CA2460114A1 - Assay buffer, compositions containing the same, and methods of using the same - Google Patents

Assay buffer, compositions containing the same, and methods of using the same Download PDF

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CA2460114A1
CA2460114A1 CA002460114A CA2460114A CA2460114A1 CA 2460114 A1 CA2460114 A1 CA 2460114A1 CA 002460114 A CA002460114 A CA 002460114A CA 2460114 A CA2460114 A CA 2460114A CA 2460114 A1 CA2460114 A1 CA 2460114A1
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composition
ecl
electrochemiluminescence
buffer
acid
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CA2460114C (en
Inventor
Michael Tsionsky
Eli N. Glezer
Selen Altunata
George Sigal
Jonathan K. Leland
Mark A. Billadeau
Svetlana Leytner
Mark Martin
Larry Helms
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Meso Scale Technologies LLC
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2458/00—Labels used in chemical analysis of biological material
    • G01N2458/30—Electrochemically active labels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2458/00—Labels used in chemical analysis of biological material
    • G01N2458/40—Rare earth chelates
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/967—Standards, controls, materials, e.g. validation studies, buffer systems
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/968—High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
    • Y10T436/108331—Preservative, buffer, anticoagulant or diluent

Abstract

Compositions, reagents, kits, systems, system components, and methods for performing assays. More particularly, the invention relates to the use of novel combinations of reagents to provide improved assay performance.

Claims (169)

  1. CLAIMS:

    A composition comprising a pH buffer and a phospho-specific antibody, the composition being substantially free of inorganic phosphate.
  2. 2. A composition suitable.for use in an assay comprising a pH buffer and a phospho-specific antibody, the composition being substantially free of inorganic phosphate.
  3. 3. A composition comprising a pH buffer, a phospho-peptide specific antibody, and a phosphopeptide that binds said phospho-peptide specific antibody, said composition being substantially free of inorganic phosphate.
  4. 4. The composition of claim 1, 2 or 3, wherein said pH buffer is selected from the group consisting of glycylglycine, tris[hydroxymethyl)aminomethane or combinations thereof.
  5. 5. The composition of claim 1, 2, 3, or 4, further comprising one or more kinases and/or one or more kinase substrates.
  6. 6. The composition of claim l, 2, 3, or 4, further comprising one or more phosphatases and/or one or more phosphatase substrates.
  7. 7. The composition of claim 1, 2, 3, 5 or 6, wherein said pH buffer is glycylglycine.
  8. 8. The composition of claim 1, 2, 3, 5 or 6, wherein said pH buffer is tris[hydroxymethyl]aminomethane.
  9. 9. The composition of any preceding claim, wherein said composition comprises one or more ECL co-reactants.
  10. 10. The composition of claim 9, wherein said ECL co-reactant comprises a tertiary amine.
  11. 11. The composition of claim 9, wherein said ECL co-reactant comprises tripropylamine (TPA).
  12. 12. The composition of any preceding claim, further comprising KOH.
  13. 13. The composition of any preceding claim, further comprising a detergent or surfactant.
  14. 14. The composition of any preceding claim, further comprising a non-ionic detergent or surfactant.
  15. 15. The composition of any one of claims 1, 2 or 4-14, further comprising a phosphopeptide that binds said phospho-peptide specific antibody.
  16. 16. The composition of any preceding claim, wherein said composition is free of inorganic phosphate.
  17. 17. The composition of any preceding claim, further comprising a stop reagent.
  18. 18. The composition of any preceding claim, further comprising ethylenedyaminetetraacetic acid (EDTA).
  19. 19. The composition of any preceding claim, further comprising a base.
  20. 20. The composition of any preceding claim, further comprising KOH.
  21. 21. The composition of any one of claims 1-18, further comprising KOH and a surfactant or a detergent.
  22. 22. The composition of any preceding claim, further comprising an electrochemiluminescent label.
  23. 23. The composition of any preceding claim, further comprising a metal-containing electrochemiluminescent label.
