one of embodiments 31 to 56, wherein gel filtration chromatography is used to perform the buffer exchange on the biological sample to produce the buffer exchanged biological sample.

[0101] Embodiment 96. The method of any one of embodiments 1 to 30 or 57 to 94, further comprising measuring the protein concentration of the biological sample prior to performing the buffer exchange.

[0102] Embodiment 97. The method of embodiment 96, wherein the protein concentration of the biological sample is measured by an assay selected from the group consisting of a fluorescent readout assay, a bicinchoninic acid (BCA) assay, a microBCA assay, a Lowry assay, and an ELISA assay.

[0103] Embodiment 98. The test sample of any one of embodiments 47-56 or 95, wherein the assay used to quantify total protein concentration is performed prior to buffer exchange of the biological sample.

[0104] Embodiment 99. The test sample of any one of embodiments 47-56, 95, or 98, wherein the assay used to quantify total protein concentration is performed after buffer exchange of the biological sample.

[0105] Embodiment 100. A method for preparing a plurality of biological samples for detecting proteins, comprising: performing buffer exchange on the plurality of biological samples with a formulation comprising a buffer, one or more salts, a chelating agent, and a non-ionic surfactant to generate a plurality of test samples; adjusting the total protein concentration of the plurality of test samples to produce a plurality of adjusted test samples, wherein each adjusted test sample has approximately the same total protein concentration; The method, wherein the method does not include concentrating the total protein of the multiple biological samples prior to performing the buffer exchange.

[0106] Embodiment 101. A method for preparing a biological sample for detecting a protein, comprising: performing buffer exchange on the biological sample with a formulation comprising a buffer, one or more salts, a chelating agent, and a non-ionic surfactant to generate a test sample; and adjusting the total protein concentration of the test sample to produce an adjusted test sample, wherein the total protein concentration in the adjusted test sample is from about 70 μg / mL to about 100 μg / mL.

[0107] Embodiment 102 The method of embodiment 100 or 101, comprising quantifying the protein concentration of the test sample or samples and / or the biological sample or samples.

[0108] Embodiment 103. A method for preparing a biological sample for detecting a protein, comprising: performing a buffer exchange on the biological sample with a formulation comprising a buffer, one or more salts, a chelating agent, and a non-ionic surfactant to generate a test sample; Quantifying the total protein concentration of the test sample; and adjusting the total protein concentration of the test sample to produce an adjusted test sample, wherein the total protein concentration in the adjusted test sample is from about 70 μg / mL to about 100 μg / mL.

[0109] Embodiment 104. The method of any one of embodiments 101 to 103, comprising generating a plurality of test samples by performing buffer exchange on a plurality of biological samples, wherein the total protein concentration of each adjusted test sample is approximately the same.

[0110] Embodiment 105. The method of any one of embodiments 100 to 103, comprising generating a plurality of test samples by performing buffer exchange on a plurality of biological samples, wherein the total protein concentration of each adjusted test sample is the same.

[0111] Embodiment 106. A method for preparing a biological sample for detecting a protein, comprising: performing a buffer exchange on the biological sample with a formulation comprising a buffer, one or more salts, a chelating agent, and a non-ionic surfactant to generate a test sample; quantitating the protein concentration of the test sample; and producing an adjusted test sample by adjusting the total protein concentration of the test sample if the total protein concentration of the test sample is not within the range of about 70 μg / mL to about 100 μg / mL, wherein the total protein concentration in the adjusted test sample is about 70 μg / mL to about 100 μg / mL.

[0112] Embodiment 107. The method of embodiment 106, wherein if the total protein concentration of the test sample is not within the range of about 70 μg / mL to about 100 μg / mL, or about 70 μg / mL to about 95 μg / mL, or about 70 μg / mL to about 90 μg / mL, or about 70 μg / mL to about 85 μg / mL, or about 70 μg / mL to about 80 μg / mL, or about 70 μg / mL to about 75 μg / mL, an adjusted test sample is produced by adjusting the total protein concentration of the test sample to said range.

[0113] Embodiment 108. The method of any one of embodiments 100-107, wherein the one or more salts are each independently selected from a sodium salt, a potassium salt, and a magnesium salt.

[0114] Embodiment 109. The method of any one of embodiments 100 to 107, wherein the one or more salts include sodium salts, potassium salts, and magnesium salts.

[0115] Embodiment 110. The method of any one of embodiments 108 or 109, wherein the sodium salt is NaCl, the potassium salt is KCl, and the magnesium salt is MgCl2.

[0116] Embodiment 111. The method of embodiment 110, wherein the NaCl in the formulation is at a concentration of about 10 mM to about 500 mM, or about 50 mM to about 250 mM, or about 100 mM to about 200 mM, or about 75 to 125 mM, or about 102 mM.

[0117] Embodiment 112. The method of embodiment 110 or 111, wherein the KCl in the formulation is at a concentration of about 0.5 mM to about 30 mM, or about 1 mM to about 20 mM, or about 2 mM to about 15 mM, or about 4 mM to about 10 mM, or about 5 mM.

[0118] Embodiment 113. The method of any one of embodiments 110 to 112, wherein the MgCl2 in the formulation is at a concentration of about 0.5 mM to about 30 mM, or about 1 mM to about 20 mM, or about 2 mM to about 15 mM, or about 4 mM to about 10 mM, or about 5 mM.

[0119] Embodiment 114. The method of any one of embodiments 100 to 113, wherein the buffer is selected from HEPES, MES, bis-trismethane, ADA, ACES, bis-trispropane, PIPES, MOPSO, cholamine chloride, MOPS, BES, TES, DIPSO, MOB, acetamidoglycine, TAPSO, TEA, POPSO, HEPPSO, EPS, HEPPS, tricine, tris, glycinamide, glycylglycine, HEPBS, bicine, TAPS, AMPB, CHES, AMP, AMPSO, CAPSO, CAPS, and CABS.

[0120] Embodiment 115. The method of embodiment 114, wherein the buffer in the formulation is at a concentration of about 4 mM to about 400 mM, or about 10 mM to about 300 mM, or about 20 mM to about 200 mM, or about 30 mM to about 100 mM, or 35 mM to about 50 mM, or about 40 mM.

[0121] Embodiment 116. The method of any one of embodiments 100 to 115, wherein the chelating agent is selected from EDTA, EGTA, DTPA, BAPTA, DMPS, and ALA.

[0122] Embodiment 117. The method of embodiment 116, wherein the chelating agent in the formulation is at a concentration of about 0.1 mM to about 10 mM, or about 0.5 mM to about 5 mM, or about 1 mM.

[0123] Embodiment 118. The method of any one of embodiments 100 to 117, wherein the nonionic surfactant is selected from polyoxyethylene (20) sorbitan monolaurate (Tween-20), polyoxyethylene (40) sorbitan monolaurate (Tween-40), and polyoxyethylene (80) sorbitan monolaurate (Tween-80).

[0124] Embodiment 119. The method of embodiment 118, wherein the nonionic surfactant is about 0.01% to about 1% of the formulation, or about 0.02% to about 0.5% of the formulation, or about 0.03% to about 0.1% of the formulation, or about 0.04% to about 0.08% of the formulation, or about 0.05% of the formulation, or about 0.1% of the formulation (volume to volume).

[0125] Embodiment 120. The method of any one of embodiments 100 to 119, wherein the total protein concentration of the adjusted test sample is from about 2 μg / mL to about 70 μg / mL, or from about 2 μg / mL to about 75 μg / mL, or from about 4 μg / mL to about 70 μg / mL, or from about 8 μg / mL to about 70 μg / mL, or from about 10 μg / mL to about 70 μg / mL, or from about 10 μg / mL to about 70 μg / mL.

[0126] Embodiment 121. The method of any one of embodiments 102 to 120, wherein the total protein concentration of the test sample or the adjusted test sample is quantified by a bicinchoninic acid (BCA) assay, and the BCA assay is optionally a microBCA assay.

[0127] Embodiment 122. The method of embodiment 121, wherein the assay comprises a bicinchoninic acid reagent or a bicinchonic acid reagent, and optionally an alkaline tartaric acid-carbonate buffer and / or a copper sulfate solution.

[0128] Embodiment 123. The method of any one of embodiments 100 to 122, wherein the formulation comprises 40 mM HEPES, 102 mM NaCl, 5 mM KCl, 5 mM MgCl2, 1 mM EDTA, and 0.05% Tween-20.

[0129] Embodiment 124. The method of any one of embodiments 100 to 123, wherein the pH of the formulation is from about pH 5 to about pH 9, or from about pH 6 to about pH 8, or from about pH 7 to about pH 7.9, or about pH 7.5.

[0130] Embodiment 125. The method of any one of embodiments 100 to 124, wherein the formulation consists of 40 mM HEPES, 102 mM NaCl, 5 mM KCl, 5 mM MgCl2, 1 mM EDTA, and 0.05% Tween-20, and has a pH of 7.5.

[0131] Embodiment 126. The method of any one of embodiments 100 to 125, wherein the buffer exchange is not performed by ultrafiltration.

[0132] Embodiment 127. The method of any one of embodiments 100 to 126, wherein the buffer exchange is performed by gel filtration chromatography.

[0133] Embodiment 128. The method of any one of embodiments 100 to 127, wherein the biological sample is a urine sample.

[0134] Embodiment 129. The method of any one of embodiments 100 to 127, wherein the biological sample is a serum sample.

[0135] Embodiment 130. The method of any one of embodiments 101 to 129, wherein the method does not include concentrating the biological sample.

[0136] Embodiment 131. A test sample comprising a buffer-exchanged biological sample, wherein the test sample has a total protein concentration of about 70 μg / mL to about 100 μg / mL, and the buffer-exchanged biological sample comprises a buffer, one or more salts, a chelating agent, and a non-ionic surfactant.

[0137] Embodiment 132. The test sample of embodiment 131, further comprising one or more protein capture reagents.

[0138] Embodiment 133. The test sample of embodiment 132, wherein each of the one or more protein capture reagents is an aptamer or an antibody.

[0139] Embodiment 134. A test sample according to any one of embodiments 131 to 133, wherein the total protein concentration of the test sample is from about 70 μg / mL to 95 μg / mL, or from about 70 μg / mL to 90 μg / mL, or from about 70 μg / mL to 85 μg / mL, or from about 70 μg / mL to 80 μg / mL, or from about 70 μg / mL to 75 μg / mL, or from about 70 μg / mL.

[0140] Embodiment 135. The test sample of any one of embodiments 131 to 134, wherein the one or more salts are each independently selected from a sodium salt, a potassium salt, and a magnesium salt.

[0141] Embodiment 136. The test sample of any one of embodiments 131 to 135, wherein the one or more salts include sodium salts, potassium salts, and magnesium salts.

[0142] Embodiment 137. The test sample of embodiment 135 or 136, wherein the sodium salt is NaCl, the potassium salt is KCl, and the magnesium salt is MgCl2.

[0143] Embodiment 138. The test sample of embodiment 137, wherein the NaCl in the test sample is at a concentration of about 10 mM to about 500 mM, or about 50 mM to about 250 mM, or about 100 mM to about 200 mM, or about 102 mM.

[0144] Embodiment 139. The test sample of embodiment 137 or 138, wherein the KCl in the test sample is at a concentration of about 0.5 mM to about 30 mM, or about 1 mM to about 20 mM, or about 2 mM to about 15 mM, or about 4 mM to about 10 mM, or about 5 mM.

[0145] Embodiment 140. The test sample of any one of embodiments 137 to 139, wherein the MgCl2 in the test sample is at a concentration of about 0.5 mM to about 30 mM, or about 1 mM to about 20 mM, or about 2 mM to about 15 mM, or about 4 mM to about 10 mM, or about 5 mM.

[0146] Embodiment 141. The test sample of any one of embodiments 131 to 140, wherein the buffer is selected from HEPES, MES, bis-trismethane, ADA, ACES, bis-trispropane, PIPES, MOPSO, cholamine chloride, MOPS, BES, TES, DIPSO, MOB, acetamidoglycine, TAPSO, TEA, POPSO, HEPPSO, EPS, HEPPS, tricine, tris, glycinamide, glycylglycine, HEPBS, bicine, TAPS, AMPB, CHES, AMP, AMPSO, CAPSO, CAPS, and CABS.

[0147] Embodiment 142. A test sample according to any one of embodiments 131 to 141, wherein the buffer in the test sample is at a concentration of about 4 mM to about 400 mM, or about 10 mM to about 300 mM, or about 20 mM to about 200 mM, or about 30 mM to about 100 mM, or 35 mM to about 50 mM, or about 40 mM.

[0148] Embodiment 143. The test sample of any one of embodiments 131 to 142, wherein the chelating agent is selected from EDTA, EGTA, DTPA, BAPTA, DMPS, and ALA.

[0149] Embodiment 144. A test sample according to any one of embodiments 131 to 143, wherein the chelating agent in the test sample is at a concentration of about 0.1 mM to about 10 mM, or about 0.5 mM to about 5 mM, or about 1 mM.

[0150] Embodiment 145. The test sample of any one of embodiments 131 to 144, wherein the nonionic surfactant is selected from polyoxyethylene (20) sorbitan monolaurate (Tween-20), polyoxyethylene (40) sorbitan monolaurate (Tween-40), and polyoxyethylene (80) sorbitan monolaurate (Tween-80).

[0151] Embodiment 146. The method of any one of embodiments 131 to 145, wherein the nonionic surfactant is about 0.01% to about 1% of the test sample, or about 0.02% to about 0.5% of the test sample, or about 0.03% to about 0.1% of the test sample, or about 0.04% to about 0.08% of the test sample, or about 0.05% of the test sample (volume to volume).

[0152] Embodiment 147. The test sample according to any one of embodiments 131 to 146, wherein the total protein concentration of the test sample is quantified by a bicinchoninic acid (BCA) assay, the BCA assay optionally being a microBCA assay.

[0153] Embodiment 148. The test sample of embodiment 147, wherein the assay comprises a bicinchoninic acid reagent or a bicinchononic acid reagent, and optionally an alkaline tartaric acid-carbonate buffer and / or a copper sulfate solution.

[0154] Embodiment 149. The test sample according to any one of embodiments 131 to 148, wherein the test sample comprises 40 mM HEPES, 102 mM NaCl, 5 mM KCl, 5 mM MgCl2, 1 mM EDTA, and 0.05% Tween-20.

[0155] Embodiment 150. A test sample described in any one of embodiments 131 to 149, wherein the pH of the test sample is about pH 5 to about pH 9, or about pH 6 to about pH 8, or about pH 7 to about pH 7.9, or about pH 7.5.

[0156] Embodiment 151. The test sample of any one of embodiments 131 to 150, wherein at least one capture reagent is an aptamer, and said at least one aptamer comprises a 5-position modified pyrimidine.

[0157] Embodiment 152 The test sample of embodiment 151, wherein the 5-modified pyrimidine comprises a hydrophobic moiety at the 5-position of the pyrimidine.

[0158] Embodiment 153. The test sample of embodiment 152, wherein the hydrophobic moiety is selected from a naphthyl moiety, a phenyl moiety, a tyrosyl moiety, an indole moiety, and a morpholino moiety.

[0159] Embodiment 154. The test sample of embodiment 152 or 153, wherein the hydrophobic moiety is covalently bonded to the 5-position of the base via a linker comprising a group selected from an amide linker, a carbonyl linker, a propynyl linker, an alkyne linker, an ester linker, a urea linker, a carbamate linker, a guanidine linker, an amidine linker, a sulfoxide linker, and a sulfone linker.

[0160] Embodiment 155. The test sample of any one of embodiments 131 to 154, wherein the biological sample is a urine sample.

[0161] Embodiment 156. The test sample of any one of embodiments 131 to 154, wherein the biological sample is a serum sample.

[0162] Embodiment 157. The test sample described in any one of embodiments 131 to 156, wherein gel filtration chromatography is used to perform the buffer exchange on the biological sample to produce the buffer exchanged biological sample.

[0163] Embodiment 158. A method for detecting a target protein in a test sample, comprising: contacting the test sample with at least one capture reagent, the at least one capture reagent capable of binding to the target protein to form a complex; incubating the test sample with the at least one capture reagent under conditions that allow for the formation of the complex; quantitating the level of the target protein in the test sample by measuring the level of the at least one capture reagent, the complex, or the protein, wherein the level of the at least one capture reagent or the complex is a surrogate for the level of the target protein; The method, wherein the test sample is generated by performing buffer exchange between a biological sample and a formulation comprising a buffer, a salt, a chelating agent, and a non-ionic surfactant, and the total protein concentration of the test sample is less than about 70 μg / mL to about 100 μg / mL.

[0164] Embodiment 159. The method of embodiment 158, wherein the at least one protein capture reagent is an aptamer or an antibody.

[0165] Embodiment 160. The method of embodiment 158 ​​or 159, comprising a plurality of capture reagents, each capture reagent being an aptamer.

[0166] Embodiment 161. The method of any one of embodiments 158 to 160, wherein the total protein concentration of the test sample is about 70 μg / mL to 95 μg / mL, or about 70 μg / mL to 90 μg / mL, or about 70 μg / mL to 85 μg / mL, or about 70 μg / mL to 80 μg / mL, or about 70 μg / mL to 75 μg / mL, or about 70 μg / mL.

[0167] Embodiment 162. The method of any one of embodiments 158-161, wherein the one or more salts are selected from sodium salts, potassium salts, and magnesium salts.

[0168] Embodiment 163. The method of any one of embodiments 158-161, wherein the one or more salts include sodium salts, potassium salts, and magnesium salts.

