Antibody complexes and methods for immunolabeling

• US 20030073149A1
• Filed: 04/05/2002
• Published: 04/17/2003
• Est. Priority Date: 10/12/2001
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Archer, Robert A., Haugland, Richard P., Beechem, Joseph M., Haugland, Rosaria, Hagen, David C.
• Granted Patent: US 8,323,903 B2
• Time in Patent Office: Days
• US Class Current: 435/7.92
• CPC Class Codes: B82Y 10/00 Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: G01N 33/53 Immunoassay; Biospecific binding assay; Materials therefor
• CPC Class Codes: G01N 33/58 involving labelled substances G01N33/53 takes precedence
• CPC Class Codes: G01N 33/6857 Antibody fragments

Abstract

The present invention provides novel immunolabeling complexes and certain components of such complexes, as well as methods of preparing and using such complexes, and kits for use in preparing labeling proteins and for immunolabeling. The pre-formed immunolabeling complexes of the invention comprise both a target-binding antibody and a labeling protein that contains covalently attached labels, where the labeling protein binds selectively and with high affinity to a selected region of the target-binding antibody. Novel labeling proteins of the invention include non-antibody peptides and proteins, such as a complex of protein G and a labeled albumin, and monovalent antibody fragments, such as labeled Fab fragments of an anti-Fc antibody. In methods of the invention, the preformed immunolabeling complexes are added to the sample alone or in combination, for purposes of labeling and optionally detecting the target of interest.

Claims

1. A method for determining the presence of a desired target in a biological sample, said method comprising:

a) combining an immunolabeling mixture with said sample wherein said immunolabeling mixture comprises a target-binding antibody bound to a labeling protein, wherein said labeling protein comprises a monovalent antibody fragment or a non-antibody peptide or protein, wherein said labeling protein is covalently linked to one or more labels; and

b) detecting the presence or absence of said label whereby the presence or absence of said target is determined.

2. The method according to claim 1, wherein said labeling mixture contains at least one target-binding antibody bound by said labeling protein wherein said target-binding antibody is a single chain fragment variable, and said labeling protein binds selectively and with high affinity to a selected peptide linker region of the target-binding antibody.

3. The method according to claim 1, wherein the labeling mixture contains two or more target-binding antibodies bound by labeling proteins, wherein one of said target-binding antibodies is bound by a first labeling protein and another said target-binding antibody is bound by a second labeling protein.

4. The method according to claim 1, wherein said target-binding antibody has two or more different labeling proteins bound to the antibody and at least one labeling protein is detectably distinct from other labeling proteins.

5. The method according to claim 1, wherein said non-antibody protein is selected from the group consisting of a protein G, a protein A, a protein L, a lectin, and a protein G bound to albumin, wherein said albumin is covalently linked to one or more labels and albumin is selected from the group consisting of human albumin, bovine serum albumin, and ovalbumin.

6. The method according to claim 1, wherein said monovalent antibody fragment is a Fab or Fab′

fragment and is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment.

7. The method according to claim 6, wherein said antibody fragment is derived from a monoclonal antibody or polyclonal antibody.

8. The method according to claim 7, wherein said polyclonal antibody is a rabbit or a goat polyclonal antibody.

9. The method according to claim 7, wherein said monoclonal antibody is classified as mouse IgM, IgG₁, IgG_2a, IgG_2b, or IgG₃ antibody, or an equivalent thereof.

10. The method according to claim 1, wherein said label is selected from the group consisting of a fluorescent dye, a phosphorescent dye, a tandem dye, a particle, an electron transfer agent, biotin and a radioisotope.

11. The method according to claim 10, wherein said fluorescent dye is selected from the group consisting of a fluorescent sulfonated xanthene dye, a fluorescent sulfonated cyanine dye, and a fluorescent phycobiliprotein dye.

12. The method according to claim 10, wherein said tandem dye is selected from the group consisting of sulfonated cyanine-phycobiliprotein tandem dye and sulfonated xanthene-phycobiliprotein tandem dye.

13. The method according to claim 1, wherein said biological sample comprises eukaryotic or prokaryotic cells, cellular extract, subcellular component, tissue culture, tissue, a bodily fluid, or a portion or combination thereof.

14. The method according to claim 1, wherein said target-binding antibody binds to an antigenic determinate present in the biological sample of claim 13.