  24. 24. The composition of any preceding claim, further comprising a Ru- or Os-containing electrochemiluminescent label.
  25. 25. The composition of any preceding claim, wherein said composition has a pH
    between 6 to 9.
  26. 26. The composition of any preceding claim, wherein said composition has a pH
    between 7 to 8.
  27. 27. The composition of any preceding claim, wherein said composition has a pH
    between 7.5 to 8.
  28. 28. The composition of any preceding claim, wherein said composition has a pH
    of about 7.8.
  29. 29. An apparatus adapted for performing electrochemiluminescence assays comprising the composition of any one of claims 1-28.
  30. 30. The apparatus of claim 29, further comprising a light detector for detecting light emitted from said composition and/or a voltage source for applying a voltage to said composition sufficient to induce electrochemiluminescence.
  31. 31. A kit comprising, in one or more containers, the composition of any one of claims 1-28 and an assay module comprising one or more working electrodes.
  32. 32. A method of performing an electrochemiluminescence method comprising applying electrochemical energy to the composition of any one of claims 1-28.
  33. 33. The method of claim 32, further comprising detecting emitted electrochemiluminescence.
  34. 34. A method of performing an assay using the composition of any one of claims 28, said method comprising forming an assay mixture comprising said composition and a kinase product and forming a complex comprising said kinase product and said phospho-specific antibody.
  35. 35. The method of claim 34, further comprising contacting a kinase with a kinase substrate to form said kinase product and detecting said complex.
  36. 36. The method of claim 34 or 35, wherein said complex is detected using electrochemiluminescence.
  37. 37. A method of performing an assay comprising forming a complex comprising a kinase product and a phospho-specific antibody, wherein said complex is not exposed to inorganic phosphate.
  38. 38. The method of claim 37, further comprising contacting a kinase with a kinase substrate to form said kinase product, contacting said kinase product with said phospho-specific antibody to form said complex and detecting said complex.
  39. 39. The method of claim 37 or 38, wherein said complex is detected using electrochemiluminescence.
  40. 40. A composition suitable for use in an electrochemiluminescence method comprising a pH buffer and an ECL co-reactant, wherein: said pH buffer is selected from the group consisting of glycylglycine, tris[hydroxymethyl]aminomethane or combinations thereof;
    and said ECL co-reactant comprises a tertiary amine.
  41. 41. The composition of claim 40, wherein said ECL co-reactant is tripropylamine
  42. 42. A composition suitable for use in an electrochemiluminescence method comprising a pH buffer, a Ru- or Os-containing ECL label and an ECL co-reactant, wherein said pH buffer is not inorganic phosphate.
  43. 43. The composition of claim 42, wherein said ECL co-reactant comprises a tertiary amine.
  44. 44. The composition of claim 43, wherein said ECL co-reactant is tripropylamine
  45. 45. A composition suitable for use in an electrochemiluminescence method comprising a pH buffer and an ECL coreactant, wherein said pH buffer is not inorganic phosphate and said coreactant is tripropylamine.
  46. 46. The composition of claim 42, 43, 44, or 45, wherein said pH buffer is a ECL
    assay buffer background reducing agent.
  47. 47. The composition of claim 40, 41, 42, 43, 44, 45 or 46, wherein the concentration of said coreactant is 50 to 1000 mM.
  48. 48. A composition suitable for use in an electrochemiluminescence method comprising: (a) tripropylamine having a concentration ranging from 50 to 1000 mM and (b) a pH
    buffer having a concentration ranging from 50 to 1000 mM, said pH buffer being selected from the group consisting of glycylglycine, tris[hydroxymethyl]-aminomethane or combinations thereof; wherein said composition has a pH ranging from 6.5 to 8.5.
  49. 49. The composition of claim 40, 41, 45 or 48, further comprising an ECL
    moiety.
  50. 50. The composition of claim 49, wherein said ECL moiety is a metal containing ECL
    moiety.
  51. 51. The composition of claim 50, wherein said ECL moiety is an Ru- or Os-containing ECL label.