[0169] Embodiment 164. The method of embodiment 162 or 163, wherein the sodium salt is NaCl, the potassium salt is KCl, and the magnesium salt is MgCl2.

[0170] Embodiment 165. The method of embodiment 164, wherein the NaCl in the formulation is at a concentration of about 10 mM to about 500 mM, or about 50 mM to about 250 mM, or about 100 mM to about 200 mM, or about 102 mM.

[0171] Embodiment 166. The method of embodiment 164 or 165, wherein the KCl in the formulation is at a concentration of about 0.5 mM to about 30 mM, or about 1 mM to about 20 mM, or about 2 mM to about 15 mM, or about 4 mM to about 10 mM, or about 5 mM.

[0172] Embodiment 167. The method of any one of embodiments 164 to 166, wherein the MgCl2 in the formulation is at a concentration of about 0.5 mM to about 30 mM, or about 1 mM to about 20 mM, or about 2 mM to about 15 mM, or about 4 mM to about 10 mM, or about 5 mM.

[0173] Embodiment 168. The method of any one of embodiments 158 to 167, wherein the buffer is selected from HEPES, MES, bis-trismethane, ADA, ACES, bis-trispropane, PIPES, MOPSO, cholamine chloride, MOPS, BES, TES, DIPSO, MOB, acetamidoglycine, TAPSO, TEA, POPSO, HEPPSO, EPS, HEPPS, tricine, tris, glycinamide, glycylglycine, HEPBS, bicine, TAPS, AMPB, CHES, AMP, AMPSO, CAPSO, CAPS, and CABS.

[0174] Embodiment 169. The method of any one of embodiments 158 to 168, wherein the buffer in the formulation is at a concentration of from 4 mM to about 400 mM, or from about 10 mM to about 300 mM, or from about 20 mM to about 200 mM, or from about 30 mM to about 100 mM, or from 35 mM to about 50 mM, or about 40 mM.

[0175] Embodiment 170. The method of any one of embodiments 158 to 169, wherein the chelating agent is selected from EDTA, EGTA, DTPA, BAPTA, DMPS, and ALA.

[0176] Embodiment 171. The method of any one of embodiments 158 to 170, wherein the chelating agent in the formulation is at a concentration of about 0.1 mM to about 10 mM, or about 0.5 mM to about 5 mM, or about 1 mM.

[0177] Embodiment 172. The method of any one of embodiments 158 to 171, wherein the nonionic surfactant is selected from polyoxyethylene (20) sorbitan monolaurate (Tween-20), polyoxyethylene (40) sorbitan monolaurate (Tween-40), and polyoxyethylene (80) sorbitan monolaurate (Tween-80).

[0178] Embodiment 173. The method of any one of embodiments 158 to 172, wherein the nonionic surfactant is from about 0.01% to about 1% of the formulation, or from about 0.02% to about 0.5% of the formulation, or from about 0.03% to about 0.1% of the formulation, or from about 0.04% to about 0.08% of the formulation, or about 0.05% of the formulation.

[0179] Embodiment 174. The method of any one of embodiments 158 to 173, wherein the total protein concentration of the test sample is measured by a bicinchoninic acid (BCA) assay, and the BCA assay is optionally a microBCA assay.

[0180] Embodiment 175. The method of embodiment 174, wherein the assay comprises a bicinchoninic acid reagent or a bicinchonic acid reagent, and optionally an alkaline tartaric acid-carbonate buffer and / or a copper sulfate solution.

[0181] Embodiment 176. The method of any one of embodiments 158 to 175, wherein the formulation comprises 40 mM HEPES, 102 mM NaCl, 5 mM KCl, 5 mM MgCl2, 1 mM EDTA, and 0.05% Tween 20.

[0182] Embodiment 177. The method of any one of embodiments 158 to 175, wherein the formulation consists of 40 mM HEPES, 102 mM NaCl, 5 mM KCl, 5 mM MgCl2, 1 mM EDTA, and 0.05% Tween-20 (pH 7.5).

[0183] Embodiment 178. The method of any one of embodiments 158 to 177, wherein the pH of the formulation is from about pH 5 to about pH 9, or from about pH 6 to about pH 8, or from about pH 7 to about pH 7.9, or about pH 7.5.

[0184] Embodiment 179. The method of any one of embodiments 158 to 178, wherein the aptamer comprises a 5-position modified pyrimidine.

[0185] Embodiment 180. The method of embodiment 179, wherein the 5-modified pyrimidine comprises a hydrophobic moiety at the 5-position of the pyrimidine.

[0186] Embodiment 181. The method of embodiment 180, wherein the hydrophobic moiety is selected from a naphthyl moiety, a phenyl moiety, a tyrosyl moiety, an indole moiety, and a morpholino moiety.

[0187] Embodiment 182. The method of embodiment 180 or 181, wherein the hydrophobic moiety is covalently attached to the 5-position of the base via a linker comprising a group selected from an amide linker, a carbonyl linker, a propynyl linker, an alkyne linker, an ester linker, a urea linker, a carbamate linker, a guanidine linker, an amidine linker, a sulfoxide linker, and a sulfone linker.

[0188] Embodiment 183. The method of any one of embodiments 158 to 182, wherein the complex is a non-covalent complex.

[0189] Embodiment 184. The method of any one of embodiments 158 to 183, wherein the at least one capture reagent is bound to a first solid support prior to contacting with the test sample, or the capture reagent is bound to a first solid support after contacting with the test sample.

[0190] Embodiment 185. The method of any one of embodiments 158 to 184, further comprising binding the complex to a first solid support via the capture reagent.

[0191] Embodiment 186. The method of embodiment 185, further comprising releasing the complex from the first solid support and binding the complex to a second solid support.

[0192] Embodiment 187. The method of embodiment 186, wherein the complex is bound to the second solid support via the protein.

[0193] Embodiment 188. The method of any one of embodiments 158 to 187, further comprising adding a competitor molecule to the test sample and / or adding a competitor molecule and diluting the test sample.

[0194] Embodiment 189. The method of embodiment 188, wherein the competitor molecule is a polyanion competitor.

[0195] Embodiment 190. The method of embodiment 189, wherein the polyanion competitor is selected from an oligonucleotide, a polydextran, DNA, heparin, and dNTPs.

[0196] Embodiment 191. The method of embodiment 190, wherein the polyanionic competitor is polydextran, and the polydextran is dextran sulfate.

[0197] Embodiment 192. The method of embodiment 190, wherein the polyanion competitor is an oligonucleotide, and the oligonucleotide contains one or more 5-position modified pyrimidines.

[0198] Embodiment 193. The method of any one of embodiments 158 to 192, wherein the biological sample is urine.

[0199] Embodiment 194. The method of any one of embodiments 158 to 192, wherein the biological sample is serum.

[0200] Embodiment 195. The method of any one of embodiments 100-130 or 158-194, wherein the buffer exchange is performed using gel filtration chromatography.

[0201] Embodiment 196. The method of any one of embodiments 100 to 130 or 158 to 195, further comprising measuring the protein concentration of the biological sample before performing the buffer exchange.

[0202] Embodiment 197. The method of embodiment 196, wherein the protein concentration of the biological sample is measured by an assay selected from the group consisting of a fluorescent readout assay, a bicinchoninic acid (BCA) assay, a microBCA assay, a Lowry assay, and an ELISA assay.

[0203] Embodiment 198. The method of embodiment 197, wherein the assay is a BCA assay or a microBCA assay.

[0204] Embodiment 199. The method of embodiment 198, wherein the assay comprises a bicinchoninic acid reagent or a bicinchonic acid reagent, and optionally an alkaline tartaric acid-carbonate buffer and / or a copper sulfate solution.

[0205] Embodiment 200. The method of embodiment 197, wherein the assay is a fluorescent readout assay.

[0206] Embodiment 201. The method of embodiment 200, wherein the fluorescent readout assay comprises a reagent selected from a merocyanine dye and epicocconone.

[0207] Embodiment 202. A method for preparing a biological sample for detecting a protein, comprising: performing buffer exchange on the biological sample with a formulation comprising a buffer, one or more salts, a chelating agent, and a non-ionic surfactant to generate a test sample; Quantifying the total protein concentration of the biological sample or the test sample; and adjusting the total protein concentration of the biological sample or the test sample, wherein the method does not include concentrating the total protein of the biological sample prior to performing the buffer exchange.

[0208] Embodiment 203. The method of embodiment 202, wherein the total protein concentration of the test sample is from about 2 μg / mL to about 60 μg / mL.

[0209] Embodiment 204. A method for preparing a biological sample for detecting a protein, comprising: Quantifying the total protein concentration of the test sample; adjusting the total protein concentration of the biological sample to about 2 μg / mL to about 60 μg / mL; and performing a buffer exchange on the biological sample with a formulation comprising a buffer, one or more salts, a chelating agent, and a non-ionic surfactant to generate a test sample.

[0210] Embodiment 205. The method of any one of embodiments 202 to 204, comprising performing buffer exchange on a plurality of biological samples to generate a plurality of test samples, wherein each test sample has approximately the same total protein concentration.

[0211] Embodiment 206. The method of any one of embodiments 202 to 205, comprising performing buffer exchange on a plurality of biological samples to generate a plurality of test samples, wherein each test sample has the same total protein concentration.

[0212] Embodiment 207. A method for preparing a biological sample for detecting a protein, comprising: Quantifying the protein concentration of the biological sample; if the total protein concentration of the biological sample is not in the range of about 2 μg / mL to about 60 μg / mL, adjusting the total protein concentration of the biological sample to about 2 μg / mL to about 60 μg / mL; and performing a buffer exchange on the biological sample with a formulation comprising a buffer, one or more salts, a chelating agent, and a non-ionic surfactant to generate a test sample.

[0213] Embodiment 208. The method of embodiment 207, wherein if the total protein concentration of the biological sample is not in the range of about 2 μg / mL to about 50 μg / mL, or about 2 μg / mL to about 45 μg / mL, or about 4 μg / mL to about 40 μg / mL, or about 8 μg / mL to about 40 μg / mL, or about 10 μg / mL to about 40 μg / mL, or about 10 μg / mL to about 30 μg / mL, or less than 40 μg / mL, or less than 30 μg / mL, or less than 20 μg / mL, or about 15 μg / mL, or about 10 μg / mL to about 20 μg / mL, the method comprises adjusting the total protein concentration of the biological sample to said range.

[0214] Embodiment 209. The method of any one of embodiments 202-208, wherein the one or more salts are each independently selected from a sodium salt, a potassium salt, and a magnesium salt.

[0215] Embodiment 210. The method of any one of embodiments 202-208, wherein the one or more salts include sodium salts, potassium salts, and magnesium salts.

[0216] Embodiment 211. The method of any one of embodiments 209 or 210, wherein the sodium salt is NaCl, the potassium salt is KCl, and the magnesium salt is MgCl2.

[0217] Embodiment 212. The method of embodiment 211, wherein the NaCl in the formulation is at a concentration of about 10 mM to about 500 mM, or about 50 mM to about 250 mM, or about 100 mM to about 200 mM, or about 102 mM.

[0218] Embodiment 213. The method of embodiment 211 or 212, wherein the KCl in the formulation is at a concentration of about 0.5 mM to about 30 mM, or about 1 mM to about 20 mM, or about 2 mM to about 15 mM, or about 4 mM to about 10 mM, or about 5 mM.

[0219] Embodiment 214. The method of any one of embodiments 211 to 213, wherein the MgCl2 in the formulation is at a concentration of about 0.5 mM to about 30 mM, or about 1 mM to about 20 mM, or about 2 mM to about 15 mM, or about 4 mM to about 10 mM, or about 5 mM.

[0220] Embodiment 215. The method of any one of embodiments 202 to 214, wherein the buffer is selected from HEPES, MES, bis-trismethane, ADA, ACES, bis-trispropane, PIPES, MOPSO, cholamine chloride, MOPS, BES, TES, DIPSO, MOB, acetamidoglycine, TAPSO, TEA, POPSO, HEPPSO, EPS, HEPPS, tricine, tris, glycinamide, glycylglycine, HEPBS, bicine, TAPS, AMPB, CHES, AMP, AMPSO, CAPSO, CAPS, and CABS.

[0221] Embodiment 216. The method of embodiment 215, wherein the buffer in the formulation is at a concentration of about 4 mM to about 400 mM, or about 10 mM to about 300 mM, or about 20 mM to about 200 mM, or about 30 mM to about 100 mM, or 35 mM to about 50 mM, or about 40 mM.

[0222] Embodiment 217. The method of any one of embodiments 202 to 216, wherein the chelating agent is selected from EDTA, EGTA, DTPA, BAPTA, DMPS, and ALA.

[0223] Embodiment 218. The method of embodiment 217, wherein the chelating agent in the formulation is at a concentration of about 0.1 mM to about 10 mM, or about 0.5 mM to about 5 mM, or about 1 mM.

[0224] Embodiment 219. The method of any one of embodiments 202 to 218, wherein the nonionic surfactant is selected from polyoxyethylene (20) sorbitan monolaurate (Tween-20), polyoxyethylene (40) sorbitan monolaurate (Tween-40), and polyoxyethylene (80) sorbitan monolaurate (Tween-80).

[0225] Embodiment 220. The method of embodiment 219, wherein the nonionic surfactant is from about 0.01% to about 1% of the formulation, or from about 0.02% to about 0.5% of the formulation, or from about 0.03% to about 0.1% of the formulation, or from about 0.04% to about 0.08% of the formulation, or about 0.05% of the formulation, or about 0.1% of the formulation (volume to volume).

[0226] Embodiment 221. The method of any one of embodiments 202 to 220, wherein the total protein concentration of the test sample is from about 2 μg / mL to about 50 μg / mL, or from about 2 μg / mL to about 45 μg / mL, or from about 4 μg / mL to about 40 μg / mL, or from about 8 μg / mL to about 40 μg / mL, or from about 10 μg / mL to about 40 μg / mL, or from about 10 μg / mL to about 30 μg / mL, or less than 40 μg / mL, or less than 30 μg / mL, or less than 20 μg / mL, or about 15 μg / mL, or from about 10 μg / mL to about 20 μg / mL.

[0227] Embodiment 222. The method of any one of embodiments 203 to 221, wherein the total protein concentration of the test sample or the biological sample is quantified by an assay selected from the group consisting of a fluorescent readout assay, a Bradford assay, a bicinchoninic acid (BCA) assay, a Lowry assay, and an ELISA assay.

[0228] Embodiment 223. The method of embodiment 222, wherein the fluorescent readout assay comprises a reagent selected from a merocyanine dye and epicocconone.

[0229] Embodiment 224. The method of any one of embodiments 202 to 223, wherein the formulation comprises 40 mM HEPES, 102 mM NaCl, 5 mM KCl, 5 mM MgCl2, 1 mM EDTA, and 0.05% Tween-20.

[0230] Embodiment 225. The method of any one of embodiments 202 to 224, wherein the pH of the formulation is from about pH 5 to about pH 9, or from about pH 6 to about pH 8, or from about pH 7 to about pH 7.9, or about pH 7.5.

[0231] Embodiment 226. The method of any one of embodiments 202 to 225, wherein the formulation consists of 40 mM HEPES, 102 mM NaCl, 5 mM KCl, 5 mM MgCl2, 1 mM EDTA, and 0.05% Tween-20, and has a pH of 7.5.

[0232] Embodiment 227. The method of any one of embodiments 202 to 226, wherein the buffer exchange is not performed by ultrafiltration.

[0233] Embodiment 228. The method of any one of embodiments 202 to 227, wherein the buffer exchange is performed by gel filtration chromatography.

[0234] Embodiment 229. The method of any one of embodiments 202 to 228, wherein the biological sample is a urine sample.

[0235] Embodiment 230. The method of any one of embodiments 202 to 228, wherein the biological sample is a serum sample.

[0236] Embodiment 231. The method of any one of embodiments 202 to 230, wherein the method does not include concentrating the biological sample.

[0237] Embodiment 232. A method for preparing a biological sample for detecting a protein, comprising: performing buffer exchange on the biological sample with a formulation comprising a buffer, one or more salts, a chelating agent, and a non-ionic surfactant to generate a test sample; Quantifying the total protein concentration of the biological sample or the test sample; and adjusting a total protein concentration of the biological sample or the test sample, wherein the method does not include concentrating the total protein of the biological sample prior to performing the buffer exchange; Optionally, (i) the total protein concentration is adjusted before performing the buffer exchange, or (ii) the total protein concentration is adjusted after performing the buffer exchange.

[0238] The method of embodiment 232 may further include any applicable features of the preceding embodiments, mutatis mutandis. Any of the preceding embodiments may be performed in the order that total protein adjustment (and measurement, if applicable) is performed before or after buffer exchange, and may be modified, if necessary, to perform total protein adjustment (and measurement, if applicable) before or after buffer exchange. Any method disclosed or claimed herein should be construed as including both orders, i.e., total protein adjustment (and measurement, if applicable) is performed before or after buffer exchange, unless the language clearly requires otherwise.

[0239] A certain way In some embodiments, the methods disclosed herein facilitate standardization of total protein concentration in a biological or test sample while limiting sample-to-sample variability in analyte measurements and expanding the linear range of the analyte in the sample. In some embodiments, the methods include performing a buffer exchange. In some such embodiments, the buffer exchange allows for both adjustment of the total protein concentration of the biological sample and control of the salt concentration of the biological sample. Variations in salt concentration in a biological sample can cause significant variability in the measurement of an analyte within a sample. The ability to adjust the total protein concentration and salt concentration limits such sample-to-sample variability. In some embodiments, the methods disclosed herein expand the linear range of analyte concentration, allowing for more reliable measurement of the analyte than measurement of the analyte in a biological or test sample that has not been buffer exchanged.

