Methods and compositions for immuno-histochemical detection

• US 20050208529A1
• Filed: 11/30/2004
• Published: 09/22/2005
• Est. Priority Date: 12/01/2003
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Dako Denmark A/S (Agilent Technologies, Inc.)
• Inventors: Lovborg, Uffe, Knoll, Anthony Lionel, Weckworth, Kellyann, Winther, Lars
• Granted Patent: US 8,632,994 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: G01N 2474/20 Immunohistochemistry assay
• CPC Class Codes: G01N 33/543 with an insoluble carrier for immobilising immunochemicals
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/58 involving labelled substances G01N33/53 takes precedence

Abstract

In certain embodiments the invention relates to methods and compositions for detecting a biological marker in a sample. In certain embodiments the biological markers are detected immunologically.

Claims

1. A method of detecting a biological marker in a sample comprising the following steps in any order:

a. contacting the sample with at least one first binding agent such that the first binding agent binds to the sample and forms a first complex;

b. contacting the first complex of a) with at least one second binding agent which specifically binds to the first binding agent such that a second complex is formed and, wherein the at least one second binding agent is optionally linked to at least one first polymer, or is polymerizable without the addition of a polymer backbone;

c. contacting the second complex of b) with at least one third binding agent wherein the third binding agent is linked to at least one second polymer, or is polymerizable without the addition of a polymer backbone, and wherein the at least one third binding agent specifically binds the second binding agent; and

d. wherein at least one detectable agent is linked to at least one of

i. the third binding agent;

ii. the at least one second polymer; and

iii. both the third binding agent and the at least one second polymer wherein the third binding agent binds to the second complex of b) and forms a third complex; and

iv. detecting the third complex of c).

2. The method of claim 1, wherein the at least one first binding agent is chosen from a primary antibody or a nucleic acid probe.

3. The method of claim 1, wherein the at least one second binding agent is a dual linked binding agent.

4. The method of claim 1, wherein the at least one second binding agent is a secondary antibody.

5. The method of claim 1, wherein the at least one second binding agent is at least one molecule chosen from fos, jun, MHC, C1q, an immunoglobulin receptor, and any molecule having a leucine zipper domain.

6. The method of claim 5, wherein the at least one second binding agent further comprises an Fc region of an antibody.

7. The method of claim 5, wherein the MHC molecule is chosen from MHC class I and MHC class II and non-conventional MHC.

8. The method of claim 1, wherein the at least one third binding agent is a tertiary antibody.

9. The method of claim 1, wherein the at least one third binding agent is a molecule chosen from fos, jun, MHC, C1q, an immunoglobulin receptor, and any molecule having a leucine zipper domain.

10. The method of claim 9, wherein the at least one third binding agent further comprises an Fc region of an antibody.

11. The method of claim 9, wherein the MHC molecule is chosen from MHCI and MHCII.

12. The method of claim 2, wherein the primary antibody is derived from any mammalian species or avian species.

13. The method of claim 12, wherein the mammalian species is chosen from rat, mouse, pig, goat, rabbit, sheep, guinea pig, donkey, lama, camel and horse.

14. The method of claim 4, wherein the secondary antibody is derived from any mammalian species except the mammalian species from which the primary antibody is derived.

15. The method of claim 14, wherein the mammalian species is chosen from rat, mouse, pig, goat, rabbit, sheep, guinea pig, donkey, lama, camel and horse.

16. The method of claim 8, wherein the tertiary antibody is derived from any mammalian species except the mammalian species from which the primary antibody and secondary antibody are derived.

17. The method of claim 16, wherein the mammalian species is chosen from rat, mouse, pig, goat, rabbit, sheep, guinea pig, donkey and horse.

18. The method of claim 1, wherein the sample is a tissue sample.

19. The method of claim 1, wherein the sample is a cell sample.

20. The method of claim 1, wherein the sample is a mammalian sample.

21. The method of claim 20, wherein the mammalian sample is a human sample.

22. The method of claim 1, wherein the first binding agent binds to a biological marker in the sample.

23. The method of claim 22, wherein the protein in the sample is expressed at higher or lower levels in a cell obtained from a subject having a disease compared to a cell obtained from a subject not having the disease.