15. The method according to claim 13, wherein said sample is immobilized on a surface.

16. The method according to claim 13, wherein said sample is in a gel or on a blot or in an array.

17. The method according to claim 1, wherein the amount of target-binding antibody is about 5 micrograms to about 0.001 micrograms.

18. The method according to claim 1, further comprising binding of unbound labeling protein prior to combining labeling mixture with sample, wherein a capture component is added to the labeling mixture and binding said unbound labeling protein.

19. The method of claim 18, wherein said capture component is an antibody or an antibody fragment, and is optionally attached to a microsphere or to agarose.

20. The method according to claim 1, wherein said labeling mixture contains at least one target-binding antibody bound to a labeling protein and

i) the labeling protein is a fragment of a rabbit or goat polyclonal antibody or of a mouse IgG₁, IgG_2a, IgG_2b, IgG3 antibody,

ii) the labeling protein is an anti-Fc antibody fragment that is a monovalent Fab or Fab′

fragment, wherein the labeling protein is covalently labeled with one or more labels that are selected from the group consisting of fluorescent sulfonated xanthene dye, fluorescent sulfonated cyanine dye, fluorescent phycobiliprotein dye, sulfonated cyanine-phycobiliprotein tandem dye, and sulfonated xanthene-phycobiliprotein tandem dye, which labels are optionally the same or different; and

the capture component is an antibody or an antibody fragment, which is optionally attached to a microsphere or to agarose.

83. A method according to claim 1, further comprising stabilizing binding of said target-binding antibody to said labeling protein.

21. A method for determining the presence of a desired target in a biological sample, said method comprising:

a) combining an immunolabeling mixture with said sample wherein said immunolabeling mixture comprises a first target-binding antibody bound to a first labeling protein and a second target-binding antibody bound to a second labeling protein, wherein said first and second labeling proteins comprise a monovalent antibody fragment or a non-antibody peptide or protein that are detectably distinct, and wherein said labeling proteins are covalently linked to one or more labels; and

b) detecting the presence or absence of said labels whereby the presence or absence of said target is determined.

22. The method according to claim 21, wherein said first labeling protein is a non-antibody peptide or protein and said second labeling protein is a non-antibody peptide or protein.

24. The method according to claim 22, wherein said first and second non-antibody proteins are independently selected from the group consisting of a protein G, a protein A, a protein L, a lectin, and a G protein bound to albumin, wherein said albumin is covalently linked to one or more labels and albumin is selected from the group consisting of human albumin, bovine serum albumin, and ovalbumin.

25. The method of claim 21, wherein said first labeling protein is a first monovalent antibody fragment and said second labeling protein is a second monovalent antibody fragment.

26. The method according to claim 25, wherein said first and second monovalent antibody fragments are independently Fab or Fab′

fragments and are selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment.

27. The method according to claim 25, wherein said first and second antibody fragments are derived from a monoclonal antibody or polyclonal antibody.

28. The method according to claim 27, wherein said polyclonal antibody is a rabbit or a goat polyclonal antibody.

29. The method according to claim 27, wherein said monoclonal antibody is classified as mouse IgM, IgG₁, IgG_2a, IgG_2b, or IgG₃ antibody, or an equivalent thereof.

30. The method according to claim 21, wherein said labels are selected from the group consisting of a fluorescent dye, a phosphorescent dye, a tandem dye, a particle, an electron transfer agent, biotin and a radioisotope.

31. The method according to claim 30, wherein said fluorescent dye is selected from the group consisting of a fluorescent sulfonated xanthene dye, a fluorescent sulfonated cyanine dye, and a fluorescent phycobiliprotein dye.

32. The method according to claim 30, wherein said tandem dye is selected from the group consisting of sulfonated cyanine-phycobiliprotein tandem dye and sulfonated xanthene-phycobiliprotein tandem dye.

33. The method according to claim 21, wherein said biological sample comprises eukaryotic or prokaryotic cells, cellular extract, subcellular component, tissue culture, tissue, a bodily fluid, or a portion or combination thereof.

34. The method according to claim 33, wherein said target-binding antibody binds to an antigenic determinate present in said biological sample.

35. The method according to claim 34, wherein the amount of target-binding antibody is about 5 micrograms to about 0.001 micrograms.