[0240] In some embodiments, the methods herein include quantifying the total protein concentration of a biological sample. In some such embodiments, the total protein concentration of a biological sample is quantified before buffer exchange of the biological sample. In some embodiments, the methods herein include quantifying the total protein concentration of a test sample. In some such embodiments, the total protein concentration of a test sample is quantified after buffer exchange of the biological sample. In some embodiments, the total protein concentration is quantified using a fluorescent readout assay. In some embodiments, the total protein concentration is quantified using a BCA assay. In some embodiments, the total protein concentration of a test sample is quantified to be at a certain value or within a certain range and is not adjusted. In some embodiments, the total protein concentration is quantified to be not at a certain value or outside a certain range and is adjusted. In some embodiments, the range is not the same in every type of assay used to quantify the total protein concentration. In some embodiments, the methods herein include generating a plurality of test samples from a plurality of biological samples, and the total protein concentrations of some test samples or some biological samples are adjusted and the total protein concentrations of other test samples or other biological samples are not adjusted.

[0241] In some embodiments, the methods herein do not include ultrafiltration or concentration of total protein of the biological sample, hi some such embodiments, buffer exchange is performed using gel filtration chromatography.

[0242] A particular test sample In some embodiments, the buffer exchanged test samples disclosed herein contain a total protein concentration of less than about 60 μg / mL, between 60 and 100 μg / mL, or between 70 and 100 μg / mL. Some such embodiments contain HEPES, NaCl, KCl, MgCl2, Tween, and EDTA.

[0243] Sample-to-sample variability in biological samples The variability of total protein concentrations from biological samples (e.g., random, untimed "spot" urine specimens) poses a challenge for proteomic analysis. The normal range of total protein in spot urine samples is approximately 1-14 mg / dL, with approximately 50-80 mg / day excreted at rest. Urinary protein concentrations tend to increase with age, exercise, and orthostatic posture. In nephrotic syndrome, total protein excretion can exceed 3.5 g / day.

[0244] Variability in protein concentration between urine samples has been reported by Thomas et al. (Thomas, C. E., Sexton W., Benson K., Sutphen R., and J. Koomen. (2010). Urine collection and processing for protein biomarker discovery and quantification. Cancer Epidemiol Biomarkers Prev 19(4):953-959). In this study, all urine voids were collected from one individual over 10 consecutive days. Total protein concentrations for each were quantified and found to vary over 100-fold between samples. The lowest coefficients of variation (CV) were observed for the 24-hour pool (39%) and the first morning void (41%). The highest CVs were observed for the second void of the day (54%) and all spot collections (61%).

[0245] Characterization of the composition and concentration of human urine. Prepared for the National Aeronautics and Space Administration by McDonnell Douglas Astronautics Company (Huntington Beach, CA), it was shown that 68 small molecule components make up 99% of the soluble solids in urine. These 68 molecules total about 36.8 grams per liter in a typical urine sample. The top 10 molecules, ranked from highest to lowest mass per unit volume, are urea, chloride, sodium, potassium, creatinine, inorganic sulfur, hippuric acid, phosphorus, citrate, and glucuronic acid. However, there is considerable variability in the absolute concentrations of any of these 68 components between urine specimens. Table 1 below summarizes some of the differences observed between several urine samples. [Table 1]

[0246] The variability of urinary total protein concentration from subject-to-subject comparisons and from sample-to-sample comparisons from the same subject, as well as variability in pH, salt concentration, and other components, makes urine a challenging matrix for proteomic measurements in biomarker discovery and diagnostics. This is because protein level measurements can be affected by the presence, and specifically the concentrations, of these urinary components (e.g., salts and other solids). Effectively, in samples with different levels of these components but the same concentration of protein, when protein levels are assayed, different protein measurements will be obtained based on the levels of the different components in the sample.

[0247] The foregoing and other objects, features, and advantages of the present invention will become more apparent from the following detailed description which proceeds with reference to the accompanying drawings. [Brief description of the drawings]

[0248] [Figure 1]A and B show the change in analyte measurements as a function of NaCl concentration. Both A and B show the same data presented differently. A shows the additional NaCl (mM) concentration to the SB17 buffer formulation (starting at 102 mM NaCl) on the x-axis and the Log2 fold change in RFU (relative fluorescence units) on the y-axis, which correlates with the amount of analyte present in the sample compared to the control buffer condition (SB17 buffer). B shows the additional NaCl (mM) concentration to the SB17 buffer (starting at 102 mM NaCl) on the x-axis and the percent of analyte that increases or decreases as a function of NaCl concentration on the y-axis. [Diagram 2] A and B show the change in analyte measurements as a function of MgCl2 concentration. Both A and B show the same data presented differently. A shows the additional MgCl2 (mM) concentration to SB17 buffer formulation (starting with 5 mM MgCl2) on the x-axis and the Log2 fold change in RFU (relative fluorescence units) on the y-axis, which correlates with the amount of analyte present in the sample compared to the control buffer condition (SB17 buffer). B shows the additional MgCl2 (mM) concentration to SB17 buffer (starting with 5 mM MgCl2) on the x-axis and the percent of analyte that increases or decreases as a function of MgCl2 concentration on the y-axis. [Diagram 3] A and B show the change in analyte measurements as a function of urea concentration. Both A and B show the same data presented differently. A shows the addition of urea (mM) concentration (starting at 0 mM) to the SB17 buffer formulation on the x-axis and the Log2 fold change in RFU (relative fluorescence units) on the y-axis, which correlates with the amount of analyte present in the sample compared to the control buffer condition (SB17 buffer). B shows the addition of urea (mM) concentration (starting at 0 mM) to the SB17 buffer on the x-axis and the percent of analyte that increases or decreases as a function of urea concentration on the y-axis. [Figure 4]A visualization of the linear range for the 2,298 samples measured in this titration is shown, with the black bars spanning the lower and upper limits of the linear range for each analyte (x-axis) as a function of the total protein concentration (μg / mL) of the sample (y-axis). [Diagram 5] The percentage of 2,298 analytes within the linear range (x-axis) is shown as a function of the total protein concentration (μg / mL) of the sample (y-axis). [Figure 6] Figure 1 shows the effect of sample buffer exchange on analyte linearity in a proteomics assay. Two different analytes were measured in the assay. One sample was subjected to buffer exchange and the other sample was not subjected to buffer exchange (control). A titration based on the total protein concentration of the sample (x-axis) was performed and the level of the analyte measured by RFU was plotted on a Log2 scale (y-axis). For the ALDO protein analyte in the control sample, a linear range is observed at relatively low total protein concentrations, but the RFU measurements decrease above approximately 30 μg / mL. When the sample is subjected to buffer exchange, the linear range of the same analyte (ALDO) extends well up to a total protein concentration of 130 μg / mL for the sample. Similarly, for the ANXA4 protein analyte in the control sample, a linear range is observed at relatively low total protein concentrations, but the RFU measurements plateau above approximately 16 μg / mL. When the sample is subjected to buffer exchange, the linear range of the same analyte (ANXA4) extends well up to a total protein concentration of 130 μg / mL of the sample. [Figure 7A] The linearity of the analytes measured in the assay is plotted as a function of the total protein concentration of the sample. Both Figures 7A and 7B show the same data presented differently. The analytes are classified as being within the linear range, above the linear range, or below the linear range. Figure 7A plots the number of analytes and their classification (above, below, or within the linear range) (y-axis) as a function of the total protein concentration of the sample. [Figure 7B]7A and 7B are graphs of the linearity of the analytes measured in the assay as a function of the total protein concentration of the sample. Both Figures 7A and 7B show the same data presented differently. The analytes are classified as being within the linear range, above the linear range, or below the linear range. Figure 7B is a graph of the percentage of analytes and their classification (above, below, or within the linear range) (y-axis) as a function of the total protein concentration of the sample. [Figure 8] Certain exemplary 5-position modified uridines and cytidines that can be incorporated into aptamers. [Figure 9] Certain exemplary modifications that may be present at the 5-position of uridine are shown. The chemical structure of the C-5 modification includes an exemplary amide bond linking the modification to the 5-position of uridine. The 5-position moieties shown include a phenyl moiety (e.g., Bn, PE, and PP), a naphthyl moiety (e.g., Nap, 2Nap, NE), a butyl moiety (e.g., iBu), a fluorobenzyl moiety (e.g., FBn), a tyrosyl moiety (e.g., Tyr), a 3,4-methylenedioxybenzyl moiety (e.g., MBn), a morpholino moiety (e.g., MOE), a benzofuranyl moiety (e.g., BF), an indole moiety (e.g., Trp), and a hydroxypropyl moiety (e.g., Thr). [Figure 10] Certain exemplary modifications may be present at the 5-position of cytidine. The chemical structures of the C-5 modifications include an exemplary amide bond that connects the modification to the 5-position of cytidine. The 5-position moieties shown include phenyl moieties (e.g., Bn, PE, and PP), naphthyl moieties (e.g., Nap, 2Nap, NE, and 2NE), and tyrosyl moieties (e.g., Tyr). [Figure 11] 1 shows a graph of the number of analytes in a urine sample that are within the linear range as measured by an aptamer-based proteomics assay as a function of total protein concentration as measured by the microBCA assay. [Figure 12A]The number of analytes in urine samples that are within the linear range, as measured by the aptamer-based proteomics assay, is shown as a function of total protein concentration from 1 to 150 μg / mL, as measured by the microBCA assay. Figure 12A shows the results for 10 individual urine samples. [Figure 12B] The average number of analytes in urine samples that are within the linear range, as measured by the aptamer-based proteomics assay, is shown as a function of total protein concentration from 1 to 150 μg / mL, as measured by the microBCA assay. Figure 12B shows the combined results for the same 10 individual urine samples. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0249] I. Terms and Methods While the present invention will be described in connection with specific exemplary embodiments, it will be understood that the invention is not limited to these embodiments, as defined by the claims.

[0250] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention, and the present invention is in no way limited to the methods and materials described.

[0251] Unless otherwise defined, technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9), Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9), and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). Although any methods, devices, and materials similar or equivalent to those described herein may be used in the practice of the present invention, certain specific methods, devices, and materials are described herein.

[0252] All publications, published patent documents, and patent applications cited in this specification are herein incorporated by reference to the same extent as if each individual publication, published patent document, or patent application was specifically and individually indicated to be incorporated by reference.

[0253] As used in this application, including the appended claims, the singular forms "a," "an," and "the" include the plural forms and may be used interchangeably with "at least one" and "one or more," unless the content clearly dictates otherwise. Thus, reference to an "aptamer" includes a mixture of aptamers, reference to a "probe" includes a mixture of probes, and so forth.

[0254] As used herein, the terms "comprise," "comprising," "include," "including," "contain," "containing," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, product-by-process, or composition that comprises, includes, or contains an element or list of elements may include other elements not expressly listed.

[0255] Moreover, it should be understood that all base or amino acid sizes and all molecular weight or mass values ​​given for the nucleic acids or polypeptides are approximate and are provided for purposes of illustration.

[0256] Additionally, ranges provided herein should be understood to be shorthand notations for all values ​​within that range, for example, a range of 1 to 50 should be understood to include any number, combination of numbers, or subrange from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, and any fraction thereof, unless the content clearly dictates otherwise.

[0257] Any concentration range, percentage range, ratio range, or integer range should be understood to include both endpoints, any integer within the recited range, and fractions thereof, where appropriate (e.g., tenths and hundredths of integers), unless otherwise indicated. Additionally, any numerical range for any physical feature described herein, e.g., polymer subunits, size, or thickness, should be understood to include any integer within the recited range, unless otherwise indicated.

[0258] The use of the alternative (eg, "or") should be understood to mean either one, both, or any combination thereof of the alternatives.

[0259] In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terminology are provided below.

[0260] About: The term "about" in the context of a numerical value or range means, unless otherwise indicated, plus or minus 10% of the recited numerical value or range.

[0261] Antibody: The term "antibody" refers to full-length antibodies of any species that retain the ability to bind antigen, as well as fragments and derivatives of such antibodies, including Fab fragments, F(ab')2 fragments, single chain antibodies, Fv fragments, and single chain Fv fragments. The term "antibody" also includes synthetically derived antibodies, such as phage display derived antibodies and fragments, affibodies, and nanobodies.

[0262] Aptamer: As used herein, "aptamer" refers to a nucleic acid having a specific binding affinity for a target molecule, where the binding of the aptamer to the target molecule does not involve Watson-Crick base pairing. Although it is recognized that affinity interactions are a matter of degree, in this context, the "specific binding affinity" of an aptamer to its target means that the aptamer binds to its target with a degree of affinity that is generally much higher than the aptamer binds to other components in the test sample. An "aptamer" is a set of copies of one type or species of nucleic acid molecule having a particular nucleotide sequence. An aptamer can contain any suitable number of nucleotides, including any number of chemically modified nucleotides. A plurality of "aptamers" refers to a set of two or more such molecules. Different aptamers can have either the same or different numbers of nucleotides. Aptamers can be DNA or RNA or chemically modified nucleic acids, and can be single-stranded, double-stranded, or contain both single-stranded and double-stranded regions, and can contain higher order structures. Aptamers may also include photoreactive or chemically reactive functional groups that allow them to be covalently bound to their corresponding targets. Any aptamer method disclosed herein may include the use of two or more aptamers that specifically bind to the same target molecule. As will be further described below, aptamers may include tags. When an aptamer includes a tag, it is not necessary that all copies of the aptamer have the same tag. Furthermore, when different aptamers each include a tag, each different aptamer can have either the same tag or different tags.

[0263] BCA Assay or Bicinchoninic Acid Assay or Bicinchoninic Acid Assay: As used herein, "BCA assay", "bicinchoninic acid assay" and "bicinchonic acid assay" are used interchangeably and refer to any assay, kit, reagent, or composition containing bicinchoninic acid that can be used to quantify protein concentration in a sample. BCA assays include, but are not limited to, microBCA assays. An exemplary microBCA assay is described in Example 6.

[0264] Biological sample or biological matrix: As used herein, "biological sample" and "biological matrix" refer to any material, solution, or mixture obtained from an organism. Biological samples or biological matrices include blood (including whole blood, white blood cells, peripheral blood mononuclear cells, plasma, and serum), sputum, exhaled breath, urine, semen, saliva, cerebrospinal fluid, amniotic fluid, glandular fluid, lymphatic fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, cells, cell extracts, and cerebrospinal fluid. Biological samples or biological matrices also include all of the above fractions isolated in an experiment. The terms "biological sample" and "biological matrix" also include materials, solutions, or mixtures that include homogenized solid materials (e.g., fecal samples, tissue samples, or tissue biopsies). The terms "biological sample" and "biological matrix" also include materials, solutions, or mixtures that are derived from cell lines, tissue cultures, cell cultures, bacterial cultures, viral cultures, or acellular biological systems (e.g., IVTT).

[0265] Level: As used herein, "target protein level," "analyte level," and "level" refer to a measurement made using any analytical method for detecting an analyte (e.g., a target protein) in a biological sample, which indicates the presence, absence, absolute amount or concentration, relative amount or concentration, titer, level, expression level, ratio of measured levels, etc., of, or corresponding to, the analyte in the biological sample. The exact nature of the "level" depends on the specific design and components of the particular analytical method used to detect the analyte.

[0266] Buffering Agent: As used herein, "buffering agent" refers to a chemical or combination of chemicals that can adjust and / or maintain the pH of a solution in which it is dissolved. In some embodiments, the buffering agent is a weak acid. In some embodiments, the buffering agent is a weak base. In some embodiments, the buffering agent is a weak acid and a weak base. In some embodiments, the buffering agent is a salt. In some such embodiments, the buffering agent is a salt of a weak acid and a weak base. In some embodiments, the buffering agent is zwitterionic.In some embodiments, the buffering agent is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), bis-trismethane, N-(2-acetamido)iminodiacetic acid (ADA), N-(carbamoylmethyl)-2-aminoethanesulfonic acid (ACES), bis-trispropane, piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO), cholamine chloride, 3-(N-morpholino)propanesulfonic acid ... Pansulfonic acid (MOPS), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES), 3-(N,N-bis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid (DIPSO), 4-(N-morpholino)butanesulfonic acid (MOB), acetamidoglycine, 2-hydroxy-3-[tris(hydroxymethyl)methylamino]-1-propanesulfonic acid (TAPSO), N,N-diethylethanamine (TEA) , piperazine-N,N'-bis(2-hydroxypropanesulfonic acid) (POPSO), 4-(2-hydroxyethyl)piperazine-1-(2-hydroxypropane-3-sulfonic acid) hydrate (HEPPSO), 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS), tricine, tris, glycinamide, glycylglycine, N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid) (HEPBS), bicine, N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid (TAPS), 2-(cyclohexylamino)ethanesulfonic acid, 2-(N-cyclohexylamino)ethanesulfonic acid (CHES), β-aminoisobutyl alcohol hydrochloride (AMP), N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid (AMPSO), N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid) (CAPSO), 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), and 4-(cyclohexylamino)-1-butanesulfonic acid (CABS).

[0267] Buffer exchanged sample: As used herein, a "buffer exchanged sample" refers to a sample that has undergone a buffer exchange step. In some embodiments, the buffer exchange step involves changing the identity and / or concentration of one or more buffer components. Such buffer components include, but are not limited to, buffers, salts, chelating agents, and surfactants. In some embodiments, the total protein concentration of the buffer exchanged sample is the same as the total protein concentration of the sample prior to performing the buffer exchange step. In some embodiments, the buffer exchanged sample is a buffer exchanged biological sample.