  52. 52. The composition of claim 42, 43, 44, 49, 50 or 51, wherein said ECL moiety which is capable of repeatedly emitting electrochemiluminescence.
  53. 53. The composition of any of claims 40-52, wherein the concentration of inorganic phosphate is less than 20 mM.
  54. 54. The composition of any of claims 40-52, wherein said composition is substantially free of inorganic phosphate.
  55. 55. The composition of any of claims 40-52, wherein said composition is free of inorganic phosphate.
  56. 56. The composition of claim 54 or 55, further comprising one or more components selected from the group consisting of kinases and kinase substrate.
  57. 57. The composition of claim 54 or 55, further comprising one or more components selected from the group consisting of phosphatase and phosphatase substrate.
  58. 58. The composition of any one of claims 40-57, wherein said pH buffer is glycylglycine.
  59. 59. The composition of any one of claims 40-57, wherein said pH buffer is tris[hydroxymethyl]aminomethane.
  60. 60. The composition of any one of claims 40-59, further comprising ethylenediaminetetraacetic acid (EDTA).
  61. 61. The composition of any one of claims 40-60, further comprising KOH.
  62. 62. The composition of any one of claims 40-61, further comprising a substance selected from the group consisting of salt, a detergent, a preservative, a chelator and an anti-foaming agent.
  63. 63. The composition of any one of claims 40-62, further comprising a surfactant.
  64. 64. The composition of any one of claims 40-63, wherein said composition has a pH
    between 6.5 to 8.5.
  65. 65. The composition of any one of claims 40-64, wherein said composition has a pH
    between 7 to 8.
  66. 66. The composition of any one of claims 40-65, wherein said composition has a pH
    between 7.5 to 8.
  67. 67. The composition of any one of claims 40-66, wherein said composition has a pH
    of about 7.8.
  68. 68. The composition of any one of claims 40-67, wherein said composition is aqueous.
  69. 69. The composition of any one of claims 40-68, wherein said composition provides a suitable environment for inducing ECL from a metal-containing ECL moiety under oxidizing conditions.
  70. 70. A reagent suitable for use in an electrochemiluminescence method comprising the composition of any one of claims 40-69.
  71. 71. A reagent comprising a pH buffer and a Ru- or Os-containing ECL moiety, wherein said pH buffer is substantially free of inorganic phosphate and said reagent is suitable for use in providing a composition for conducting an ECL assay wherein electromagnetic radiation is emitted by an assay composition comprising members selected from the group consisting of:
    (i) a metal-containing ECL moiety capable of being converted to an excited state from which electromagnetic radiation is emitted;
    (ii) an ECL co-reactant which when oxidized forms a strong reducing agent; and (iii) an electrolyte capable of functioning as a medium in which said ECL
    moiety and said amine or amine moiety can be oxidized.
  72. 72. The reagent of claim 71, wherein said reagent further comprises said pH
    buffer, an amine or amine moiety and one of the other two members of said group (i)-(iii).
  73. 73. The reagent of claim 71, further comprising a surfactant or a detergent.
  74. 74. The reagent of claim 71, further comprising an ECL co-reactant.
  75. 75. The reagent of claim 71, further comprising EDTA.
  76. 76. A kit suitable for use in an electrochemiluminescence method comprising, in one or more containers, a pH buffer and an ECL co-reactant, wherein said pH buffer is selected from the group consisting of glycylglycine, tris[hydroxymethyl]aminomethane or combinations thereof, and said ECL co-reactant comprises a tertiary amine.
  77. 77. A kit suitable for use in an electrochemiluminescence method comprising, in one or more containers, a pH buffer, an Ru- or Os-containing ECL label and an ECL
    co-reactant, said composition being substantially free of inorganic phosphate.
  78. 78. A kit suitable for use in an electrochemiluminescence method comprising, in one or more containers, a pH buffer and tripropylamine, said composition being substantially free of inorganic phosphate.