24. The method of claim 1, wherein the sample is provided on a solid support.

25. The method of claim 1, wherein the solid support is chosen from a microscope slide, a solid support which may be inspected under a microscope, and a solid support which may be used to produce photomicrographs.

26. The method of claim 1, wherein the at least one second binding agent is linked to at least one first polymer or is polymerizable without the addition of a polymer backbone.

27. The method of claim 4, wherein the at least one secondary antibody linked to the at least one second polymer comprises two different secondary antibodies linked to two different polymers, each of the two secondary antibodies comprising an antigen binding domain which specifically binds to a primary antibody derived from a different mammalian source.

28. The method of claim 27, wherein the different mammalian source is chosen from rat, mouse, pig, goat, rabbit, sheep, guinea pig, donkey and horse.

29. The method of claim 26, further comprising at least one second detectable agent linked to at least one of

a. the first polymer;

b. the at least one second binding agent; and

c. both the first polymer and the at least one second binding agent.

30. The method of claim 29, wherein the first and second detectable agent are the same agent.

31. The method of claim 29, wherein the first and second detectable agent are different agents.

32. The method of claim 29, wherein the first and second detectable agent are chosen from fluorescent labels, enzyme labels, radioisotopes, chemiluminescent labels, bioluminescent labels, polymers, metal particles, haptens, antibodies, and dyes.

33. The method of claim 32, wherein the detectable agent is chosen from 5-(and 6)-carboxyfluorescein, 5- or 6-carboxyfluorescein, 6-(fluorescein)-5-(and 6)-carboxamido hexanoic acid, fluorescein isothiocyanate, rhodamine, tetramethylrhodamine, Cy2, Cy3, Cy5, AMCA, PerCP, R-phycoerythrin (RPE) allophycoerythrin (APC), Texas Red, Princeton Red, Green fluorescent protein (GFP) coated CdSe nanocrystallites, DNP, biotin, digoxiginin, horse radish peroxidase (HRP), alkaline phosphatase (AP), beta-galactosidase (GAL), glucose-6-phosphate dehydrogenase, beta-N-acetylglucosamimidase, β

-glucuronidase, invertase, Xahthine Oxidase, firefly luciferase, glucose oxidase (GO), luminol, isoluminol, acridinium esters, 1,2-dioxetanes and pyridopyridazines, and radioactive isotopes of hydrogen, carbon, sulfur, iodide, cobalt, selenium, tritium, and phosphorous.

34. The method of claim 1, wherein at least one of the first binding agent, the second binding agent, and the third binding agent is an antibody.

35. The method of claim 34 wherein the antibody is a monoclonal antibody.

36. The method of claim 1, further comprising 2 second binding agents where each second binding agent is individually linked to at least one separate polymer, or is polymerizable without the addition of a polymer backbone.

37. The method of claim 8, wherein the tertiary antibody binds to a hapten.

38. The method of claim 37, wherein the hapten is chosen from FITC, DNP, Digoxigenin, nitrotyrosine biotin, avidin, strepavidin and an anti-dye antibody.

39. The method of claim 1, wherein at least one of the first polymer and the second polymer is chosen from:

polysaccharides including dextrans, carboxy methyl dextran, dextran polyaldehyde, carboxymethyl dextran lactone, and cyclodextrins;

pullulans, schizophyllan, scleroglucan, xanthan, gellan, O-ethylamino guaran, chitin, chitosans including 6-O-carboxymethyl chitin, N-carboxymethyl chitosan;

derivatized cellolosics including carboxymethyl cellulose, carboxymethyl hydroxyethyl cellulose, hydroxyethyl cellulose, 6-amino-6-deoxy cellulose and O-ethylamine cellulose;

hydroxylated starch, hydroxypropyl starch, hydroxyethyl starch, carrageenans, alginates, and agarose;

synthetic polysaccharides including ficoll and carboxymethylated ficoll;

vinyl polymers including poly(acrylic acid), poly(acryl amides), poly(acrylic esters), poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(maleic acid), poly(maleic anhydride), poly(acrylamide), poly(ethyl-co-vinyl acetate), poly(methacrylic acid), poly(vinylalcohol), poly(vinyl alcohol-co-vinyl chloroacetate), aminated poly(vinyl alcohol), and co block polymers thereof;

poly ethylene glycol (PEG) or polypropylene glycol or poly(ethylene oxide-co-propylene oxides) containing polymer backbones including linear, comb-shaped or branched dendrimers;

poly amino acids including polylysines, polyglutamic acid, polyurethanes, poly(ethylene imines), pluriol;

proteins including albumins, immunoglobulins, and virus-like proteins (VLP); and

polynucleotides, DNA, PNA, LNA, oligonucleotides and oligonucleotide dendrimer constructs.