[0268] C-5 modified pyrimidine: As used herein, the term "C-5 modified pyrimidine" refers to a pyrimidine having a modification at the C-5 position. Examples of C-5 modified pyrimidines include those described in U.S. Patent Nos. 5,719,273, 5,945,527, 9,163,056, and Dellafiore et al., 2016, Front. Chem., 4:18. Examples of C-5 modifications include replacement of deoxyuridine at the C-5 position with a substituent independently selected from benzylcarboxamide (alternatively benzylaminocarbonyl) (Bn), naphthylmethylcarboxamide (alternatively naphthylmethylaminocarbonyl) (Nap), tryptaminocarboxamide (alternatively tryptaminocarbonyl) (Trp), phenethylcarboxamide (alternatively phenethylaminocarbonyl) (Pe), thiophenylmethylcarboxamide (alternatively thiophenylmethylaminocarbonyl) (Th), and isobutylcarboxamide (alternatively isobutylaminocarbonyl) (iBu), as exemplified immediately below.

[0269] Chemical modifications of C-5 modified pyrimidines can also be combined, alone or in any combination, with 2' sugar modifications, modifications with exocyclic amines, and 4-thiouridine substitutions.

[0270] Representative C-5 modified pyrimidines include 5-(N-benzylcarboxamido)-2'-deoxyuridine (BndU), 5-(N-benzylcarboxamido)-2'-O-methyluridine, 5-(N-benzylcarboxamido)-2'-fluorouridine, 5-(N-isobutylcarboxamido)-2'-deoxyuridine (iBudU), 5-(N-isobutylcarboxamido)-2'-O-methyluridine, 5-(N-phenethylcarboxamido)-2'-deoxyuridine (PedU), 5-(N-thiophenylmethylcarboxamido)-2'-deoxyuridine (ThdU), 5-(N-isobutylcarboxamido)-2'-fluorouridine, 5-(N-tryptaminocarboxamido)-2'-deoxyuridine (Tht ... Examples of such uridine derivatives include 5-(N-naphthylmethylcarboxamido)-2'-O-methyluridine, 5-(N-naphthylmethylcarboxamido)-2'-fluorouridine, 5-(N-[1-(3-trimethylammonium)propyl]carboxamido)-2'-deoxyuridine chloride, 5-(N-naphthylmethylcarboxamido)-2'-deoxyuridine (NapdU), 5-(N-naphthylmethylcarboxamido)-2'-O-methyluridine, 5-(N-naphthylmethylcarboxamido)-2'-fluorouridine, and 5-(N-[1-(2,3-dihydroxypropyl)]carboxamido)-2'-deoxyuridine).

[0271] Nucleotides can be modified before or after the synthesis of oligonucleotide.The sequence of nucleotides in oligonucleotide can be interrupted by one or more non-nucleotide components.Modified oligonucleotides can be further modified after polymerization, for example, by conjugation with any suitable labeling component.

[0272] As used herein, the term "at least one pyrimidine," when referring to modification of a nucleic acid, refers to one, some, or all of the pyrimidines in a nucleic acid, indicating that any or all occurrences of any or all C, T, or U in a nucleic acid may be modified or unmodified.

[0273] Capture Reagent: As used herein, a "capture agent" or "capture reagent" refers to a molecule capable of specifically binding to an analyte (e.g., a biomarker, a protein, and / or a peptide). A "target protein capture reagent" refers to a molecule capable of specifically binding to a target protein. Non-limiting exemplary capture reagents include aptamers, antibodies, adnectins, ankyrins, other antibody mimetics and other protein scaffolds, autoantibodies, chimeras, small molecules, nucleic acids, lectins, ligand-binding receptors, imprinted polymers, avimers, peptidomimetics, hormone receptors, cytokine receptors, synthetic receptors, as well as modifications or fragments of any of the above capture reagents. In some embodiments, the capture reagent is selected from an aptamer and an antibody.

[0274] Chelating Agent: As used herein, "chelating agent" refers to a chemical that can form multiple bonds to a single metal ion. In some embodiments, the chelating agent is selected from ethylenediaminetetraacetic acid (EDTA), ethyleneglycoltetraacetic acid (EGTA), diethylenetriaminepentaacetic acid (DTPA), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), 2,3-dimercaptopropanesulfonic acid (DMPS).

[0275] Control level: "Control level" of a target molecule refers to the level of the target molecule in the same sample type from an individual who does not have a disease or condition, or from an individual who is not suspected or at risk of having a disease or condition, or from an individual who has a non-progressive form of the disease or condition. Furthermore, "control level" can refer to a reference material based on the average, or what is considered within normal or healthy parameters. "Control level" can also refer to a previously adopted reference level, which is used to compare with the subsequent measured or detected level of the target. For example, the level of the target can be detected at time A, and then at time B, where time B is after time A. In a more specific example, time point A is considered to be 0 hours or day 0, and time point B is considered to be how many minutes after time point A (e.g., 10, 20, 30, 40, 50, or 60 minutes after time point A), how many hours after (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours after time point A), or how many days after (e.g., 1, 2, 3, 4, 5, 6, or 7 days after time point A). or 7 days later), weeks later (e.g., 1, 2, 3, or 4 weeks after time point A), months later (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after time point A), and even years later (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 years after time point A). The "control level" of the target molecule does not need to be quantified every time the method of the present invention is performed, but may be a pre-quantified level used as a reference or threshold to determine whether the level of a particular sample is higher or lower than a normal level.

[0276] Corresponding correlation: "corresponding correlation" or "corresponding correlation coefficient" measures the agreement between two continuous variables X and Y (e.g., predicted, estimated or determined, and actual values). "Corresponding correlation" evaluates the extent to which a pair lies on a 45-degree line, and includes a measure of accuracy and precision (or "Lin's agreement"). Further information can be found in Lin, Biometric, Vol. 45, No. 1 (March, 1989), 255-268 (incorporated herein by reference). Other methods for quantifying correlation that can be used herein include, but are not limited to, Pearson correlation coefficient, paired t-test, least squares analysis of slope (=1) and intercept (=0), coefficient of variation, and intraclass correlation coefficient. In certain embodiments, the corresponding correlation is quantified by a method selected from Lin's agreement, Pearson correlation coefficient, paired t-test, least squares analysis of slope (=1) and intercept (=0), coefficient of variation, and intraclass correlation coefficient.

[0277] Detection: As used herein, "detecting" or "quantifying" includes the use of both an instrument used to observe and record a signal corresponding to an analyte level as well as the materials necessary to generate that signal. In various embodiments, the level is detected using any suitable method, including fluorescence, chemiluminescence, surface plasmon resonance, surface acoustic waves, mass spectrometry, infrared spectroscopy, Raman spectroscopy, atomic force microscopy, scanning tunneling microscopy, electrochemical detection, nuclear magnetic resonance, quantum dots, and the like.

[0278] Diagnosis: "diagnose", "diagnosing", "diagnosis" and variations thereof refer to detecting, quantifying, or recognizing the health state or condition of an individual based on one or more signs, symptoms, data, or other information associated with that individual. An individual's health state may be diagnosed as healthy / normal (i.e., a diagnosis of the absence of a disease or condition) or diseased / abnormal (i.e., a diagnosis of the presence or evaluation of the characteristics of a disease or condition). The terms "diagnose", "diagnosing", "diagnosis" and the like, with respect to a particular disease or condition, encompass early detection of disease, characterization or classification of disease, detection of disease progression, remission, or recurrence, and detection of disease response following administration of a treatment or therapy to an individual.

[0279] Dilution: "Dilution", "dilution series", and variations thereof encompass several different types of dilution, including but not limited to stepwise dilution, serial dilution, and combinations thereof. As an example of a serial dilution, if the dilution factor is 1000 (1:1000 dilution), the user may first perform a 1:10 dilution (dilution factor 10), then a 1:100 dilution (dilution factor 100), and diluting from the 1:10 dilution with 1 part solute and 99 parts diluent to get to a dilution factor of 1000 or a 1:1000 dilution of solute. Serial dilution involves a series of stepwise dilutions, each with the same dilution factor, where the diluted material from the previous step is used to make the subsequent dilutions. As an example of a serial dilution, to make a 5-point 1:2 serial dilution, one would need to use 1 part solute and combine with 1 part diluent to make the first dilution (point 1 of 5), then use 1 part solute from the first dilution and combine with 1 part diluent to make the second dilution (point 2 of 5), and so on until the 5th consecutive serial dilution is reached.

[0280] Dilution Factor: "Dilution factor" refers to the ratio of parts of solute to parts of diluent. For example, a dilution factor of 2 means a 1:2 dilution, with 1 part solute and 1 part diluent for a total of 2 parts. A dilution factor of 10 means a 1:10 dilution, with 1 part solute and 9 parts diluent for a total of 10 parts.

[0281] Evaluate: "Evaluate," "assessing," "evaluation," and variations thereof encompass both "diagnosis" and "prognosis," and include the judgment or prediction of the future course of a disease or condition in an individual who has a disease or condition, as well as the judgment and prediction of the likelihood of a disease or condition occurring in an individual who has not been previously diagnosed with the disease or condition, and the judgment or prediction of the likelihood of a disease or condition recurring in an individual who is in remission or thought to have been cured of the disease. The term "evaluating" also encompasses evaluating an individual's response to a therapy, e.g., predicting whether an individual is likely to respond favorably or unlikely to respond to a therapeutic agent (or, for example, experience toxicity or other undesirable side effects), selecting a therapeutic agent for administration to the individual, or monitoring or determining an individual's response to a therapy that has been or is being administered to the individual.

[0282] Individual: As used herein, "individual" and "subject" are used interchangeably to refer to a test subject or patient. An individual may be a mammal or a non-mammal. In various embodiments, the individual is a mammal. A mammalian individual may be a human or a non-human. In various embodiments, the individual is a human. A healthy or normal individual is one in which the disease or condition of interest is not detected by conventional diagnostic methods.

[0283] Linear Regression: As used herein, the term "linear regression" refers to an approach for modeling the relationship between a scalar dependent variable y and one or more explanatory variables, denoted x. In the case of one explanatory variable, it is called simple linear regression. For multiple explanatory variables, it is called multiple linear regression. In general, linear regression can be used to fit a predictive model to an observed data set of y and x values. After developing such a model, if a value of x is added without the accompanying value of y, the fitted model can be used to predict the value of y.

[0284] Marker: As used herein, "marker" and "biomarker" are used interchangeably and refer to a target molecule (or analyte) that indicates or is indicative of a normal or abnormal process in an individual, or of a disease or other condition in an individual. More specifically, a "marker" or "biomarker" is an anatomical, physiological, biochemical, or molecular parameter that is associated with the presence of a particular physiological state or process, whether normal or abnormal, and if abnormal, whether chronic or acute. Biomarkers are detectable and measurable by a variety of methods, including laboratory assays and medical imaging. In some embodiments, the biomarker is a target protein.

[0285] Modification: As used herein, the terms "modify", "modified", "modification", and any variations thereof, when used in relation to an oligonucleotide, mean that at least one of the four constituent nucleotide bases (i.e., A, G, T / U, and C) of the oligonucleotide is an analog or ester of a natural nucleotide. In some embodiments, the modified nucleotide confers nuclease resistance to the oligonucleotide. In some embodiments, the modified nucleotide provides predominantly hydrophobic interactions between the aptamer and the protein target, resulting in high binding efficiency and stable co-crystal complexes. A pyrimidine with a substitution at the C-5 position is an example of a modified nucleotide. Modifications can include backbone modifications, methylation, rare base pairing combinations, such as the isobases isocytidine and isoguanidine. Modifications can also include 3' and 5' modifications, such as capping. Other modifications can include substitution of one or more natural nucleotides with analogs, internucleotide modifications such as uncharged (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and charged (e.g., phosphorothioates, phosphorodithioates, etc.) linkages, intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, boron, metal oxides, etc.), alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.). Additionally, any of the hydroxyl groups normally present on the sugar of the nucleotide can be replaced with phosphonate or phosphate groups, protected by standard protecting groups, or activated to provide for additional linkages to additional nucleotides or to solid supports.The 5' and 3' terminal OH groups may be phosphorylated or substituted with amines, organic capping group moieties of about 1 to about 20 carbon atoms, polyethylene glycol (PEG) polymers (in some embodiments, in the range of about 10 to about 80 kDa), PEG polymers (in some embodiments, in the range of about 20 to about 60 kDa), or other hydrophilic or hydrophobic biopolymers or synthetic polymers. In one embodiment, the modification is at the C-5 position of the pyrimidine. Such modifications may be made via an amide bond directly at the C-5 position or by other types of bonds.

[0286] Polynucleotides can also include analogous forms of ribose or deoxyribose sugars that are well known in the art, including 2'-O-methyl-, 2'-O-allyl, 2'-fluoro or 2'-azido-ribose, carbocyclic analogs, α-anomeric sugars, epimeric sugars (e.g., arabinose, xylose, or lyxose), pyranose sugars, furanose sugars, sedoheptulose, acrylic acid analogs, and abasic nucleoside analogs (e.g., methyl riboside).As noted above, one or more phosphodiester linkages may be replaced with alternative linking groups. Such alternative linking groups include embodiments in which phosphate is replaced with P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), where each R or R' is independently H or substituted or unsubstituted alkyl (1-20C) (optionally containing ether (-O-) linkages), aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all linkages in a polynucleotide need be identical. Substitution of similar forms of sugars, purines, and pyrimidines may be advantageous in the design of the final product, as may alternative backbone structures, such as, for example, polyamide backbones.

[0287] Nucleic acid: As used herein, "nucleic acid", "oligonucleotide", and "polynucleotide" are used interchangeably to refer to polymers of nucleotides, including DNA, RNA, DNA / RNA hybrids, and modifications of these types of nucleic acids, oligonucleotides, and polynucleotides, including various entities or moieties attached to the nucleotide units at any position. The terms "polynucleotide", "oligonucleotide", and "nucleic acid" include triple helix molecules in addition to double-stranded or single-stranded molecules. Nucleic acid, oligonucleotide, and polynucleotide are broader terms than the term aptamer, and thus the terms nucleic acid, oligonucleotide, and polynucleotide include polymers of nucleotides that are aptamers, but the terms nucleic acid, oligonucleotide, and polynucleotide are not limited to aptamers.

[0288] Least Squares: As used herein, "Least Squares" or "OLS" or "Linear Least Squares" refers to a method for estimating unknown parameters in a linear regression model. The method minimizes the sum of squared vertical distances between the observed responses in a data set and the responses predicted by a linear fit. The resulting estimator can be expressed as a single simple equation, especially when there is a single regressor on the right hand side.

[0289] Prognosis: "Predict," "prognosis," "prognosis," and variations thereof refer to a prediction of the future course of a disease or condition in an individual having the disease or condition (e.g., a prediction of how long a patient will survive), and such terms encompass assessment of disease response during and / or after administration of a treatment or therapy to an individual.

[0290] SELEX: The terms "SELEX" and "SELEX method" are used interchangeably herein and generally refer to the combination of (1) the selection of aptamers that interact with a target molecule in a desired manner (e.g., bind with high affinity to a protein) and (2) the amplification of the selected nucleic acids. The SELEX method can be used to identify aptamers that have high affinity for a particular analyte, such as a target protein.

[0291] Sequence identity: As used herein in relation to two or more nucleic acid sequences, sequence identity is a function of the number of identical nucleotide positions shared by the sequences (i.e., identity %=number of identical positions / total number of positions in the reference sequence×100), taking into account the number of gaps and the length of each gap that needs to be introduced to optimize the alignment of two or more sequences. Comparison of sequences and quantification of percent identity between two or more sequences can be accomplished using mathematical algorithms such as BLAST and Gapped BLAST programs with their default parameters (e.g., Altschul et al., J. Mol. Biol. 215:403, 1990; also see BLASTN at www.ncbi.nlm.nih.gov / BLAST). For sequence comparison, typically one sequence serves as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence(s), based on the designated program parameters.Optimal alignment of sequences for comparison can be performed, for example, by the homology alignment of Smith and Waterman, Adv. Appl. Numerical., 2:482, 1981, by the homology alignment of Needleman and Wunsch, J. Mol. Biol., 48:443, 1970, by the similarity method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TAFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel, FM et al., Current Protocols in Molecular Biology (published by Greene Publishing Assoc. and Wiley-Interscience (1987))). As used herein, when describing the percent identity of a nucleic acid (e.g., an aptamer) whose sequence is at least about 95% identical to a reference nucleotide sequence, it is intended that the nucleic acid sequence is identical to the reference sequence except that it may contain up to 5 point mutations per 100 nucleotides of the reference nucleic acid sequence. In other words, to obtain a desired nucleic acid sequence that is at least about 95% identical in sequence to a reference nucleic acid sequence, up to 5% of the nucleotides in the reference sequence can be deleted or replaced with another nucleotide, or a number of nucleotides up to 5% of the total number of nucleotides in the reference sequence can be inserted (referred to as insertion herein) into the reference sequence. These mutations of the reference sequence to generate a desired sequence can occur at the 5' or 3' terminal position of the reference nucleotide sequence, or anywhere between these terminal positions, and can be interspersed between nucleotides in the reference sequence individually or in one or more consecutive groups in the reference sequence.