  79. 79. A kit suitable for use in an electrochemiluminescence method comprising, in one or more containers: (a) tripropylamine having a concentration ranging from 50 to 1000 mM and (b) a pH buffer having a concentration ranging from 50 to 1000 mM, said pH
    buffer being selected from the group consisting of glycylglycine, tris[hydroxymethyl]-aminomethane or combinations thereof.
  80. 80. The kit of claim 78, comprising tripropylamine in a concentration ranging from 50 to 1000 mM.
  81. 81. The kit of claim 76, 77, 78, 79 or 80, wherein said kit is free of inorganic phosphate.
  82. 82. The kit of claim 76, 77, 78, 79, 80 or 81, further comprising one or more components selected from the group consisting of kinases and kinase substrate.
  83. 83. The kit of claim 76, 77, 78, 79, 80 or 81, further comprising one or more components selected from the group consisting of phosphatase and phosphatase substrate.
  84. 84. The kit of any one of claims 76-83, wherein said pH buffer is glycylglycine.
  85. 85. The kit of any one of claims 76-83, wherein said pH buffer is tris[hydroxymethyl]aminomethane.
  86. 86. The kit of any one of claims 77 or 81-83, wherein said ECL co-reactant comprises a tertiary amine.
  87. 87. The kit of claim 76 or 77, wherein said ECL co-reactant comprises tripropylamine (TPA).
  88. 88. The kit of any one of claims 76-87, further comprising ethylenedyaminetetraacetic acid (EDTA).
  89. 89. The kit of claim 76, 77, or 78, wherein said pH buffer is glycylglycine and said composition further comprises ethylenedyaminetetraacetic acid (EDTA).
  90. 90. The kit of any one of claims 76-89, further comprising KOH.
  91. 91. The kit of any one of claims 76 or 78-90, further comprising an ECL
    moiety.
  92. 92. The kit of any one of claims 76 or 78-90, further comprising an ECL moiety which is capable of repeatedly emitting electrochemiluminescence.
  93. 93. The kit of any one of claims 76 or 78-90, further comprising a metal containing ECL moiety.
  94. 94. The kit of any one of claims 76 or 78-90, further comprising a Ru- or Os-containing ECL label.
  95. 95. The kit of any one of claims 76-94, further comprising one or more substances selected from the group consisting of a salt, a detergent, a preservative, a chelator and an anti-foaming agent.
  96. 96. The kit of any one of claims 76-95, further comprising a surfactant.
  97. 97. The kit of any one of claims 76-96, wherein said kit is aqueous.
  98. 98. The kit of any one of claims 76, 78-89 or 95-97, wherein said kit further comprises at least one separate component in which one or more members of the group consisting of an ECL moiety (i) and electrolyte (ii) is included.
  99. 99. A method of effecting a specific-binding assay, either qualitatively or quantitatively, in a composition comprising: (a) a pH buffer substantially free of inorganic phosphate and (b) a phospho-specific antibody.
  100. 100. The method of claim 99, wherein said composition further comprises an ECL
    co-reactant.
  101. 101. The method of claim 99, wherein said composition further comprises an ECL
    label.
  102. 102. The method of claim 101, wherein said composition further comprises a phospho-specific antibody.
  103. 103. A method of effecting a specific-binding non-wash assay, either qualitatively or quantitatively, in a well having one or more assay domains with binding reagents immobilized thereon using a composition comprising a pH buffer substantially free of inorganic phosphate.
  104. 104. A method of performing a kinase assay using a composition comprising an electrochemiluminescence label and at least one reagent selected from the group consisting of a kinase, a kinase substrate, a kinase product and combinations therefore, said composition being substantially free of inorganic phosphate.
  105. 105. The method of claim 104, further comprising applying electrochemical energy effective to induce the electrochemiluminescent label to emit electromagnetic radiation; and detecting emitted electrochemiluminescence.
  106. 106. The method of claim 104 or 105, further comprising contacting said composition with a phospho-specific antibody.