40. The method of claim 39, wherein the polymer is a mixed polymer comprised of two or more of the following:

polysaccharides including dextrans, carboxy methyl dextran, dextran polyaldehyde, carboxymethyl dextran lactone, and cyclodextrins;

pullulans, schizophyllan, scleroglucan, xanthan, gellan, O-ethylamino guaran, chitin, chitosans including 6-O-carboxymethyl chitin, N-carboxymethyl chitosan;

derivatized cellolosics including carboxymethyl cellulose, carboxymethyl hydroxyethyl cellulose, hydroxyethyl cellulose, 6-amino-6-deoxy cellulose and O-ethylamine cellulose;

hydroxylated starch, hydroxypropyl starch, hydroxyethyl starch, carrageenans, alginates, and agarose;

synthetic polysaccharides including ficoll and carboxymethylated ficoll;

vinyl polymers including poly(acrylic acid), poly(acryl amides), poly(acrylic esters), poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(maleic acid), poly(maleic anhydride), poly(acrylamide), poly(ethyl-co-vinyl acetate), poly(methacrylic acid), poly(vinylalcohol), poly(vinyl alcohol-co-vinyl chloroacetate), aminated poly(vinyl alcohol), and co block polymers thereof;

poly ethylene glycol (PEG) or polypropylene glycol or poly(ethylene oxide-co-propylene oxides) containing polymer backbones including linear, comb-shaped or StarBurst™

dendrimers;

poly amino acids including polylysines, polyglutamic acid, polyurethanes, poly(ethylene imines), pluriol;

proteins including albumins, immunoglobulins, and virus-like proteins (VLP); and

polynucleotides, DNA, PNA, LNA, oligonucleotides and oligonucleotide dendrimer constructs.

41. The method of claim 1, wherein at least one of the first polymer and the second polymer is comprised of dextran.

42. A method of detecting a biological marker in a sample comprising

a. contacting the sample with at least one primary antibody, chosen from a mouse antibody and a rabbit antibody such that the at least one primary antibody binds to the sample forming a first complex;

b. contacting the complex of a) with

i. at least one first secondary goat antibody linked to a first dextran polymer, the at least one first secondary goat antibody comprising an antigen binding domain which specifically binds to a mouse antibody; and

ii. at least one second secondary goat antibody linked to a second dextran polymer, the at least one second secondary goat antibody comprising an antigen binding domain which specifically binds to a rabbit antibody, such that at least one secondary antibody binds to the complex of a) forming a second complex;

c. contacting the second complex of b) with

i. at least one tertiary rabbit antibody linked to a third dextran polymer, the at least one tertiary rabbit antibody comprising an antigen binding domain which specifically reacts with a goat antibody;

wherein at least one horse radish peroxidase molecule is linked to at least one of

1. the tertiary antibody;

2. the at least one second polymer; and

3. both the tertiary antibody and the at least one second polymer wherein the tertiary antibody binds to the second complex of b) and forms a third complex; and

d. detecting the third complex of c)

43. A method of detecting a biological marker in a sample comprising

a. contacting the sample with at least one primary mouse antibody such that the at least one primary mouse antibody binds to the sample forming a first complex;

b. contacting the first complex of a) with at least one rabbit antibody comprising an antigen binding domain which specifically reacts with a mouse antibody such that the at least one rabbit antibody specifically binds to the complex of a) forming a second complex;

c. contacting the second complex of b) with

i. at least one tertiary goat antibody linked to a dextran polymer, the at least one tertiary goat antibody comprising an antigen binding domain which specifically binds to a rabbit antibody;

wherein at least one horse radish peroxidase molecule is linked to at least one of

1. the tertiary antibody;

2. the at least one second polymer; and

3. both the tertiary antibody and the at least one second polymer such that the tertiary antibody binds to the second complex of b) and forms a third complex; and

d. detecting the third complex of c).