[0292] SOMAmer: As used herein, SOMAmer or SOMAmer reagent refers to an aptamer with improved off-rate characteristics. SOMAmer reagents are alternatively referred to as slow off-rate modified aptamers and can be selected via the improved SELEX method described in U.S. Patent Publication No. 20090004667, entitled "Method for Generating Aptamers with Improved Off-Rates," which is incorporated by reference in its entirety. In some embodiments, slow off-rate aptamers (including aptamers that contain at least one nucleotide with hydrophobic modification) have an off-rate (t1 / 2) of 2 minutes or more, 4 minutes or more, 5 minutes or more, 8 minutes or more, 10 minutes or more, 15 minutes or more, 30 minutes or more, 60 minutes or more, 90 minutes or more, 120 minutes or more, 150 minutes or more, 180 minutes or more, 210 minutes or more, or 240 minutes or more.

[0293] Substantially equivalent: As used herein, the term "substantially equivalent" refers to two values ​​that are sufficiently similar that a person skilled in the art would consider the difference between the two values ​​to have little or no biological and / or statistical significance within the context of the characteristics measured by the two values. More specifically, the difference between the two values ​​(e.g., between the reference value and the regression model reference value) is preferably less than about 25%, or less than about 20%, or less than about 15%, or less than about 10%, or less than about 5%, or less than about 4%, or less than about 3%, or less than about 2.5%, or less than about 2%, or less than about 1%.

[0294] Target molecule: "target", "target molecule", and "analyte" are used interchangeably herein and refer to any molecule of interest that may be present in a sample. The term may include any slight change in a particular molecule, e.g., in the case of a protein, a slight change in the amino acid sequence, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, e.g., conjugation with a labeling moiety, that does not substantially change the identity of the molecule. "Target molecule", "target", or "analyte" refers to a set of copies of one type or species of molecule or multimolecular structure. "Target molecule", "target", and "analyte" refer to two or more types or species of molecule or multimolecular structure. Exemplary target molecules include proteins, polypeptides, nucleic acids, carbohydrates, lipids, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies, affibodies, antibody mimetics, viruses, pathogens, toxic substances, substrates, metabolites, transition state analogs, cofactors, inhibitors, drugs, dyes, nutrients, growth factors, cells, tissues, and any fragments or portions of any of the above. In some embodiments, the target molecule is a protein, in which case the target molecule may be referred to as a "target protein."

[0295] Test sample: As used herein, a "test sample" refers to a material, solution, or mixture that contains or is derived from a biological sample. In some embodiments, a test sample is generated from a biological sample. In some embodiments, a test sample is generated from a biological sample or a solution containing a biological sample by performing buffer exchange on the biological sample or a solution containing a biological sample. As used herein, an "adjusted test sample" is a test sample that has been subjected to an adjustment, such as a change in total protein concentration.

[0296] Total protein: As used herein, "total protein" in a solution, buffer, or sample refers to all the proteins in the solution, buffer, or sample. The concentration, level, or amount of total protein takes into account all the different types of proteins in a solution, buffer, or sample. In contrast, a "target protein" or "analyte protein" refers to one specific protein in a solution, buffer, or sample.

[0297] The foregoing and other objects, features, and advantages of the present invention will become more apparent from the following detailed description which proceeds with reference to the accompanying drawings.

[0298] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0299] Detection and Quantification of Analytes and Analyte Levels The analyte levels of the analytes described herein can be detected using any of a variety of known analytical methods. In one embodiment, the analyte levels are detected using a capture reagent. In various embodiments, the capture reagent can be exposed to the analyte in solution or while the capture reagent is immobilized on a solid support. In some embodiments, the capture reagent includes a feature that is reactive to a secondary feature on the solid support. In such embodiments, the capture reagent can be exposed to the analyte in solution, and then the feature on the capture reagent can be used in conjunction with the secondary feature on the solid support to immobilize the analyte on the solid support. The capture reagent is selected based on the type of analysis to be performed. Capture reagents include, but are not limited to, aptamers, antibodies, adnectins, ankyrins, other antibody mimetics and other protein scaffolds, chimeras, small molecules, F(ab')2 fragments, single chain antibody fragments, Fv fragments, single chain Fv fragments, nucleic acids, lectins, ligand binding receptors, affibodies, nanobodies, imprinted polymers, avimers, peptidomimetics, hormone receptors, cytokine receptors, and synthetic receptors, including modifications and fragments of any of these.

[0300] In some embodiments, the analyte level is detected using an analyte / capture reagent complex.

[0301] In some embodiments, the analyte level originates from the analyte / capture reagent complex and is detected indirectly (e.g., as a result of a reaction following analyte / capture reagent interaction) but is dependent on the formation of the analyte / capture reagent complex.

[0302] In some embodiments, the analytes are detected using a multiplexing format that allows for the simultaneous detection of two or more analytes in a biological sample. In some embodiments of the multiplexing format, the capture reagents are immobilized directly or indirectly, covalently or non-covalently, at individual locations on a solid support. In some embodiments, the multiplexing format uses individual solid supports, where each solid support has a unique capture reagent (e.g., quantum dots) associated with it. In some embodiments, individual devices are used to detect each one of the multiple analytes to be detected in the biological sample. The individual devices can be configured to allow each analyte in the biological sample to be processed simultaneously. For example, a microtiter plate can be used such that each well in the plate is used for the analysis of one or more of the multiple analytes to be detected in the biological sample.

[0303] In one or more embodiments described herein, a fluorescent tag can be used to label a component of an analyte / capture reagent complex to allow detection of the analyte level. In various embodiments, a fluorescent label can be conjugated to a capture reagent specific for any of the analytes described herein using known techniques, and the fluorescent label can then be used to detect the corresponding analyte level. Suitable fluorescent labels include rare earth chelates, fluorescein and its derivatives, rhodamine and its derivatives, dansyl, allophycocyanin, PBXL-3, Qdot 605, Lissamine, phycoerythrin, Texas Red, and other such compounds.

[0304] In some embodiments, the fluorescent label is a fluorescent dye molecule. In some embodiments, the fluorescent dye molecule comprises at least one substituted indolium ring system, where the substituent on the 3-carbon of the indolium ring comprises a chemically reactive group or a conjugation agent. In some embodiments, the dye molecule comprises an AlexFluor molecule, such as AlexaFluor 488, AlexaFluor 532, AlexaFluor 647, AlexaFluor 680, or AlexaFluor 700. In some embodiments, the dye molecule comprises a first type and a second type of dye molecule, such as two different AlexaFluor molecules. In some embodiments, the dye molecule comprises a first type and a second type of dye molecule, where the two dye molecules have different emission spectra.

[0305] Fluorescence can be measured using a variety of instrumentation to accommodate a wide range of assay formats. For example, spectrofluorometers are designed to analyze microtiter plates, microscope slides, printed arrays, cuvettes, etc. See Principles of Fluorescence Spectroscopy (J.R. Lakowicz, Springer Science + Business Media, Inc., 2004). See Bioluminescence & Chemiluminescence: Progress & Current Applications; Philip E. Stanley and Larry J. Kricka editors, World Scientific Publishing Company, January 2002.

[0306] In one or more embodiments, a chemiluminescent tag can optionally be used to label components of the analyte / capture complex to allow detection of the analyte level. Suitable chemiluminescent materials include oxalyl chloride, rhodamine 6G, Ru(bipy)3 2+, TMAE (tetrakis(dimethylamino)ethylene), pyrogallol (1,2,3-trihydroxybenzene), lucigenin, peroxyoxalates, aryloxalates, acridinium esters, dioxetanes, and the like.

[0307] In some embodiments, the detection method involves an enzyme / substrate combination that generates a detectable signal corresponding to the analyte level. In general, the enzyme catalyzes a chemical change of a chromogenic substrate, which can be measured using a variety of techniques, including spectrophotometry, fluorescence, and chemiluminescence. Suitable enzymes include, for example, luciferase, luciferin, malate dehydrogenase, urease, horseradish peroxidase (HRPO), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, uricase, xanthine oxidase, lactoperoxidase, microperoxidase, and the like.

[0308] In some embodiments, detection methods can be fluorescent, chemiluminescent, radionuclide combinations, and / or enzyme / substrate combinations that generate a measurable signal. In some embodiments, multimodal signaling can have unique and advantageous features in analyte assay formats.

[0309] In some embodiments, analyte levels of the analytes described herein can be detected using any analytical method, including singleplex aptamer assays, multiplexed aptamer assays, singleplex or multiplexed immunoassays, mRNA expression profiling, miRNA expression profiling, mass spectrometry, histological / cytological methods, etc., as discussed below.

[0310] Quantifying analyte levels using aptamer-based assays The analyte levels of the analytes described herein can be detected using any of a variety of aptamer-based assays. Assays directed to the detection and quantification of physiologically important molecules in biological and other samples are important tools in the fields of scientific research and health care. One class of such assays involves the use of microarrays that include one or more aptamers immobilized on a solid support. Each aptamer can bind to a target molecule in a highly specific manner and with very high affinity. See, for example, U.S. Pat. No. 5,475,096, entitled "Nucleic Acid Ligands." See also, for example, U.S. Pat. Nos. 6,242,246, 6,458,543, and 6,503,715, each entitled "Nucleic Acid Ligand Diagnostic Biochip." Once the microarray is contacted with the sample, the aptamers bind to the respective target molecules present in the sample, thereby allowing quantification of the analyte levels corresponding to the analyte.

[0311] In one embodiment, an aptamer can comprise up to about 100 nucleotides, up to about 95 nucleotides, up to about 90 nucleotides, up to about 85 nucleotides, up to about 80 nucleotides, up to about 75 nucleotides, up to about 70 nucleotides, up to about 65 nucleotides, up to about 60 nucleotides, up to about 55 nucleotides, up to about 50 nucleotides, up to about 45 nucleotides, up to about 40 nucleotides, up to about 35 nucleotides, up to about 30 nucleotides, up to about 25 nucleotides, and up to about 20 nucleotides. In related embodiments, the aptamer is about 25 to about 100 nucleotides in length (i.e., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, , 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides in length) or about 25-50 nucleotides in length (i.e., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length).

[0312] Aptamers can be identified using any known method, including SELEX. Once identified, aptamers can be prepared or synthesized according to any known method, including chemical synthesis and enzymatic synthesis. In some embodiments, aptamers contain at least one nucleotide with a hydrophobic modification (e.g., a hydrophobic base modification), which allows hydrophobic contact with the target protein. Such hydrophobic contacts, in some embodiments, contribute to increased affinity and / or slower off-rate binding by the aptamer. In some embodiments, aptamers contain at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotides with hydrophobic modifications, each of which may be the same or different from the others. In some embodiments, the hydrophobic base modification is a C-5 modified pyrimidine. Non-limiting exemplary C-5 modified pyrimidines are described herein and / or known in the art.

[0313] In some assay formats, the aptamer is immobilized on a solid support before contacting with a sample. However, under certain circumstances, immobilization of the aptamer before contacting with a sample may not provide an optimal assay. For example, in some instances, pre-immobilization of the aptamer may cause inefficient mixing of the aptamer with the target molecule on the surface of the solid support, which may lead to a longer reaction time, and therefore the incubation period may be extended to allow the aptamer to efficiently bind to its target molecule. Furthermore, when photoaptamers are used in the assay, and depending on the material utilized as the solid support, the solid support may tend to scatter or absorb the light used to affect the formation of covalent bonds between the photoaptamer and its target molecule. Furthermore, depending on the method used, the detection of the target molecule bound to the aptamer may be inaccurate. Because the surface of the solid support may also be exposed to and affected by any labeling agent used. Finally, immobilization of aptamers onto a solid support generally involves a step of preparing the aptamer (i.e., immobilization) prior to exposing the aptamer to a sample, and this preparation step may affect the activity or functionality of the aptamer.

[0314] Aptamer assays or "aptamer-based assay(s)" have also been described that allow an aptamer to capture its target in solution and then use a separation step designed to remove certain components of the aptamer-target mixture prior to detection (see, for example, U.S. Patent Publication No. 2009 / 0042206, entitled "Multiplexed Analyses of Test Samples"). The aptamer assays described allow for the detection and quantification of non-nucleic acid targets (e.g., protein targets) in a test sample by detecting and quantifying a nucleic acid (i.e., an aptamer). The methods described create a nucleic acid surrogate (i.e., an aptamer) for detecting and quantifying a non-nucleic acid target, thus allowing a wide variety of nucleic acid techniques, including amplification, to be applied to a wider range of desired targets, including protein targets.

[0315] Aptamers can be constructed to facilitate separation of assay components from aptamer-analyte complexes (or photoaptamer-analyte covalent complexes) and allow isolation of the aptamer for detection and / or quantification. In one embodiment, such constructs can include cleavable or releasable elements in the aptamer sequence. In other embodiments, additional functionality can be introduced into the aptamer, such as a label or detectable moiety, a spacer moiety, or a specific binding tag or immobilization element. For example, an aptamer can include a tag connected to the aptamer via a cleavable moiety, a label, a spacer moiety that separates the label, and a cleavable moiety. In one embodiment, the cleavable element is a photocleavable linker. The photocleavable linker can be attached to a biotin moiety and a spacer moiety, can include an NHS group for derivatization of amines, and can be used to introduce a biotin group into the aptamer, which allows for release of the aptamer later in the assay.

[0316] Homogeneous assays, which in some embodiments are performed with all assay components in solution, may not require separation of sample and reagents prior to detection of a signal. These methods are fast and easy to use.

[0317] In some embodiments, the method for signal generation utilizes anisotropic signal change due to the interaction of a fluorophore-labeled capture reagent with its specific analyte target. When the labeled capture agent reacts with its target, the increase in molecular weight causes rotational motion of the fluorophore bound to the complex, which changes the anisotropy value much slower. By monitoring the anisotropy change, the binding event can be used to quantitatively measure the analyte in solution. Other methods include fluorescence polarization assays, molecular beacon methods, time-resolved fluorescence quenching, chemiluminescence, fluorescence resonance energy transfer, etc.

[0318] An exemplary solution-based aptamer assay that can be used to detect analyte levels in a biological sample includes: (a) contacting the biological sample with an aptamer that includes a first tag and has a specific affinity for the analyte, such that if the analyte is present in the sample, an aptamer affinity complex is formed; (b) exposing the mixture to a first solid support that includes a first capture element, causing the first tag to associate with the first capture element; (c) removing components of the mixture that are not associated with the first solid support; (d) detecting the analyte level of the aptamer affinity complex. (e) binding a second tag to the light component; (f) exposing the released aptamer affinity complex to a second solid support containing a second capture element, and associating the second tag with the second capture element; (g) removing uncomplexed aptamers from the mixture by partitioning the uncomplexed aptamers from the aptamer affinity complex; (h) eluting the aptamer from the solid support; (i) detecting the analyte by detecting the aptamer component of the aptamer affinity complex. For example, the protein concentration or level in the sample can be expressed as relative fluorescence units (RFU), which can be the product of detecting the aptamer component of the aptamer affinity complex (e.g., an aptamer complexed with a target protein creates an aptamer affinity complex). That is, in an aptamer-based assay, the protein concentration or level correlates with the RFU.

[0319] A non-limiting exemplary method for detecting an analyte in a biological sample using aptamers is described in Kraemer et al., PLoS One 6(10):e26332.

[0320] Quantifying analyte levels using immunoassays In some embodiments, the levels of target or analyte proteins are quantified using immunoassays. Immunoassay methods are based on the reaction of antibodies to the corresponding targets or analytes, and can detect the analytes in a sample depending on the specific assay format. To improve the specificity and sensitivity of immunoreactivity-based assay methods, monoclonal antibodies and their fragments are often used due to their specific epitope recognition. Polyclonal antibodies have also been successfully used in various immunoassays due to their higher affinity for targets compared to monoclonal antibodies. Immunoassays are designed for use with a wide range of biological sample matrices. Immunoassay formats are designed to provide qualitative, semi-quantitative, and quantitative results.

[0321] Quantitative results are obtained by using a standard curve generated with known concentrations of the particular analyte to be detected. The response or signal from an unknown sample is plotted against the standard curve to establish the corresponding amount or level of the target in the unknown sample.

[0322] A number of immunoassay formats have been designed. ELISA or EIA can be quantitative in the detection of the analyte. The method relies on the attachment of a label to either the analyte or an antibody, where the label component includes an enzyme, either directly or indirectly. ELISA tests can be formatted for direct, indirect, competitive, or sandwich detection of the analyte. Other methods use, for example, radioisotopes (I 125 ) or rely on labels such as fluorescence. Additional techniques include, for example, agglutination, nephelometry, turbidity, Western blot, immunoprecipitation, immunocytochemistry, immunohistochemistry, flow cytometry, Luminex assays, etc. (see ImmunoAssay: A Practical Guide (ed. Brian Law, published by Taylor & Francis, Ltd., 2005 edition)).

[0323] Exemplary assay formats include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays, fluorescence, chemiluminescence, and fluorescence resonance energy transfer (FRET) or time-resolved FRET (TR-FRET) immunoassays. Exemplary procedures for detecting analytes include analyte immunoprecipitation followed by quantitative methods that allow size and peptide level discrimination (e.g., gel electrophoresis, capillary electrophoresis, planar electrochromatography, etc.).

[0324] The method of detecting and / or quantifying the detectable label or signal generating material depends on the nature of the label. The product of the reaction catalyzed by the appropriate enzyme (when the detectable label is an enzyme (see above)) can be, but is not limited to, fluorescent, luminescent, or radioactive, or such product can absorb visible or ultraviolet light. Examples of detectors suitable for detecting such detectable labels include, but are not limited to, X-ray film, radioactivity counters, scintillation counters, spectrophotometers, colorimeters, fluorometers, luminometers, and densitometers.