  107. 107. A method of performing an assay comprising:
    (a) forming a complex comprising a kinase product and a phospho-specific antibody, wherein said complex is not exposed to inorganic phosphate;
    (b) inducing a metal-containing ECL moiety to emit electromagnetic radiation;
    and (c) detecting emitted electromagnetic radiation.
  108. 108. The method of claim 107, wherein said complex further comprises said metal-containing ECL moiety.
  109. 109. A method for inducing electrochemiluminescence wherein said electrochemiluminesence is induced in the presence of a composition comprising an ECL label, an ECL coreactant and a pH buffer, wherein said pH buffer is not phosphate.
  110. 110. The method of claim 109, wherein said pH buffer is an ECL assay buffer background reducing agent.
  111. 111. The method of claim 109, wherein said pH buffer is selected from the group consisting of glycylglycine, tris[hydroxymethyl]aminomethane or combinations thereof.
  112. 112. The method of claim 109, 110 or 111, wherein said composition is substantially free of inorganic phosphate.
  113. 113. The method of claim 109, 110, 111 or 112, wherein said ECL co-reactant is a tertiary amine.
  114. 114. The method of claim 109, wherein said coreactant is present at a concentration ranging from 50 to 1000 mM, said pH buffer is present at a concentration ranging from 50 to 1000 mM, said pH is between 6.5 and 8.5, said ECL coreactant is tripropylamine, and said pH
    buffer is selected from the group consisting of glycylglycine, tris[hydroxymethyl]-aminomethane or combinations thereof.
  115. 115. The method of any one of claims 109-114, wherein said ECL label is an organometallic complex of ruthenium or osmium.
  116. 116. The method of any one of claims 109-115, wherein said method further comprises:
    (a) exposing said composition to electrochemical energy to induce the electrochemiluminescence composition to emit electromagnetic radiation; and (b) detecting emitted electromagnetic radiation.
  117. 117. The method of claim 116, wherein said electrochemical energy is generated by applying an oxidizing potential to an electrode.
  118. 118. The method of claim 117, wherein said ECL label is attached to said electrode.
  119. 119. The method of claim 117 or 118, wherein said electrode comprises elemental carbon.
  120. 120. An electrochemiluminescence assay buffer comprising an ECL co-reactant and at least one non-phosphate-based pH buffer, wherein said ECL co-reactant is a tertiary amine.
  121. 121. The electrochemiluminescence assay buffer of claim 120, wherein said co-reactant is TPA.
  122. 122. The electrochemiluminescence assay buffer of claim 120, wherein said non-phosphate-based pH buffer is selected from tris-[hydroxymethyl]aminomethane (Tris), oligo(glycines) or combinations thereof.
  123. 123. The electrochemiluminescence assay buffer of claim 120, wherein said non-phosphate-based pH buffer is glycyl-glycine (Gly-Gly).
  124. 124. The electrochemiluminescence assay buffer of any one of claims 120-123, further comprising one or more substances selected from the group consisting of a chelator, a detergent, a salt, an anti-foaming agent and a preservative.
  125. 125. The electrochemiluminescence assay buffer of any of claims 120-124, further comprising a phospho-specific antibody.
  126. 126. A method of generating electrochemiluminescence comprising contacting an ECL
    label with the assay buffer of any one of claims 120-125, inducing electrochemiluminescence and detecting emitted electrochemiluminescence.
  127. 127. A composition comprising an electrochemiluminescent label, a non-TPA
    electrochemiluminescence coreactant and at least one phenyl ether-containing detergent.
  128. 128. The composition of claim 127, wherein said non-TPA
    electrochemiluminescence coreactant is PIPES.