36. The method of claim 21, wherein said sample is immobilized on a solid surface.

37. The method of claim 21, wherein said sample is in a gel, or on a blot or in an array.

38. The method according to claim 21, further comprising binding of unbound labeling protein prior to combining labeling mixture with sample, wherein a capture component is added to the labeling mixture binding said unbound labeling protein.

39. The method of claim 38, wherein said capture component is an antibody or an antibody fragment, and is optionally attached to a microsphere or to agarose.

40. A method according to claim 21 wherein said labeling mixture contains at least two target-binding antibodies each bound by at least one labeling protein;

and

a) said first and second labeling proteins are independently a fragment of a rabbit or goat polyclonal antibody or a mouse IgG₁, IgG_2a, IgG_2b, IgG₃ antibody,

b) said first and second labeling proteins are an anti-Fc antibody fragment that is a monovalent Fab or Fab′

fragment, wherein said first and second labeling proteins are covalently labeled with one or more labels that are independently selected from the group consisting of fluorescent sulfonated xanthene dye, fluorescent sulfonated cyanine dye, fluorescent phycobiliprotein dye, sulfonated cyanine-phycobiliprotein tandem dye, and sulfonated xanthene-phycobiliprotein tandem dye, which labels are optionally the same or different; and

the capture component is an antibody or an antibody fragment, which is optionally attached to a microsphere or to agarose.

41. A method for determining the presence of a desired target in a biological sample, said method comprising:

a) combining an immunolabeling mixture with said sample wherein said immunolabeling mixture comprises a target-binding antibody bound to a labeling protein, wherein said labeling protein comprises a monovalent antibody fragment or a non-antibody peptide or protein, and wherein said labeling protein is covalently linked to one or more labels; and

b) adding an enzymatic substrate, which is selected from the group consisting of a chromogenic, a fluorogenic and chemiluminescent substrate to the sample combined with said immunolabeled mixture; and

c) detecting the presence or absence of said label whereby the presence or absence of said target is determined.

42. The method according to claim 41, wherein said substrate is a peroxidase substrate, a luciferase substrate, a phosphatase substrate, a glycosidase substrate or a luciferase substrate..

43. The method according to claim 42, wherein said substrate is activated by an enzyme, which is selected from the group consisting of peroxidase, phosphatase, glycosidase, and luciferase.

44. The method according to claim 43, wherein said peroxidase is horseradish peroxidase, Coprinus cinereus peroxidase, or Arthromyces ramosus peroxidase.

45. The method according to claim 43, wherein said phosphatase is alkaline phosphatase.

46. The method according to claim 43, wherein said glycosidase is β

-galactosidase or β

-glucuronidase.

47. The method according to claim 43, wherein said luciferase is protein A luciferase or protein G luciferase.

48. An isolated immunolabeling complex consisting essentially of:

a target-binding antibody bound by one or more labeling proteins that is a non-antibody peptide or protein, or a monovalent antibody fragment, wherein said labeling protein(s) bind selectively and with high affinity to a selected region of the target-binding antibody, and wherein said labeling protein is covalently linked to one or more labels.

49. The immunolabeling complex according to claim 48, wherein at least one labeling protein is detectably distinct from the other labeling proteins.

50. An isolated immunolabeling complex consisting essentially of:

a target-binding antibody bound by one or more monovalent labeling proteins that is a non-antibody immunoglobulin-binding peptide or protein, or is a monovalent antibody fragment, wherein said labeling protein binds selectively and with high affinity to a selected region of the target-binding antibody, and where each labeling protein is covalently labeled with an enzyme.

51. An isolated immunolabeling complex consisting essentially of:

a target-binding antibody bound by one or more monovalent labeling proteins that is a non-antibody immunoglobulin-binding peptide or protein, or is a monovalent antibody fragment, wherein said labeling protein binds selectively and with high affinity to a selected region of the target-binding antibody, and where each labeling protein is covalently labeled with a hapten.

52. An isolated monovalent labeling protein consisting essentially of a monovalent fragment generated from an antibody and capable of binding selectively and with high affinity to a selected region of a second antibody, wherein said monovalent protein is covalently labeled with one or more labels, and which labels are optionally the same or different.