[0325] Any method for detection can be carried out in any format that allows any suitable preparation, processing, and analysis of the reaction. This can be, for example, in a multi-well assay plate (e.g., 96-well or 386-well) or any suitable array or microarray. Stock solutions for various agents can be made manually or robotically, and all subsequent pipetting, dilution, mixing, dispensing, washing, incubation, sample readout, data collection, and analysis can be performed robotically using commercially available analysis software, robotics, and detection instruments that can detect detectable labels.

[0326] Quantifying analyte levels using gene expression profiling In some embodiments, target mRNA levels are quantified using the gene expression profiling methods described herein. Measurement of mRNA in a biological sample can be used in some embodiments as a substitute for detection of the level of the corresponding protein in a biological sample. Thus, in some embodiments, an analyte or analyte panel described herein can be detected by detecting the appropriate RNA.

[0327] In some embodiments, mRNA expression levels are measured by reverse transcription quantitative polymerase chain reaction (RT-PCR followed by qPCR). RT-PCR is used to generate cDNA from mRNA. The cDNA can be used in qPCR assays to generate fluorescence as the DNA amplification process proceeds. By comparing to a standard curve, qPCR can produce absolute measurements such as the number of copies of mRNA per cell. Northern blots, microarrays, invader assays, and RT-PCR combined with capillary electrophoresis have all been used to measure the expression levels of mRNA in samples. See Gene Expression Profiling: Methods and Protocols, Richard A. Shimkets, editor, Humana Press, 2004.

[0328] Quantitation of analyte levels using mass spectrometry In some embodiments, the target protein or analyte protein levels are quantified using mass spectrometry. Analyte levels can be detected using mass spectrometers in various configurations. Several types of mass spectrometers are available or can be manufactured in various configurations. Generally, mass spectrometers have the following major components: sample inlet, ion source, mass analyzer, detector, vacuum system, and instrument control system, and data system. The differences in the sample inlet, ion source, and mass analyzer generally define the type of instrument and its capabilities. For example, the inlet can be a capillary column liquid chromatography source, or a direct probe or stage such as used in matrix-assisted laser desorption. Common ion sources are, for example, electrospray (including nanospray and microspray) or matrix-assisted laser desorption. Common mass analyzers include quadrupole mass filters, ion trap mass analyzers, and time-of-flight mass analyzers. Additional mass spectrometry methods are well known in the art (see Burlingame et al. Anal. Chem. 70:647 R-716R (1998); Kinter and Sherman, New York (2000)).

[0329] Protein analytes and analyte levels can be detected and measured by any of the following: electrospray ionization mass spectrometry (ESI-MS), ESI-MS / MS, ESI-MS / (MS)n, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS), surface-enhanced laser desorption / ionization time-of-flight mass spectrometry (SELDI-TOF-MS), desorption / ionization on silicon (DIOS), secondary ion mass spectrometry (SIMS), quadrupole time-of-flight (Q-TOF), tandem time-of-flight (TOF / TOF) technology (called ultraflex III TOF / TOF), atmospheric pressure chemical ionization mass spectrometry (APCI-MS), APCI-MS / MS, APCI-(MS). N , atmospheric pressure photoionization mass spectrometry (APPI-MS), APPI-MS / MS and APPI-(MS) N, quadrupole mass spectrometry, Fourier transform mass spectrometry (FTMS), quantitative mass spectrometry, and ion trap mass spectrometry.

[0330] Sample preparation strategies are used to label and enrich samples prior to mass spectrometric characterization of protein analytes and quantification of analyte levels. Labeling methods include, but are not limited to, isobaric tags for relative and absolute quantification (iTRAQ) and stable isotope labeling with amino acids in cell culture (SILAC). Capture reagents used to selectively enrich samples for candidate analyte proteins prior to mass spectrometric analysis include, but are not limited to, aptamers, antibodies, nucleic acid probes, chimeras, small molecules, F(ab')2 fragments, single-chain antibody fragments, Fv fragments, single-chain Fv fragments, nucleic acids, lectins, ligand-binding receptors, affibodies, nanobodies, ankyrin, domain antibodies, alternative antibody scaffolds (e.g., diabodies, etc.), imprinted polymers, avimers, peptidomimetics, peptoids, peptide nucleic acids, threose nucleic acids, hormone receptors, cytokine receptors, and synthetic receptors, as well as modifications and fragments thereof.

[0331] A specific detection method Aptamer Assay Methods As used herein, an "aptamer assay method" comprises or consists of the steps described below. In some embodiments, the aptamer-based assay is an aptamer assay method. Unless otherwise indicated, all steps were performed at room temperature.

[0332] Preparation of aptamer master mix solution. The aptamers were grouped into three unique mixtures, Dil1, Dil2, and Dil3, which corresponded to sample dilutions of 20%, 0.5%, and 0.005%, respectively. The assignment of aptamers to the mixtures was determined empirically by assaying a dilution series of plasma and serum samples matched with each aptamer and identifying the sample dilution that showed the greatest linear range of signal. Separation of the aptamers and mixing with different dilutions of the sample (20%, 0.5%, or 0.005%) allowed the assay to be performed at 10% of the protein concentration. 7 The aptamer master mix was prepared in HE-Tween buffer (10 mM HEPES, pH 7.5, 1 mM EDTA, 0.05% Tween 20) at 4 nM of each aptamer and stored frozen at -20°C. Approximately 4271 aptamers were mixed in the Dil1 mix, approximately 828 aptamers in Dil2, and approximately 173 aptamers in the Dil3 mix. Prior to use, the stock solutions were diluted with HE-Tween buffer to a working concentration of 0.55 nM for each aptamer and aliquoted into individual working aliquots. Prior to using the aptamer master mix to prepare catch-0 plates, the working solutions were heated and cooled prior to use to refold the aptamers by incubating at 95°C for 10 min and then at 25°C for at least 30 min.

[0333] Preparation of catch-0 plates. 60 μL of streptavidin magnetic sepharose 10% slurry (GE Healthcare, 28-9857) was dispensed into each well of a 96-well plate (Thermo Scientific, AB-0769). The beads were washed once with 175 μL of assay buffer containing 40 mM HEPES, 100 or 102 mM NaCl, 5 mM KCl, 5 mM MgCl2, 1 mM EDTA, and 0.05% or 0.1% (v / v) Tween-20 (pH 7.5), and then 100 μL of heated and cooled aptamer master mix was added to each well. The plate was incubated at 25° C. for 30 min with shaking at 850 rpm on a ThermoMixer C shaker (Eppendorf). After 30 min incubation, 6 μL of MB Block Buffer (50 mM D-biotin in 50 mM Tris-HCl, pH 8, 0.01% Tween) was added to each well of the plate and the plate was incubated for an additional 2 min with shaking. The plate was then washed with 175 μL of Assay Buffer and subjected to a 1 min wash cycle with shaking at 850 rpm on a ThermoMixer C, followed by separation on a magnet for 30 s. After removing the wash solution, the beads were resuspended in 175 μL of Assay Buffer and stored at −20° C. until use.

[0334] Preparation of Catch-2 beads. Prior to starting the robotic processing of the assay, a 10 mg / mL bead slurry of MyOne Streptavidin C1 beads (Dynabeads, product no. 35002D, Thermo Scientific) used for the Catch-2 step of the multiplex aptamer assay was washed once in bulk for 5 min with MB Prep buffer (10 mM Tris-HCl, pH 8, 1 mM EDTA, 0.4% SDS) followed by two washes with assay buffer. After the final wash, the beads were resuspended at a concentration of 10 mg / mL and 75 μL of bead slurry was dispensed into each well of the Catch-2 plate. At the start of the assay, the Catch-2 plate was placed in the aluminum adaptor and placed in the appropriate position on the Fluent deck.

[0335] Sample binding step. Catch-0 plates were prepared by immobilizing the aptamer mix on streptavidin magnetic sepharose beads as described above. Frozen plates were thawed for 30 min at 25° C. and washed once with 175 μL of assay buffer. 100 μL of each sample dilution (20%, 0.5%, and 0.005%) was added to the plate containing the beads with the three different aptamer master mixes (Dil1, Dil2, and Dil3, respectively). The catch-0 plate was then sealed with an aluminum foil seal (Microseal 'F' Foil, Bio-Rad) and placed in a four-plate rotary shaker (PHMP-4, Grant Bio) set at 850 rpm and 28° C. The sample binding step was carried out for 3.5 h.

[0336] Multiplexed aptamer assay processing on a Fluent robot. After the sample binding step was completed, the catch-0 plate was placed in an aluminum plate adaptor and placed on the robot deck. The magnetic bead washing step was performed using a temperature controlled plate. For all robotic processing steps, the plate was set at a temperature of 25° C., except for the catch-2 wash as described below. The plate was washed four times with 175 μL of assay buffer, with each wash cycle programmed to shake the plate at 1000 rpm for at least 1 minute, followed by allowing the magnetic beads to separate for at least 30 seconds before aspirating the buffer. During the last wash cycle, tag reagent was prepared by diluting 100x tag reagent (EZ-Link NHS-PEG4-biotin, product no. 21363, Thermo, 100 mM solution prepared in anhydrous DMSO) 1:100 with assay buffer and poured into a trough on the robot deck. 100 μL of tag reagent was added to each of the wells of the plate and incubated with shaking at 1200 rpm for 5 minutes to biotinylate the proteins captured on the bead surface. The biotinylation reaction was quenched by adding 175 μL of quench buffer (20 mM glycine in assay buffer) to each well. The plate was left to incubate for 3 minutes and then washed 4 times with 175 μL of assay buffer, with the washes carried out under the same conditions as described above.

[0337] Photocleavage and kinetic challenges. After the plate was washed a final time, 90 μL of photocleavage buffer (2 μM of oligonucleotide competitor in assay buffer, competitor oligonucleotide (also called Z-block) has the nucleotide sequence 5'-(AC-Bn-Bn)7-AC-3', where Bn represents a 5-position benzyl substituted deoxyuridine residue) was added to each well of the plate. The plate was transferred to the photocleavage substation on the Fluent deck. The substation consists of a BlackRay light source (UVP XX series bench lamp, 365 nm) and three Bioshake 3000-T shakers (Q Instruments). The plate was irradiated for 20 minutes with shaking at 1000 rpm.

[0338] Catch-2 bead capture. At the end of the photocleavage process, the buffer was removed from the Catch-2 plate by magnetic separation and the plate was washed once with 100 μL of assay buffer. The photocleavage eluate containing the aptamer-protein complex was removed from each Catch-0 plate starting from the dilution 3 plate. All 90 μL of solution was first transferred to the Catch-1 eluate plate placed on a shaker and the magnet was raised to capture any streptavidin magnetic sepharose beads that may have been attracted. The solution was then transferred to the Catch-2 plate and the plate was incubated for 3 minutes with shaking at 1400 rpm and 25 °C. After 3 minutes of incubation, the magnetic beads were allowed to separate for 90 seconds, the solution was removed from the plate, and the photocleaved Dil2 plate solution was added to the plate. After an identical process, the solution from the Dil1 plate was added and incubated for 3 minutes. At the end of the 3 minutes of incubation, 6 μL of MB block buffer was added to the magnetic bead suspension and the beads were incubated for 2 minutes with shaking at 1200 rpm and 25 °C. After this incubation, the plate was transferred to another shaker preset at a temperature of 38°C. The magnetic beads were allowed to separate for 2 minutes before removing the solution. The Catch-2 plate was then washed 4 times with 175 μL of MB wash buffer (20% glycerol in assay buffer), with each wash cycle programmed to shake the beads at 1200 rpm for 1 minute and allow the beads to separate on the magnet for 3.5 minutes. During the final bead separation step, the shaker temperature was set to 25°C. The beads were then washed once with 175 μL of assay buffer. In this wash step, the beads were shaken at 1200 rpm for 1 minute and then allowed to separate on the magnet for 2 minutes. After the wash steps, the aptamers were eluted from the purified aptamer-protein complexes using elution buffer (1.8 M NaClO4, 40 mM PIPES, pH 6.8, 1 mM EDTA, 0.05% Triton X-100). Elution was performed with 75 μL of elution buffer for 10 min at 25° C. while shaking the beads at 1250 rpm. 70 μL of the eluate was transferred to an archive plate and separated on a magnet to split any magnetic beads that may have been attracted.10 μL of the eluted material was transferred to a black half-area plate, diluted 1:5 in assay buffer, and used to measure the Cy3 fluorescent signal monitored as an internal assay QC. 20 μL of the eluted material was transferred to a plate containing 5 μL of hybridization blocking solution (Oligo aCGH / ChIP-on-chip Hybridization Kit, large volume, Agilent Technologies 5188-5380, containing spikes of cyanine 3-labeled DNA sequences complementary to the corner marker probes on the Agilent array). This plate was removed from the robot deck and further processed for hybridization (see below). Archive plates with remaining eluate were heat sealed using aluminum foil and stored at -20 °C.

[0339] Hybridization. 25 μL of 2x Agilent hybridization buffer (Oligo aCGH / ChIP-on-chip Hybridization Kit, Agilent Technologies, product number 5188-5380) was manually pipetted into each well of the plate containing the eluted samples and blocking buffer. 40 μL of this solution was manually pipetted into each "well" of a hybridization gasket slide (Hybridization Gasket Slide - 8 microarrays per slide format, Agilent Technologies). Custom SurePrint G3 8x60k Agilent microarray slides containing 10 probes per array, complementary to each aptamer, were placed on the gasket slide following the manufacturer's protocol. Each assembly (Hybridization Chamber Kit - SureHyb compatible, Agilent Technologies) was clamped securely and placed in a hybridization oven for 19 hours at 55°C, rotating at 20 rpm.

[0340] Post-hybridization washes. Slide washing was performed using a Little Dipper Processor (Model 650C, Scigene). Approximately 700 mL of Wash Buffer 1 (Oligo aCGH / ChIP-on-chip Wash Buffer 1, Agilent Technologies) was poured into a large glass staining dish and used to separate the microarray slides from the gasket slide. Once disassembled, the slides were quickly transferred to a slide rack in a bath containing Wash Buffer 1 on the Little Dipper. The slides were washed with Wash Buffer 1 for 5 minutes while mixing with a magnetic stir bar. The slide rack was then transferred to a bath containing Wash Buffer 2 (Oligo aCGH / ChIP-onchip Wash Buffer 2, Agilent Technologies) at 37°C and incubated for 5 minutes with agitation. The slide rack was slowly removed from the second bath and then transferred to a bath containing acetonitrile and incubated for 5 minutes with agitation.

[0341] Microarray imaging. Microarray slides were imaged using a microarray scanner (Agilent G4900DA Microarray Scanner System, Agilent Technologies) in the cyanine 3 channel at 3 μm resolution, 100% PMT setting, and 20-bit option enabled. The resulting tiff images were processed using Agilent feature extraction software (version 10.7.3.1 or higher) using the GE1_1200_Jun14 protocol.

[0342] Collection and preparation of certain biological samples In some embodiments, the biological sample is prepared by performing sample collection, buffer exchange, and / or protein standardization as described below.

[0343] Urine sample collection and preparation Urine samples were collected from individuals via midstream urine collected in a tube and then immediately stored in a -80° C. freezer. Frozen urine samples were processed as follows: Buffer exchange: Samples were thawed in a 37°C water bath for approximately 15 minutes to ensure all cryoprecipitates were dissolved. The thawed samples were removed from the water bath and placed in a biological safety cabinet. The caps were removed and the contents mixed by pipetting up and down three times with a 200 μL multichannel pipette. Samples were aliquoted into new tubes at 216 μL each and 1 M Tris (tris(hydroxymethyl)aminomethane) pH=8 was added to each aliquot of thawed / mixed urine using a 20 μL single channel pipette to give a final Tris concentration of 40 mM (4%). The Tris-treated aliquots were mixed by gently pipetting up and down three times with a 200 μL multichannel pipette. 15 μL of the Tris-treated urine samples were transferred to the corresponding wells in a PCR plate (96-well PCR plate (Fisher Scientific #NC0508659)). The 15 μL aliquots were stored at −80° C. until quantification of protein concentration was performed using a NanoOrange® kit containing a merocyanine dye (see, e.g., Jones et al. “Development and Characterization of the NanoOrange® Protein Quantitation Assay: A Fluorescence-Based Assay of Proteins in Solution.” BioTechniques. 34(4):850-4 (2003)).