  129. 129. The composition of claim 127, wherein said non-TPA
    electrochemiluminescence coreactant is selected from the group consisting of 3-(di-n-propylamino)-propanesulfonic acid; 4-(di-n-propylamino)-butanesulfonic acid; 4-[bis-(2-hydroxyethane)-amino]-butanesulfonic acid;
    piperidine-N-(3-propanesulfonic acid); azepane-N-(3-propanesulfonic acid);
    piperidine-N-(3-propionic acid) (PPA); 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO); 3-morpholinepropanesulfonic acid (MOPS); N-(2-hydroxyethyl)piperazine-N'-3-propanesulfonic acid (EPPS); N-(2-hydroxyethyl)piperazine-N'-3-ethanesulfonic acid (BES);
    piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES); triethanolamine; N-2-hydroxypiperazine-N-2-ethanesulfonic acid (HEPES); piperazine-N,N'-bis-4-butanesulfonic acid; homopiperidine-N-3-propanesulfonic acid; piperazine-N,N'-bis-3-propanesulfonic acid; piperidine-N-3-propanesulfonic acid;
    piperazine-N-2-hydroxyethane-N'-3-methylpropanoate; piperazine-N,N'-bis-3-methylpropanoate; 1,6-diaminohexane-N,N,N',N'-tetraacetic acid; N,N-bis propyl-aminobutanesulfonic acid; N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid (TES);

    1,3-bis[tris(hydroxymethyl)methylamino]propane (bis-Tris propane); 3-dimethylamino-1-propanol; 3-dimethylamino-2-propanol; N,N,N',N'-tetrapropylpropane-1,3,-diamine (TPA
    dimer); piperazine-N,N'-bis(2-hydroxypropane)sulfonic acid (POPSO) and 2-hydroxy-3-[4-(2-hydroxyethyl)piperazin-1-yl]propane-1-sulfonic acid (HEPPSO).
  130. 130. The composition of claim 127, 128 or 129, wherein said composition is in contact with a working electrode.
  131. 131. The composition of claim 127, 128 or 129, wherein said composition is in contact with a carbon-based working electrode.
  132. 132. The composition of any of claims 127-131, wherein said electrochemiluminescent label is a Ru- or Os- containing ECL label.
  133. 133. A composition comprising 3-(di-n-propylamino)-propanesulfonic acid or 4-(di-n-propylamino)-butanesulfonic acid.
  134. 134. The composition of claim 133, further comprising an ECL label.
  135. 135. The composition of claim 133, further comprising a metal-containing ECL
    label.
  136. 136. The composition of claim 133, further comprising a Ru- or Os- containing ECL
    label.
  137. 137. A method of inducing electrochemiluminescence using the composition of any one of claims 133-136.
  138. 138. A method of generating electrochemiluminescence comprising:
    (a) contacting an electrochemiluminescent label with the composition of claim 136;

    (b) inducing said electrochemiluminescent label to emit electrochemiluminescence; and (c) detecting said electrochemiluminescence.
  139. 139. A method of generating electrochemiluminescence comprising:
    (a) inducing the composition of any one of claims 133-137 to emit electrochemiluminescence; and (b) detecting said electrochemiluminescence.
  140. 140. The method of claim 138 or 139, wherein said electrochemiluminescent label is attached to a working electrode.
  141. 141. The method of claim 138 or 139, wherein said ECL induction is achieved in a composition substantially free of detergent.
  142. 142. A kit comprising, in one or more containers, an electrochemiluminescent label, a non-TPA electrochemiluminescence co-reactant and at least one phenyl ether-containing detergent.
  143. 143. A kit containing, in one or more containers, one or more ECL assay buffers comprising at least one trialkylamine non-TPA coreactant.
  144. 144. A kit comprising, in one or more containers, a metal-containing electrochemiluminescent label and at least one coreactant selected from the group consisting of 3-(di-n-propylamino)-propanesulfonic acid, 4-(di-n-propylamino)-butanesulfonic acid, and combinations thereof.
  145. 145. A method of generating electrochemiluminescence comprising:

    (a) contacting an electrochemiluminescent label with an electrochemiluminescence coreactant;
    (b) inducing said electrochemiluminescent label to emit electrochemiluminescence; and (c) detecting said electrochemiluminescence, wherein said electrochemiluminescence coreactant is a tertiary amine other than TPA.