53. An isolated labeling mixture comprising one or more immunolabeling complexes, wherein at least one immunolabeling complex consists essentially of:

a target-binding antibody bound by a labeling protein, that is a non-antibody peptide or protein, or is a monovalent antibody fragment, wherein said labeling protein binds selectively and with high affinity to a selected region of the target-binding antibody, and the labeling protein is covalently labeled with one or more labels, which labels are optionally the same or different.

54. The isolated labeling mixture according to claim 53, further comprising a capture component that is an antibody or antibody fragment and is optionally attached to a microsphere or to agarose.

55. An isolated labeling mixture comprising one or more immunolabeling complexes, wherein at least one immunolabeling complex consist essentially of a target-binding antibody bound by a labeling protein that is a protein G complexed with albumin, wherein said albumin is covalently labeled with one or more labels, and which labels are optionally the same or different.

56. The isolated labeling mixture according to claim 55, wherein said albumin is human albumin, bovine serum albumin, or ovalbumin.

57. An isolated protein G complex consisting essentially of protein G and albumin, wherein said albumin is covalently labeled with one or more labels, which labels are optionally the same or different.

58. The isolated protein G complex according to claim 57, wherein said albumin is human albumin, bovine serum albumin, or ovalbumin.

59. A kit for preparing an immunolabeling complex, said kit comprising:

a) a labeling mixture consisting essentially of one or more monovalent antibody fragments that bind selectively and with high affinity to a specific region of one or more target-binding antibodies, where each fragment is covalently labeled with one or more labels, and which said labels are optionally the same or different and a capture component to which the antibody fragments bind, that is optionally attached to a microsphere or to agarose.

60. A kit for preparing an immunolabeling complex, said kit comprising:

a) a protein G complex with albumin, wherein said albumin is covalently labeled with one or more labels, and which labels are optionally the same or different; and

b) a capture component to which the protein G complexed with albumin binds, wherein said capture component is optionally attached to a microsphere or to agarose.

61. A kit for immunolabeling a desired target in a biological sample, said kit comprising:

a) a labeling mixture of one or more immunolabeling complexes, wherein at least one immunolabeling complex comprises a target-binding antibody bound by a protein G complexed with albumin, wherein said albumin is covalently labeled with one or more labels, and which detectable moieties are optionally the same or different; and

b) a buffer.

62. The kit according to claim 61, wherein said albumin is human albumin, bovine serum albumin, or ovalbumin.

63. A kit for immunolabeling a target in a sample, said kit comprising:

a) a labeling mixture of one or more immunolabeling complexes, wherein at least one immunolabeling complex comprises a target-binding antibody bound by a labeling protein that is a non-antibody peptide or protein, or a monovalent antibody fragment, wherein said labeling protein binds selectively and with high affinity to a selected region of the target-binding antibody, and where the labeling protein is covalently labeled with one or more labels, which directly detectable moieties are optionally the same or different; and

b) a buffer.

64. An isolated monovalent fragment for labeling antibodies made by the process comprising the steps of:

a) generating a labeling antibody against an antigen, where said antigen is a Fc region of an antibody from a different species;

b) digesting said labeling antibody to generate multiple monovalent fragments;

c) covalently labeling said monovalent fragments with one or more labels, where said labels on each monovalent fragment are optionally the same or different; and

d) isolating said monovalent fragments before or after covalent labeling.

65. The isolated monovalent fragment for labeling target-binding antibodies made by the process according to claim 64, wherein said species is selected from the group consisting of mouse, rabbit and goat.

66. The isolated monovalent fragment for labeling target-binding antibodies made by the process according to claim 64, wherein said labeling antibody is digested with papain to yield multiple Fab fragments.

67. The isolated monovalent fragment for labeling target-binding antibodies made by the process according to claim 64, wherein said fragments are covalently labeled with one or more directly detectable fluorescent dyes, phosphorescent dyes, dye tandems, fluorescent proteins, particles, or electron transfer agents, or combinations thereof.

68. The isolated monovalent fragment for labeling target-binding antibodies made by the process according to claim 64, wherein;

a) before said fragments are covalently labeled, said fragments are immobilized by binding to one or more complementary antibodies, where such binding protects the binding region of such fragments;

b) after said fragments are covalently labeled, isolating said monovalent fragments by dissociating such fragments from such complementary antibodies.