[0344] Aliquots were buffer exchanged on two Zeba™ 7 kDa MWCO Spin Desalting Plates (Thermo Scientific #89808) or two Harvard G25 1.5 kDa MWCO 96-Well SpinColumns™ (Harvard Apparatus #74-5652) following the respective manufacturer's instructions. For Zeba™ Desalting Plates, see the "Procedure for Buffer-Exchange" section of the manufacturer's protocol. Briefly, the Zeba™ Spin Desalting Plates were incubated until they reached room temperature. The assembly was centrifuged at 1,000×g for 2 minutes using a 96-well plate-carrier rotor to remove the storage buffer. The flow-through was discarded. A 250 μL volume of buffer was added to the top of the resin bed. The plate was again centrifuged at 1,000×g for 2 minutes and the flow-through was discarded. This step was repeated three more times. The washing plates were rinsed three times with deionized water, dried, and stored for future use. Each desalting plate was stacked on top of the sample collection plate, aligning the alphanumeric headings on the plates. 100 μL of sample was applied to the center of the resin bed of the first desalting plate. Another 100 μL sample of the same sample was applied to the center of the resin bed of the second desalting plate. The plates were centrifuged at 1000×g for 2 minutes to collect the processed samples. Using a 200 μL multichannel pipette, 100 μL of each Tris-treated / desalted sample was transferred to each of the two matrix racks, maintaining the orientation of the samples. The matrix tubes were recapped, and now these tubes contained 100 μL of Tris-treated / desalted sample. The aliquot tubes were transferred to dry ice and stored at −80°C. For the Harvard G25 96-Well SpinColumn™, see the "For Gel Filtration / Ion Exchange Columns" section of the manufacturer's protocol. Briefly, the 96-Well SpinColumn™ was placed into one of the collection tubes and 200 μL of buffer was added to all open wells. The tubes were incubated for 15 minutes to allow for hydration. The plate was centrifuged at 2000×g for 2 minutes. The 96-Well SpinColumn™ was removed from the collection plate and the outside of the column was blotted dry. A 100 μL volume of sample was transferred to the top of the wells of the first 96-Well SpinColumn plate. Another 100 μL volume of the same sample was added to the center of the wells of the second 96-Well SpinColumn plate. Each column plate was added to a new collection plate and spun at 2000×g for 2 minutes, and 100 μL of each Tris-treated / desalted sample was transferred to each of the two matrix racks. The matrix tubes were recapped. These tubes now contain 100 μL of Tris-treated / desalted sample. The rack of completed aliquot tubes was transferred to dry ice and stored at -80°C. Protein normalization to 15μg / mL total protein: Buffer-exchanged urine samples were standardized to a total protein concentration of 15 μg / mL in 120 μL as follows: Buffer-exchanged urine specimens were thawed in a 28-37 °C water bath for 10 min. The required volume of buffer-exchanged specimen was calculated by the following formula: 0.12 mL × 15 μg / mL / urine [protein] (μg / mL) = V* (urine sample volume). If V* was greater than 0.108 mL by this formula, V* was adjusted to 0.108 mL. 1x assay buffer was added to the tube by the following formula: 0.108 mL - V* = volume of assay buffer. A 0.012 mL volume of 1x assay buffer containing 50 μM Z-block (10x Z-block) was added. The sample was gently mixed. A 100 μL volume was transferred to a single well of a 96-well catch-0 plate for aptamer-based assays. All three plasma master mixes (Dil1, Dil2, and Dil3) were combined in each well.

[0345] kit Any combination of analytes described herein can be detected using a suitable kit, e.g., a kit for use in practicing the methods disclosed herein. Additionally, any kit can include one or more detectable labels (e.g., fluorescent moieties, etc.) as described herein.

[0346] In the above description, numerous details have been set forth. However, it will be apparent to one skilled in the art having the benefit of this disclosure that the present disclosure may be practiced without such specific details. In some instances, well-known structures and devices are shown in block diagram form, rather than in detail, in order to avoid obscuring the present disclosure.

[0347] Some portions of the detailed description are presented in terms of steps leading to one or more desired results. Generally, such steps are steps requiring physical manipulations of physical quantities. Usually, though not necessarily, such quantities take the form of electrical or magnetic signals capable of being stored, transferred, combined, compared, and otherwise manipulated. It has proven convenient at times, for reasons of common usage, to refer to these signals as bits, values, elements, symbols, characters, terms, numbers, or the like.

[0348] It should be borne in mind, however, that all of these and similar terms are to be associated with the appropriate physical quantities and are merely convenient labels applied to these quantities. As will become apparent from the discussion that follows, unless expressly stated otherwise, throughout the description, the discussion utilizing terms such as "calculate," "compare," "apply," "create," "rank," "categorize," and the like, will be understood to refer to the operations and processes of a specialized computer system or similar specialized electronic computing device that manipulates and transforms data represented as physical (e.g., electronic) quantities in the registers and memory of a computer system into other data similarly represented as physical quantities in the memory or registers of a computer system, or in other such information storage, transmission, or display devices.

[0349] Certain examples of the present disclosure also relate to apparatus for performing the operations herein. The apparatus may be constructed for an intended purpose or may include specialized computer hardware and / or specialized computer programs selectively installed, activated, or configured to perform an intended purpose. Such computer programs may be stored on a computer-readable storage medium, such as, but not limited to, floppy disks, optical disks, CD-ROMs, and magneto-optical disks, read-only memory (ROM), random access memory (RAM), EPROM, EEPROM, magnetic or optical cards, or any type of medium suitable for storing electronic instructions.

[0350] It is to be understood that the above description is intended to be illustrative, and not limiting. Many other examples will become apparent to those of ordinary skill in the art upon reading and understanding the above description. Thus, the scope of the present disclosure should be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled. EXAMPLES

[0351] The following examples are presented to illustrate certain particular features and / or embodiments, and should not be construed as limiting the disclosure to the particular features or embodiments described.

[0352] Example 1: Variability of urinary total protein and composition To determine inter-sample variability in urine samples, the NanoOrange® protein quantification method (Invitrogen; Molecular Probes NanoOrange® Protein Quantification Kit; Catalog No. N6666) was used on three different urine collections, resulting in a wide range of protein concentrations from spot urine samples. Group 1 consisted of 46 volunteers, group 2 consisted of 170 volunteers, and group 3 consisted of 183 volunteers. Generally, urine samples were collected from individuals via midstream urine collected in a tube and then immediately stored in a -80°C freezer.

[0353] Protein concentrations in the three different urine collection groups ranged from about 4 μg / mL to about 4423 μg / mL, resulting in an approximately 1,000-fold difference between the most dilute and most concentrated samples. The median (interquartile range) protein concentrations at each collection time were 46.5 (23-74.5) μg / mL for subjects in group 1, 57.5 (27.2-89.8) μg / mL for subjects in group 2, and 75.0 (51-114) μg / mL for subjects in group 3.

[0354] In general, the method used to quantify urinary total protein concentration using the NanoOrange kit was as follows: 1x Protein Quantification Diluent was made by diluting 10x concentrated NanoOrange® Protein Quantification Diluent 10x with distilled water. 15mL of 1x Protein Quantification Diluent was required for each of the 96-well plates. 1.5mL of NanoOrange® Protein Quantification Diluent was added to 13.5mL of distilled or deionized water. 13mL of 1.04x NanoOrange® Reagent Working Solution was prepared for each of the 96-well plates. NanoOrange® Protein Quantification Reagent (Component A) was diluted 481x into 1x Protein Quantification Diluent. 0.027mL of NanoOrange® Protein Quantification Reagent (Component A) was added to 13mL of 1x Protein Quantification Diluent in a foil-wrapped tube. The 1x NanoOrange® working solution was protected from light to prevent photodegradation of the NanoOrange® dye. The NanoOrange® kit includes a 2 mg / mL sample of BSA (component B) that was used to prepare the standard curve. The standard curve was generated at 25x to cover a final range of 10.25-0.402 μg / mL. An initial 25x stock solution of BSA (final 10.25 μg / mL) in the final 1x protein quantification diluent was prepared. 39 μL of 10x NanoOrange® protein quantification diluent (supplied by the manufacturer) and 50 μL of 2 mg / mL BSA were added to 302 μL of distilled water. Eight 1.5x serial dilutions of the 25x BSA solution in 1x NanoOrange® protein quantification diluent were prepared. 200 μL of the previous standard solution was added to 100 μL of 1x NanoOrange® protein quantification diluent. 25x QC#1 in 1x Protein Quantification Diluent was prepared at 205 μg / mL (8.2 μg / mL final). 48.8 μL of 10x NanoOrange® Protein Quantification Diluent (supplied by manufacturer) and 50 μL of 2 mg / mL BSA were added to 389.2 μL of distilled water. 25x QC#2 in 1x Protein Quantification Diluent was prepared at 132.5 μL (5.3 μg / mL final).226 μL of QC#1 was added to 123.7 μL of 1× NanoOrange® Protein Quantification Diluent. 25× QC#3 was prepared at 30 μg / mL (1.2 μg / mL final) in 1× Protein Quantification Diluent. 79 μL of QC#2 was added to 269.9 μL of 1× NanoOrange® Protein Quantification Diluent. Each 25× Tris-treated urine sample (see Processing Notes below) was diluted into 1.04× NanoOrange® Reagent Working Solution. In a non-skirted PCR plate (Abgene #AB-0600-L), 6 μL of raw urine sample was added to 144 μL of 1.04× NanoOrange® Reagent Working Solution. Each 25× QC sample was diluted in duplicate into 1.04× NanoOrange® Reagent Working Solution. In a non-skirted PCR plate (Abgene #AB-0600-L), 6 μL of QC samples were added in duplicate to 144 μL of 1.04x NanoOrange® reagent working solution. Each 25x standard curve sample was diluted into 1.04x NanoOrange® reagent working solution. In a non-skirted PCR plate (Abgene #AB-0600-L), 6 μL of standard curve sample was added to 144 μL of 1.04x NanoOrange® reagent working solution. Two "blanks" were included.

[0355] In a non-skirted PCR plate (Abgene no. AB-0600-L), 6 μL of 1x NanoOrange® Protein Quantification Diluent was added to 144 μL of 1.04x NanoOrange® Reagent Working Solution. All sample-containing wells were capped with MicroAmp Optical 8-cap strips (Applied BioSystems no. N8010560). Samples were incubated at 95°C for 10 minutes in a thermocycler with a heated lid, protected from light, and then cooled to room temperature for 30 minutes in either the thermocycler or on the benchtop, protected from light. Using a multichannel pipette, 100 μL of each sample was transferred to a black clear bottom half area 96-well plate (Corning no. 3880). Fluorescence was measured at 470 nm on a SpectraMax M5 (or similar instrument). SpectraMax parameters were set as follows: Fluorescence Endpoint; Top Read: 470 nm excitation / 590 nm emission / 570 cutoff; Automix: off; Calibrate: on; PMT: Auto; Settle Time: off; Column Priority; Speed: Normal; Reads / Well: 6.

[0356] Protein concentrations of unknown samples were quantified by first subtracting the fluorescence value of the reagent blank from the fluorescence values ​​of all samples including QCs and standards. A standard curve with a four-parameter fit was used to quantitate protein concentrations of unknown samples and QCs. CV% (<15%) and precision (<15%) of duplicate QC samples were calculated to confirm plate passage.

[0357] Additionally, the effect of adding Tris-HCl (pH=8) on the NanoOrange protein quantification assay was evaluated in 17 independent urine specimens. Fresh frozen urine aliquots were thawed in a 37°C water bath for 15 min, centrifuged at 14,000g for 10 min, and the clarified supernatant was transferred to a fresh tube. A 6 microliter aliquot was removed from the clarified supernatant for protein quantification in the NanoOrange assay. Tris pH 8.0 (1M) was then added to the remaining 44 μL of supernatant to a final concentration of 40 mM. Six microliters of these Tris-treated samples were similarly run in the NanoOrange assay, and protein concentrations were compared to untreated counterparts.

[0358] Briefly, 6 μL of urine was added to 144 μL of 1x NanoOrange working solution in a PCR plate. Samples were capped and heated to 95°C for 10 minutes in a thermocycler and cooled on the benchtop for 30 minutes, protected from light. 100 μL of the hot-cooled sample was transferred to a clear bottom / black half area UV plate and fluorescence was measured using 470 nm excitation / 590 nm emission wavelengths. Protein concentrations were quantified using a standard curve included on each plate.

[0359] This example confirms that urine specimens from both multiple subjects and a single subject across multiple collection times show variability in total protein measurements. Additionally, this example demonstrates that urine sample composition and characteristics (e.g., pH and salt content) can vary from subject to subject.

[0360] Example 2: Analyte measurements are sensitive to differential concentrations of salt and urea in proteomic assays This example demonstrates that analyte measurements in proteomic analyses are sensitive to changes in the concentrations of salt and urea, both of which are components of urine.

[0361] The effect of various concentrations of urea, NaCl, and / or MgCl2 on an aptamer-based proteomics assay was measured. The control (or "true standard") formulation used in this example is SB17 assay buffer. SB17 is an assay buffer made by combining 102 mM NaCl, 5 mM KCl, 5 mM MgCl2, 40 mM HEPES, 1 mM EDTA, and 0.1% Tween-20 (v / v) at pH 7.5. Formulations of SB17 buffer with various salt and / or urea concentrations were made (see formulations 1-28 in Table 3) and spiked with one protein selected from the 201 recombinant proteins listed in Table 2 at a specific protein concentration. [Table 2] TIFF0007604386000003.tif192170 [Table 3]

[0362] As shown in Table 3, the final concentration of NaCl in the formulations ranged from about 100 mM to about 420 mM, more specifically, the concentrations of NaCl were 112 mM, 122 mM, 142 mM, 182 mM, 262 mM, 342 mM, and 422 mM. The final concentration of MgCl2 in the formulations ranged from about 5 mM to about 13 mM, more specifically, the concentrations of MgCl2 were 5.25 mM, 5.5 mM, 6 mM, 7 mM, 9 mM, 11 mM, and 13 mM. The final concentration of urea in the formulations ranged from about 0 mM to about 320 mM, more specifically, the concentrations of urea were 0 mM, 20 mM, 20 mM, 40 mM, 80 mM, 160 mM, 240 mM, and 320 mM.

[0363] The concentrations of 201 recombinant proteins in SB17 buffer and formulations 1-28 were measured using an aptamer assay method.

[0364] Data on the effect of increasing concentrations of NaCl on the measurements of 201 recombinant proteins in an aptamer-based assay are shown in Figure 1. The Log2 fold change in RFU (relative fluorescence units), which corresponds to the relative level of protein present in the assay, for the 201 protein analytes compared to the control (or baseline comparative measurement) indicates that with increasing NaCl concentrations, the assay measured different levels of the individual protein analytes. In general, these data indicate that when two or more samples (e.g., urine samples) with the same concentration of one or more proteins but different concentrations of NaCl are used in an aptamer-based assay, the levels of one or more proteins obtained from the two or more samples will differ, which may result in any results related to biomarker discovery and / or diagnosis.

[0365] Data on the effect of increasing concentrations of MgCl2 on measurements of 201 recombinant proteins in an aptamer-based assay are shown in Figure 2. The Log2 fold change in RFU (relative fluorescence units), which corresponds to the relative level of protein present in the assay, for the 201 protein analytes compared to the control (or baseline comparative measurement) indicates that with increasing concentrations of MgCl2, the assay measured different levels of the individual protein analytes. Overall, these data indicate that when two or more samples (e.g., urine samples) with the same concentration of one or more proteins but different concentrations of MgCl2 are used in an aptamer-based assay, the levels of one or more proteins obtained from the two or more samples will be different.

[0366] Data on the effect of increasing concentrations of urea on the measurements of 201 recombinant proteins in an aptamer-based assay are shown in Figure 3. The Log2 fold change in RFU (relative fluorescence units), which corresponds to the relative level of protein present in the assay, for the 201 protein analytes compared to the control (or baseline comparative measurement) indicates that with increasing concentrations of urea, the assay measured different levels of the individual protein analytes. Overall, these data indicate that when two or more samples (e.g., urine samples) with the same concentration of one or more proteins but different concentrations of urea are used in an aptamer-based assay, the levels of one or more proteins obtained from the two or more samples will be different.

[0367] Taken together, these data indicate that varying concentrations of salt affected protein level measurements in aptamer-based assays. Such variation complicates biomarker discovery and diagnostic testing, especially for biological samples (e.g., urine) where salt concentrations are known to vary between samples. Thus, changes in salt concentration must be mitigated in order to compare levels of a protein or set of proteins derived from biological samples and derive meaningful health-related information and / or clinical decision tools (e.g., diseased vs. non-diseased).

[0368] Example 3: Use of fixed urinary protein concentration to measure analytes in aptamer-based assays This example demonstrates that total protein concentration in urine affects the number of analytes within the linear range in an aptamer-based assay.

[0369] The linear range of a binding curve is the region where a change in target concentration corresponds to a proportional (i.e. linear) change in relative fluorescence unit (RFU) signal (i.e. the rate of change in concentration is equal to the rate of change in RFU, the unit of measurement for hybridization-based readout in aptamer-based assays). In general, the preferred total protein concentration or concentration range for any type of sample is defined as the concentration that maximizes the number of analytes within the linear range.

[0370] To quantify the preferred urinary total protein concentration or concentration range, an 8-point titration was performed with a urine sample with a high protein concentration, e.g., 147 μg / mL. The starting protein concentration of the sample was approximately 80% of the analyte's 147 μg / mL concentration, i.e., 118 μg / mL, since the aptamer assay buffer occupies approximately 20% of the reaction volume. A 6-point 1:2 dilution series to 1.8 μg / mL was then performed, with one more point at 88 μg / mL (i.e., 75% of the starting protein concentration) added to slightly increase the resolution at higher concentrations. The samples were assayed with the aptamer-based assays described herein. All samples were assayed in duplicate with 20 μM Z-block.

[0371] A total of 2,298 analytes were observed to have 3 or more points of dilution linearity, but this linearity was dependent on the starting total protein concentration. The linear ranges are shown in Figure 4. The X-axis identifies the number of analytes (0-2298 analytes) and the Y-axis identifies the total protein concentration (2 μg / mL-128 μg / mL). The black bars span the lower and upper limits of the linear range for each analyte as a function of total protein concentration. There were analytes that fell within the linear range at each total protein concentration, but when samples were normalized to a total protein concentration of 15 μg / mL, more than 60% of the analytes had a linear range. Thus, linear ranges for the analytes were observed at total protein concentrations of 2, 4, 8, 16, 32, 64, and 128 μg / mL. An alternative view of the same data is shown in Figure 5.