  146. 146. A method of inducing electrochemiluminescence comprising contacting an electrochemiluminescent label with an electrochemiluminescence coreactant, wherein said electrochemiluminescence coreactant is a tertiary amine other than tri-n-propylamine and said electrochemiluminescent label is attached to a working electrode.
  147. 147. A method of performing an electrochemiluminescence assay comprising:
    (a) forming a composition comprising an electrochemiluminescent label and at least one electrochemiluminescence coreactant, wherein said electrochemiluminescence coreactant is a tertiary amine other than tri-n-propylamine and said electrochemiluminescent label is attached to a working electrode;
    (b) applying electrochemical energy effective to induce the electrochemiluminescent label to emit electrochemiluminescence; and (c) detecting emitted electrochemiluminescence.
  148. 148. A method of performing an electrochemiluminescence assay comprising:
    (a) forming a composition comprising electrochemiluminescent labels and at least one electrochemiluminescence coreactant, wherein said electrochemiluminescence coreactant is a tertiary amine other than tri-n-propylamine and wherein a first portion of said electrochemiluminescent labels is attached to a working electrode and a second portion of said electrochemiluminescent labels is in solution;
    (b) applying electrochemical energy effective to induce the first portion of said electrochemiluminescent labels to emit electrochemiluminescence; and (c) selectively detecting electrochemiluminescence emitted from said first portion of electrochemiluminescent labels.
  149. 149. A method of conducting an assay for an analyte or activity of interest comprising (a) forming a composition comprising a sample, an assay reagent comprising an ECL
    label and an electrode, wherein the presence of said analyte or activity in said sample leads to the attachment or dissociation of said assay reagent from said electrode;
    (b) contacting said electrode with an ECL assay buffer comprising an ECL
    coreactant, wherein said coreactant is a tertiary amine other than TPA;
    (c) applying electrochemical energy to said electrode under conditions suitable to induce ECL labels on said electrode to induce ECL; and (d) measuring emitted ECL.
  150. 150. A method for measuring a binding event comprising:
    (a) contacting a binding reagent immobilized on an electrode with an assay reagent comprising an ECL label, wherein said binding reagent specifically binds said assay reagent;
    (b) binding said assay reagent to said binding reagent so as to attach said label to said electrode;
    (c) contacting said electrode with an ECL assay buffer comprising an ECL
    coreactant, wherein said coreactant is a tertiary amine other than TPA;

    (d) applying electrochemical energy to said electrode under conditions suitable to induce ECL labels on said electrode to induce ECL; and (e) measuring emitted ECL.
  151. 151. The method of any of claims 145-150, wherein said coreactant has one or more of the following properties: i) it is oxidized on carbon-based electrodes in a one electrode oxidation to give an amine radical cation which can subsequently lose a proton to form a radical reductant;
    ii) has an oxidation potential on carbon-based electrodes that is comparable or greater than that of Ru(II)(bpy)3; iii) can be oxidized at a potential less than that required to breakdown water at a carbon-based electrode; iv) the energy released by the reaction of the radical reductant with Ru(III)(bpy)3 to produce Ru(II)(bpy)3 is sufficient to produce Ru(In(bpy)3 in a luminescent excited state; and v) the lifetimes of the amine radical canon and/or radical reductant are less than the corresponding TPA-derived species.
  152. 152. The method of any one of claims 145-151, wherein said coreactant forms an amine radical cation and/or radical reductant and the diffusion distance of the amine radical cation and/or radical reductant is less than 100 nm.
  153. 153. The method of any one of claims 145-152, wherein said method is performed without a wash step.
  154. 154. The method of any one of claims 145-153, wherein the replacement of the ECL
    coreactant with TPA leads to an at least two folds drop in the selectivity for inducing ECL from labels attached to the electrode relative to labels in solution.
  155. 155. The method of any of claims 145-154, wherein said electrochemiluminescence coreactant is PIPES.