69. An isolated monovalent fragment for labeling target-binding antibodies made by the process comprising the steps of:

a) generating a labeling antibody against an antigen, where said antigen is a selected peptide linker region of a single-chain variable protein fragment;

b) digesting said labeling antibody to generate multiple monovalent fragments;

c) covalently labeling said monovalent fragments with one or more labels, where said labels on each monovalent fragment are optionally the same or different; and

d) isolating said monovalent fragments before or after covalent labeling.

70. An isolated immunolabeling complex made by a process comprising:

a) combining, in vitro, one or more target-binding antibodies with an amount of labeling protein in excess of a ratio of at least one labeling protein to each target-binding antibody, under conditions suitable for the labeling protein to bind to the target-binding antibodies, and where each labeling protein is a non-antibody peptide or protein, or is a monovalent antibody fragment, wherein said labeling protein binds selectively and with high affinity to a selected region of said target-binding antibody to form an immunolabeling complex, and each labeling protein is covalently labeled with one or more labels, where the said labels on each labeling protein are optionally the same or different;

b) isolating said immunolabeling complex after said labeling protein binds to the target-binding antibodies.

71. The isolated immunolabeling complex made by the process according to claim 70, wherein said target-binding antibodies that are combined with the labeling protein are present in a heterogeneous protein mixture.

72. The isolated immunolabeling complex made by the process according to claim 70, wherein said heterogeneous protein mixture is ascites fluid or hybridoma culture supernatant.

73. The isolated immunolabeling complex made by the process according to claim 70, wherein said labeling protein is in excess of a ratio of 3 moles of labeling protein for each mole of target-binding antibody.

74. The isolated immunolabeling complex made by the process according to claim 70, wherein at least one labeling protein is a monovalent antibody fragment, and said monovalent fragment is covalently labeled with one or more fluorescent dyes, chemiluminescent dyes, tandem dyes, fluorescent proteins, or combinations thereof.

75. The isolated immunolabeling complex made by the process according to claim 70, wherein said target-binding antibody binds to an antigenic determinate present in a biological sample.

76. The isolated immunolabeling complex made by the process according to claim 70, wherein at least one labeling protein is a monovalent fragment that has been made by the process consisting essentially of:

a) generating a labeling antibody against an antigen, where said antigen is a Fc region of an antibody from a different species;

b) digesting said labeling antibody to generate multiple monovalent fragments;

c) covalently labeling said monovalent fragments with one or more labels, where said labels on each monovalent fragment are optionally the same or different; and

d) isolating said monovalent fragments before or after covalent labeling.

77. The isolated monovalent fragment made by the process according to claim 76, wherein said labeling protein that is not bound to the target-binding antibodies are bound to a capture component, which is optionally attached to a microsphere or to agarose.

78. The monovalent fragment made by the process according to claim 76, wherein the step of covalently labeling said monovalent fragments is accomplished by immobilizing said monovalent fragments by binding to one or more complementary antibodies, where such binding protects the binding region of such fragments;

and

wherein the step of isolating said monovalent fragments is accomplished by dissociating such fragments from such complementary antibodies after covalent labeling.

79. The isolated monovalent fragment made by the process according to claim 76, wherein said species is selected from the group consisting of mouse, rabbit or goat.

80. The isolated monovalent fragment made by the process according to claim 76, wherein said labeling antibody is digested with papain to yield multiple Fab fragments.

81. The isolated immunolabeling complex made by the process according to claim 70, wherein the target-binding antibody is a single-chain variable protein fragment wherein at least one labeling protein is a monovalent fragment that has been made by the process consisting essentially of:

a) generating a labeling antibody against an antigen, wherein said antigen is a selected peptide linker region of said single-chain variable protein fragment;

b) digesting said labeling antibody to generate multiple monovalent fragments;

c) covalently labeling said monovalent fragments with one or more labels, where said labels on each monovalent fragment are optionally the same or different; and

d) isolating said monovalent fragments before or after covalent labeling.

82. The isolated monovalent fragment for labeling antibodies made by the process according to claim 81, wherein said labeling antibody is digested with papain to yield multiple Fab fragments.

84. A kit for preparing labeling proteins, said kit comprising:

a) a monovalent antibody fragment that binds selectively and with high affinity to a specific region of one or more target-binding antibodies,

b) a chemically reactive dye; and

c) an immobilization matrix comprising the binding domain of the labeling protein.