[0372] Figure 5 shows the percentage of the total number of analytes (n=2,298) measured within the linear range as a function of total protein concentration. Starting at 0 μg / mL, a local maximum of the analytes within the linear range occurs at approximately 7 μg / mL, and then a slight decrease is observed to approximately 30 μg / mL. An increase was observed at a total protein concentration of 59 μg / mL, but the measurement signal (i.e., RFU level) was relatively low (data not shown) and therefore may have been difficult to measure consistently.

[0373] Taken together, these data show that while linear ranges can be obtained for analytes with total protein concentrations ranging from about 2 μg / mL to about 128 μg / mL, the preferred total protein concentration ranges that yielded the greatest number of analytes in the linear range with a relatively "strong" RFU signal were about 2 μg / mL to 50 μg / mL, more preferably about 5 μg / mL to 40 μg / mL, and most preferably about 10 μg / mL to 30 μg / mL as measured by NanoOrange. More precise total protein concentrations that yielded the greatest number of analytes in the linear range were about 7 μg / mL, 10 μg / mL, and 15 μg / mL.

[0374] Although these data show that adjusting the total protein concentration in a sample to a standardized total protein concentration prior to measuring the protein analytes in the sample can increase the number of analytes in the linear range, complex matrices such as urine still contain various components such as salts, which can hinder the ability to accurately and consistently measure protein levels in the sample.

[0375] Example 4: Use of buffer exchange in urine samples to measure analytes in aptamer-based assays This example demonstrates that applying a buffer exchange method to urine samples affects the number of analytes with a linear range that can be measured by an aptamer-based assay.

[0376] Titrations were performed on urine samples without buffer exchange (control) and after buffer exchange with SB17 assay buffer. Buffer exchange was accomplished using Zeba™ 7 kDa MWCO spin desalting columns according to the manufacturer's protocol. The dilution series was 4, 8.1, 16.3, 32.5, 65, and 130 μg / mL total protein in both control and buffer exchanged urine samples. 2,298 analytes in each of the control and buffer exchanged samples were measured individually with the aptamer-based assay as outlined in Example 3.

[0377] The results of the aptamer-based assay in a total protein titration series for two analytes (ALDOA and ANXA4) are shown in Figure 6. Buffer exchanged urine samples (indicated as "Desalted" in Figure 6) and non-buffer exchanged urine samples are plotted with the total protein concentration of the sample on the x-axis and the Log 10 The RFU values ​​were used as a surrogate for the relative levels of protein in the sample. Figure 6 shows that buffer exchange of urine samples prior to measuring the analyte in the aptamer-based assay extended the linear range of the analyte, especially when the total protein was above 16 μg / mL.

[0378] Other analytes tested showed similar results. These data indicate that high total protein concentrations, likely indicating high urine content (less dilute sample) and therefore high concentrations of assay "interfering substances" (e.g., urine salts), affected the analyte measurements in the assay. Furthermore, these data indicate that performing buffer exchange on the urine samples prior to assaying the level of analyte in the samples mitigated the impact of such "interfering substances" on the ability to consistently and reproducibly measure a particular analyte in a urine sample.

[0379] Thus, buffer exchange of urine samples prior to measuring analyte levels in an aptamer-based assay reduced the effect of urinary "interfering substances", especially in urine samples with protein concentrations above about 16 μg / mL (e.g., ALDOA protein) and 32 μg / mL (e.g., ANXA4 protein).

[0380] Example 5: Combined buffer exchange and total protein concentration normalization of urine samples This example demonstrates that the combined application of buffer exchange and total protein concentration normalization to urine samples improves the reproducibility of analyte measurements and the number of analytes in the sample, thereby increasing the ability to perform biomarker discovery and provide diagnostic or prognostic type assays for individuals using urine samples with proteomic assays.

[0381] A titration experiment was performed to determine the total protein concentration of buffer-exchanged urine samples that yielded the most numerous analytes with a linear range. Eleven urine samples from human donors were buffer-exchanged into SB17 buffer using 2 mL Zeba spin desalting columns (400 μL urine load). Titration was performed by serial 2-fold dilutions starting with 80% "urine" while maintaining the oligonucleotide competitor (Z-block) concentration at 5 μM. A titration curve for each sample was fitted and the number of analytes in the linear range in the total protein space measured by NanoOrange®, as well as those above and below the linear range, were calculated and plotted.

[0382] FIG. 7 shows the number of analytes within, above, and below the linear range. The X-axis represents the total protein concentration of the sample (ranging from about 0 μg / mL to about 50 μg / mL), and in FIG. 7A, the Y-axis is the number of analytes measured, while in FIG. 7B, the same data is plotted but the Y-axis shows the percentage of analytes. In general, the data in FIG. 7 shows that the majority of analytes remained within the linear range when samples were normalized to a total protein concentration of about 10 μg / mL to 50 μg / mL and subjected to buffer exchange with assay buffer before measuring the analytes in the aptamer-based assay.

[0383] In summary, the data described herein demonstrate that test samples with total protein concentrations significantly greater than about 100 μg / mL (e.g., those described in Russell et al. “Potential of High-Affinity, Slow Off-Rate Modified Aptamer (SOMAmer) Reagents for Mycobacterium tuberculosis Proteins as Tools for Infection Models and Diagnostic Application.” J. Clin. Microbiol. 55(10):3072-3088 (2017)) have a reduced number of analytes within the linear range compared to test samples with total protein concentrations of 60 μg / mL or less.

[0384] Example 6: Use of the BCA assay to measure total protein in urine samples Protein concentrations of urine specimens were measured using the MicroBCA™ Protein Assay Kit (ThermoFisher, Cat. No. 23235). The assay was run in a semi-automated protocol using a Tecan Fluent 780 liquid handling platform. Protein standards were prepared using bovine serum albumin (BSA) and quality control (QC) samples were purchased commercially from Millipore Sigma (Cat. No. NIST927E).

[0385] 60 mL of microBCA working reagent was prepared using 30 mL of Reagent A, 28.8 mL of Reagent B, and 1.2 mL of Reagent C. Protein standards and QC samples were prepared in triplicate tubes by diluting known concentrations ranging from 4 to 64 µg / mL with 0.05x assay buffer to a final volume of 500 µL per tube. Urine samples were pH adjusted and buffer exchanged, then diluted 20x with deionized water.

[0386] Protein standards and QC samples were analyzed in triplicate, and "blank" buffer samples were analyzed in duplicate. Urine samples of unknown protein concentration were analyzed as single measurements, but each sample was tested at four different dilutions.

[0387] The operator initiated the Tecan Fluent liquid handling protocol, which included performing several sample dilution steps and mixing the microBCA working reagent with the 1:1 diluted sample. After completion of the automated protocol, the operator wrapped the sample mixed with working reagent in foil and placed it in a 37°C incubator for 2 hours. After the 2-hour incubation was completed, the operator centrifuged the samples at 1,000 x g for 1 minute and then measured the absorbance of all samples, including the standards.

[0388] An unweighted quadratic fit was used to generate a calibration curve correlating the absorbance of the standards with their known concentrations. The calibration curve was used to calculate the protein concentrations of the QC and urine samples. To verify the accuracy of the results, the concentrations of duplicate QC samples were calculated and confirmed to be within 20% of their known values.

[0389] Example 7: Use of the BCA assay in combination with an aptamer-based assay As described in Example 3, the total protein concentration in a urine sample affects the number of analytes that are within the linear range of an aptamer-based proteomic assay.

[0390] To quantify the preferred total protein concentration or concentration range in urine as measured by the BCA assay, a titration series of 10 urine samples was analyzed substantially as described in Example 3, and protein concentrations were measured using the BCA assay as described in Example 6. The number of aptamers (e.g., SOMAmer reagents) that gave a signal within the estimated linear range were counted for each total protein concentration value measured.

[0391] FIG. 11 shows the results, plotted as the number of aptamers (e.g., SOMAmer reagents) in the linear range versus protein concentration as measured by the microBCA assay described in Example 6. The optimal protein concentration in a urine test sample is the lowest concentration at which there appears to be no further increase in linear counts. Based on the results shown in FIG. 11, that point appears to be 100 μg / mL or even higher. However, concentrations above 100 μg / mL may be impractical to obtain in samples with variability that is typically at lower concentrations than this. Therefore, the optimal range was determined to be 70-100 μg / mL for the adjusted test samples.

[0392] The same data viewed in an alternative manner is shown in Figure 12. The number of aptamers within the linear range was quantified for protein concentrations ranging from 1 to 150 μg / mL. Figure 12A shows a plot of the counts as a function of protein concentration for each of the 10 samples tested. Figure 12B shows the average of these counts across all 10 samples as a function of protein concentration. As shown in Figure 12B, the maximum average count was achieved at 96 μg / mL, but there is a small increase in the number of aptamers within the linear range for protein concentrations of approximately 70 to 85 μg / mL and above.

[0393] Taken together, these data indicate that while for many analytes the linear range could be obtained at total protein concentrations ranging from about 2 μg / mL to about 200 μg / mL, the preferred total protein concentration range that yielded the greatest number of analytes within the linear range along with relatively "strong" RFU signals was about 70 μg / mL to 100 μg / mL as measured by microBCA.

Claims

1. 1. A method for preparing a plurality of biological samples for detecting proteins, comprising: performing buffer exchange on the plurality of biological samples with a formulation comprising a buffer, one or more salts, a chelating agent, and a non-ionic surfactant to generate a plurality of test samples; adjusting the total protein concentration of the plurality of test samples to produce a plurality of adjusted test samples, wherein each adjusted test sample has a total protein concentration of about 70 μg / mL to about 100 μg / mL; The method, wherein the method does not include concentrating the total protein of the multiple biological samples prior to performing the buffer exchange.

2. 1. A method for preparing a biological sample for detecting a protein, comprising: performing buffer exchange on the biological sample with a formulation comprising a buffer, one or more salts, a chelating agent, and a non-ionic surfactant to produce a test sample; and adjusting the total protein concentration of the test sample to produce an adjusted test sample, wherein the total protein concentration in the adjusted test sample is from about 70 μg / mL to about 100 μg / mL.

3. 3. The method of claim 1 or 2, comprising quantifying the protein concentration of the test sample or samples and / or the biological sample or samples.

4. 1. A method for preparing a biological sample for detecting a protein, comprising: generating a test sample by performing buffer exchange on the biological sample with a formulation comprising a buffer, one or more salts, a chelating agent, and a non-ionic surfactant; Quantifying the total protein concentration of the test sample; and adjusting the total protein concentration of the test sample to produce an adjusted test sample, wherein the total protein concentration in the adjusted test sample is from about 70 μg / mL to about 100 μg / mL.

5. 5. The method of any one of claims 1 to 4, comprising performing buffer exchange on a plurality of biological samples to generate a plurality of test samples, wherein each adjusted test sample has approximately the same total protein concentration.

6. 1. A method for preparing a biological sample for detecting a protein, comprising: generating a test sample by performing buffer exchange on the biological sample with a formulation comprising a buffer, one or more salts, a chelating agent, and a non-ionic surfactant; quantitating the protein concentration of the test sample; and if the total protein concentration of the test sample is not within the range of about 70 μg / mL to about 100 μg / mL, adjusting the total protein concentration of the test sample to produce an adjusted test sample, wherein the total protein concentration in the adjusted test sample is about 70 μg / mL to about 100 μg / mL. The method.

7. The method according to any one of claims 2 to 6, wherein the method does not include concentrating the biological sample.

8. The method of any one of claims 1 to 7, wherein the one or more salts are each independently selected from a sodium salt, a potassium salt, and a magnesium salt.

9. The method of any one of claims 1 to 8, wherein the one or more salts comprise sodium, potassium, and magnesium salts.

10. The sodium salt is NaCl, the potassium salt is KCl, and the magnesium salt is MgCl ２ The method according to claim 8 or 9,

11. 11. The method of claim 10, wherein the NaCl in the test sample is at a concentration of about 10 mM to about 500 mM, or about 50 mM to about 250 mM, or about 100 mM to about 200 mM, or about 102 mM.

12. 11. The method of claim 10, wherein the KCl in the test sample is at a concentration of about 0.5 mM to about 30 mM, or about 1 mM to about 20 mM, or about 2 mM to about 15 mM, or about 4 mM to about 10 mM, or about 5 mM.

13. The MgCl in the test sample ２ 11. The method of claim 10, wherein the concentration of is from about 0.5 mM to about 30 mM, or from about 1 mM to about 20 mM, or from about 2 mM to about 15 mM, or from about 4 mM to about 10 mM, or about 5 mM.

14. 14. The method of any one of claims 1 to 13, wherein the buffer is selected from HEPES, MES, bis-trismethane, ADA, ACES, bis-trispropane, PIPES, MOPSO, cholamine chloride, MOPS, BES, TES, DIPSO, MOB, acetamidoglycine, TAPSO, TEA, POPSO, HEPPSO, EPS, HEPPS, tricine, tris, glycinamide, glycylglycine, HEPBS, bicine, TAPS, AMPB, CHES, AMP, AMPSO, CAPSO, CAPS, and CABS.

15. 15. The method of any one of claims 1 to 14, wherein the buffer in the test sample is at a concentration of about 4 mM to about 400 mM, or about 10 mM to about 300 mM, or about 20 mM to about 200 mM, or about 30 mM to about 100 mM, or 35 mM to about 50 mM, or about 40 mM.

16. 16. The method of any one of claims 1 to 15, wherein the chelating agent is selected from EDTA, EGTA, DTPA, BAPTA, DMPS, and ALA.

17. 17. The method of any one of claims 1 to 16, wherein the chelating agent in the test sample is at a concentration of about 0.1 mM to about 10 mM, or about 0.5 mM to about 5 mM, or about 1 mM.

18. 18. The method according to any one of claims 1 to 17, wherein the non-ionic surfactant is selected from polyoxyethylene (20) sorbitan monolaurate (Tween-20), polyoxyethylene (40) sorbitan monolaurate (Tween-40), and polyoxyethylene (80) sorbitan monolaurate (Tween-80).

19. 19. The method of any one of claims 1-18, wherein the non-ionic surfactant is about 0.01% to about 1% of the test sample, or about 0.02% to about 0.5% of the test sample, or about 0.03% to about 0.1% of the test sample, or about 0.04% to about 0.08% of the test sample, or about 0.05% of the test sample (volume to volume).

20. The method of any one of claims 1 to 19, wherein the total protein concentration of the test sample is quantified by a bicinchoninic acid (BCA) assay or a microBCA assay.

21. 21. The method of claim 20, wherein the assay comprises (a) a bicinchoninic acid reagent, or (b) a bicinchoninic acid reagent, an alkaline tartaric acid-carbonate buffer, and a copper sulfate solution, or (c) a bicinchoninic acid reagent and (i) an alkaline tartaric acid-carbonate buffer, or (ii) a copper sulfate solution.

22. The test sample contained 40 mM HEPES, 102 mM NaCl, 5 mM KCl, 5 mM MgCl ２ 22. The method of any one of claims 1 to 21, comprising 1 mM EDTA, and 0.05% Tween-20.

23. 23. The method of any one of claims 1 to 22, wherein the pH of the test sample is from about pH 5 to about pH 9, or from about pH 6 to about pH 8, or from about pH 7 to about pH 7.9, or about pH 7.

5.

24. The method according to any one of claims 1 to 23, wherein the biological sample is a urine sample.

25. The method according to any one of claims 1 to 24, wherein the biological sample is a serum sample.

26. The method according to any one of claims 1 to 25, wherein the buffer exchange is not performed by ultrafiltration.

27. 27. The method according to any one of claims 1 to 26, wherein gel filtration chromatography is used for performing said buffer exchange on said biological sample in order to produce said buffer exchanged biological sample.

28. A method for detecting a target protein in a test sample prepared by the method of any one of claims 1 to 27, comprising: a) contacting the test sample with at least one capture reagent, the at least one capture reagent capable of binding to the target protein to form a complex; b) incubating the test sample with the at least one capture reagent under conditions that allow the formation of the complex; c) quantifying the level of the target protein in the test sample by measuring the level of the at least one capture reagent, the complex, or the protein, wherein the level of the at least one capture reagent or the complex is a surrogate for the level of the target protein; the test sample is generated by performing buffer exchange on the biological sample with a formulation comprising a buffer, a salt, a chelating agent, and a non-ionic surfactant; The method, wherein the total protein concentration of the test sample is greater than or equal to about 70 μg / mL and less than about 100 μg / mL.

29. The method of any one of claims 1 to 28, comprising measuring the protein concentration of the biological sample prior to performing the buffer exchange.

30. 30. The method of claim 29, wherein the protein concentration of the biological sample is measured by an assay selected from the group consisting of a fluorescent readout assay, a bicinchoninic acid (BCA) assay, a microBCA assay, a Lowry assay, and an ELISA assay.

31. 31. The method of claim 30, wherein the assay is a BCA assay or a microBCA assay.

32. 31. The method of claim 30, wherein the assay comprises (a) a bicinchoninic acid reagent, or (b) a bicinchoninic acid reagent, an alkaline tartaric acid-carbonate buffer, and a copper sulfate solution, or (c) a bicinchoninic acid reagent and (i) an alkaline tartaric acid-carbonate buffer, or (ii) a copper sulfate solution.

33. 31. The method of claim 30, wherein the assay is a fluorescent readout assay.

34. 34. The method of claim 33, wherein the fluorescent readout assay comprises a reagent selected from a merocyanine dye and epicocconone.

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