45. The method of claim 43, further comprising at least one horse radish peroxidase molecule linked to

a. the first secondary rabbit antibody;

or

b. the first dextran polymer;

or

c. both the first secondary rabbit antibody and the first dextran polymer.

44. A method of detecting a biological marker in a sample comprising

a. contacting the sample with at least one primary mouse antibody such that the at least one primary mouse antibody binds to the sample forming a first complex;

b. contacting the complex of a) with at least one secondary rabbit antibody linked to a first dextran polymer, the at least one secondary rabbit antibody comprising an antigen binding domain which specifically reacts with a mouse antibody such that at least one secondary antibody binds to the complex of a) forming a second complex;

c. contacting the second complex of b) with

i. at least one first tertiary goat antibody linked to a second dextran polymer, the at least one first tertiary goat antibody comprising an antigen binding domain which specifically binds to a rabbit antibody; and

ii. at least one second tertiary goat antibody linked to a third dextran polymer, the at least one second tertiary goat antibody comprising an antigen binding domain which specifically reacts with a mouse antibody;

wherein at least one horse radish peroxidase molecule is linked to at least one of

1. any tertiary antibody;

2. at least one second dextran polymer;

3. at least one third dextran polymer;

4. both at least one second dextran molecule and at least one third dextran molecule; and

5. any tertiary antibody and both at least one second dextran molecule and at least one third dextran molecule such that the tertiary antibody binds to the second complex of b) and forms a third complex; and

d. detecting the third complex of c).

46. A method of detecting a biological marker in a sample comprising

a. contacting the sample with at least one primary antibody, chosen from a mouse antibody and a rabbit antibody such that the at least one primary antibody binds to the sample forming a first complex;

b. contacting the complex of a) with

i. at least one first secondary goat antibody linked to a first dextran polymer, the first secondary goat antibody comprising an antigen binding domain which specifically binds to a mouse antibody;

ii. at least one second secondary goat antibody linked to a second dextran polymer, the second secondary goat antibody comprising an antigen binding domain which specifically binds to a rabbit antibody; and

iii. at least one hapten molecule linked to at least one of

1. a secondary goat antibody;

2. a dextran molecule; and

3. a secondary goat antibody and at least one dextran molecule such that at least one secondary antibody binds to the complex of a) forming a second complex;

c. contacting the second complex of b) with

i. at least one tertiary antibody, chosen from a rabbit antibody, a mouse antibody, a rat antibody, a pig antibody and a goat antibody, linked to a third dextran polymer, the at least one tertiary antibody comprising an antigen binding domain which specifically binds to at least one hapten molecule of b);

wherein at least one horse radish peroxidase molecule is linked to at least one of

1. the tertiary antibody;

2. the at least one second polymer; and

3. both the tertiary antibody and the at least one second polymer such that the tertiary antibody binds to the second complex of b) and forms a third complex; and

d. detecting the third complex of c).

47. A composition comprising:

a. at least one second binding agent which specifically binds to at least one first binding agent, where the at least one second binding agent is optionally linked to the at least one first polymer or optionally is polymerizable without the addition of a polymer backbone;

b. at least one third binding agent, which specifically binds to the at least second binding agent, where the at least one third binding agent is optionally linked to the at least one second polymer or is polymerizable without the addition of a polymer backbone;

wherein a detectable substance is linked to at least one of

i. the at least one third binding agent and optionally the at least one second binding agent;

ii. the at least one second polymer and optionally the at least one first polymer; and

iii. both the at least one third binding agent and at the least one second polymer and optionally both the at least one second binding agent and the at least one first polymer.

48. The composition of claim 47 further comprising at least one first binding agent which specifically binds to a biological marker in a sample, where the at least one first binding agent is optionally linked to a polymer or optionally is polymerizable without the addition of a polymer backbone.

49. The composition of claim 48, wherein the at least one first binding agent is chosen from a primary antibody or a nucleic acid probe.

50. The composition of claim 47, wherein the at least one second binding agent is a secondary antibody.

51. The composition of claim 47, wherein at least one of the binding agents is at least one molecule chosen from fos, jun, MHC, C1q, an immunoglobulin receptor and any molecule having a leucine zipper domain.