  156. 156. The method of any one of claims 145-154, wherein said coreactant is an amine having a structure NR1R2R3, wherein R1, R2 and R3 are alkyl groups comprising at least 2 carbons and wherein one or more of R1, R2 and R3 are functionalized with a hydrophilic functional group
  157. 157. The method of claim 156, wherein said hydrophilic functional group comprises a charged group
  158. 158. The method of claim 157, wherein said hydrophilic functional group comprises a negatively charged group.
  159. 159. The method of claim 157, wherein said hydrophilic functional group includes hydroxyl, dialkylamino, sulfate, sulfonate, carboxylate and/or carboxylic acid ester.
  160. 160. The method of any one of claims 145-154, wherein said coreactant has the structure (n-Pr)2N(CH2)n R, wherein n is greater than or equal to 2 and R is a hydrophilic functional group.
  161. 161. The method of any one of claims 145-154, wherein said coreactant has the formula below and wherein i) X is -(CH2)- or a heteroatom; ii) R and R1 are alkyl groups comprising 2 or more carbons; iii) n and m are each greater than or equal to 1; and iv) R (and, optionally R1) comprise a hydrophilic functional group.
  162. 162. The method of any one of claims 145-154, wherein said coreactant is selected from the group consisting of: 3-(di-n-propylamino)-propanesulfonic acid; 4-(di-n-propylamino)-butanesulfonic acid; 4-[bis-(2- hydroxyethane)-amino]-butanesulfonic acid;
    piperidine-N-(3-propanesulfonic acid); azepane-N-(3-propanesulfonic acid); piperidine-N-(3-propionic acid) (PPA); 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO); 3-morpholinepropanesulfonic acid (MOPS); N-(2-hydroxyethyl)piperazine-N'-3-propanesulfonic acid (EPPS); N-(2-hydroxyethyl)piperazine-N'-3-ethanesulfonic acid (BES); piperazine-N,N'-bis(2-ethanesulfonic acid)(PIPES); triethanolamine; N-2-hydroxypiperazine-N-2-ethanesulfonic acid (HEPES);
    piperazine-N,N'-bis-4-butanesulfonic acid; homopiperidine-N-3-propanesulfonic acid;
    piperazine-N,N'-bis-3-propanesulfonic acid; piperidine-N-3-propanesulfonic acid; piperazine-N-2-hydroxyethane-N'-3-methylpropanoate; piperazine-N,N'-bis-3-methylpropanoate;
    1,6-diaminohexane-N,N,N',N'-tetraacetic acid; N,N-bis propyl-N-4-aminobutanesulfonic acid; N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid (TES); 1,3-bis[tris(hydroxymethyl)methylamino]propane (bis-Tris propane); 3-dimethylamino-1-propanol;
    3-dimethylamino-2-propanol; N,N,N',N'-tetrapropylpropane-1,3,-diamine (TPA
    dimer);
    piperazine-N,N'-bis(2-hydroxypropane)sulfonic acid (POPSO) and 2-hydroxy-3-[4-(2-hydroxyethyl)piperazin-1-yl]propane-1-sulfonic acid (HEPPSO).
  163. 163. The method of any one of claims 145-154, wherein said coreactant is selected from the group consisting of: piperidine-N-(3-propionic acid) and piperazine-N,N'-bis(2-ethanesulfonic acid).
  164. 164. The method of any one of claims 145-163, wherein said electrochemiluminescence is induced using one or more carbon-based working electrodes.
  165. 165. The method of claim 164, wherein said one or more carbon-based working electrodes comprise carbon particles or carbon nanotubes.
  166. 166. The method of any one of claims 145-165, wherein said ECL label is also contacted with at least one phenyl ether-containing detergent.
  167. 167. The method of any one of claims 145-166, wherein the concentration of said coreactant is between 10 and 800 mM.
  168. 168. The method of any one of claims 146-167, further comprising contacting said label with a pH buffering agent.
  169. 169. The method of claim 168, wherein the concentration of the pH buffering agent is preferably between 0 and 800 mM.
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