52. The composition of claim 51, wherein the immunoglobulin receptor is a Fc receptor.

53. The composition of claim 51, wherein the MHC molecule is chosen from MHC class I and MHC class II and non-conventional MHC.

54. The composition of claim 47, wherein at least one of the binding agents is a dual linked binding agent.

55. The composition of claim 47, wherein the at least one third binding agent is a tertiary antibody.

56. The composition of claim 49, wherein the primary antibody is derived from any mammalian or avian species.

57. The composition of claim 56, wherein the mammalian species is chosen from rat, mouse, pig, goat, rabbit, sheep, guinea pig, donkey, lama, camel and horse.

58. The composition of claim 56, wherein the avian species is chosen from chicken and duck.

59. The composition of claim 50, wherein the secondary antibody is derived from any mammalian or avian species except the mammalian or avian species from which the primary antibody is derived.

60. The composition of claim 59, wherein the mammalian species is chosen from rat, mouse, pig, goat, rabbit, sheep, guinea pig, donkey, lama, camel and horse.

61. The composition of claim 59, wherein the avian species is chosen from chicken and duck.

62. The composition of claim 55, wherein the tertiary antibody is derived from any mammalian or avian species except the mammalian or avian species from which the primary antibody and secondary antibody are derived.

63. The composition of claim 62, wherein the mammalian species is chosen from rat, mouse, pig, goat, rabbit, sheep, guinea pig, donkey, lama, camel and horse.

64. The composition of claim 62, wherein the avian species is chosen from chicken and duck.

65. The composition of claim 47, wherein the at least one second binding agent is linked to the at least one first polymer or the at least one second polymer or is polymerizable without the addition of a polymer backbone.

66. The composition of claim 50, comprising two different secondary antibodies each of the two secondary antibodies comprising an antigen binding domain which specifically binds to a different antibody.

67. The composition of claim 47, further comprising at least one second detectable substance linked to at least one of

a. a first polymer;

b. at least one second binding agent; and

c. both the first polymer and the at least one second binding agent.

68. The composition of claim 67, wherein the first and second detectable substances are the same substance.

69. The composition of claim 67, wherein the first and second detectable substances are different substances.

70. The composition of claim 47, wherein the first and second detectable substances are chosen from fluorescent labels, enzyme labels, radioisotopes, chemiluminescent labels, bioluminescent labels, polymers, metal particles, haptens, antibodies, and dyes.

71. The composition of claim 47, wherein the detectable substance is chosen from 5-(and 6)-carboxyfluorescein, 5- or 6-carboxyfluorescein, 6-(fluorescein)-5-(and 6)-carboxamido hexanoic acid, fluorescein isothiocyanate, rhodamine, tetramethylrhodamine, Cy2, Cy3, Cy5, AMCA, PerCP, R-phycoerythrin (RPE) allophycoerythrin (APC), Texas Red, Princeton Red, Green fluorescent protein (GFP) coated CdSe nanocrystallites, DNP, biotin, digoxiginin, horse radish peroxidase (HRP), alkaline phosphatase (AP), beta-galactosidase (GAL), glucose-6-phosphate dehydrogenase, beta-N-acetylglucosamin idase, β

-glucuronidase, invertase, xanthine oxidase, firefly luciferase, glucose oxidase (GO), luminol, isoluminol, acridinium esters, 1,2-dioxetanes and pyridopyridazines, and radioactive isotopes of hydrogen, carbon, sulfur, iodide, cobalt, selenium, tritium, and phosphor.

72. The composition of claim 48, wherein at least one of the primary antibody, the secondary antibody or the tertiary antibody is a monoclonal antibody.

73. The composition of claim 72, wherein at least one of the primary antibody, the secondary antibody and the tertiary antibody is an IgG.

74. The composition of claim 50, comprising a first secondary antibody and a second secondary antibody where each antibody is linked to the same polymer or individually linked to at least one separate polymer or is polymerizable without the addition of a polymer backbone.

75. The composition of claim 64, wherein the first polymer and the second polymer is comprised of dextran.

76. The composition of claim 64, where the first and second polymer are each chosen from polysaccharides including dextrans, carboxy methyl dextran, dextran polyaldehyde, carboxymethyl dextran lactone, and cyclodextrins;

pullulans, schizophyllan, scleroglucan, xanthan, gellan, O-ethylamino guaran, chitin, chitosans inducing 6-O-carboxymethyl chitin, N-carboxymethyl chitosan;

derivatized cellolosics including carboxymethyl cellulose, carboxymethyl hydroxyethyl cellulose, hydroxyethyl cellulose, 6-amino-6-deoxy cellulose and O-ethylamine cellulose;

hydroxylated starch, hydroxypropyl starch, hydroxyethyl starch, carrageenans, alginates, and agarose;

synthetic polysaccharides including ficoll and carboxymethylated ficoll;

vinyl polymers including poly(acrylic acid), poly(acryl amides), poly(acrylic esters), poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(maleic acid), poly(maleic anhydride), poly(acrylamide), poly(ethyl-co-vinyl acetate), poly(methacrylic acid), poly(vinylalcohol), poly(vinyl alcohol-co-vinyl chloroacetate), aminated poly(vinyl alcohol), and co block polymers thereof;

poly ethylene glycol (PEG) or polypropylene glycol or poly(ethylene oxide-co-propylene oxides) containing polymer backbones including linear, comb-shaped or branched dendrimers;

poly amino acids including polylysines, polyglutamic acid, polyurethanes, poly(ethylene imines), pluriol;

proteins including albumins, immunoglobulins, and virus-like proteins (VLP); and

polynucleotides, DNA, PNA, LNA, oligonucleotides and oligonucleotide dendrimer constructs.

77. The composition of claim 64, where at least one of the first and second polymer is a mixed polymer comprised of at least two of the following:

polysaccharides including dextrans, carboxy methyl dextran, dextran polyaldehyde, carboxymethyl dextran lactone, and cyclodextrins;

pullulans, schizophyllan, scleroglucan, xanthan, gellan, O-ethylamino guaran, chitin, chitosans including 6-O-carboxymethyl chitin, N-carboxymethyl chitosan;

derivatised cellolosics including carboxymethyl cellulose, carboxymethyl hydroxyethyl cellulose, hydroxyethyl cellulose, 6-amino-6-deoxy cellulose and O-ethylamine cellulose;

hydroxylated starch, hydroxypropyl starch, hydroxyethyl starch, carrageenans, alginates, and agarose;

synthetic polysaccharides including ficoll and carboxymethylated ficoll;

vinyl polymers including poly(acrylic acid), poly(acryl amides), poly(acrylic esters), poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(maleic acid), poly(maleic anhydride), poly(acrylamide), poly(ethyl-co-vinyl acetate), poly(methacrylic acid), poly(vinylalcohol), poly(vinyl alcohol-co-vinyl chloroacetate), aminated poly(vinyl alcohol), and co block polymers thereof;

poly ethylene glycol (PEG) or polypropylene glycol or poly(ethylene oxide-co-propylene oxides) containing polymer backbones including linear, comb-shaped or branched dendrimers;

poly amino acids including polylysines, polyglutamic acid, polyurethanes, poly(ethylene imines), pluriol;

proteins including albumins, immunoglobulins, and virus-like proteins (VLP); and

polynucleotides, DNA, PNA, LNA, oligonucleotides and oligonucleotide dendrimer constructs.

81. The composition of claim 68, further comprising at least one horse radish peroxidase molecule linked to

a. the at least one first secondary rabbit antibody;

or

b. the first dextran polymer;

or

c. both the at least one first secondary rabbit antibody and the first dextran polymer.

85. The kit of claim 73, comprising at least one first secondary antibody linked to a first polymer and at least one second secondary antibody linked to a second polymer.

86. The kit of claim 73, further comprising a second detectable agent linked to at least one of

a. the at least one secondary antibody;

b. the second polymer; and

c. both the at least one secondary antibody and the second polymer.

87. The kit of claim 75, wherein the first and second detectable agents are different.

88. The kit of claim 75, wherein the first and second detectable agents are the same.

89. The kit of claim 73, further comprising at least one hapten molecule linked to at least one of

a. the at least one secondary antibody;

b. the second polymer; and

c. both the at least one secondary antibody and the second polymer.

78. A composition comprising;

a. at least one primary antibody, chosen from a mouse antibody and a rabbit antibody;

b. at least one first secondary goat antibody linked to a first dextran polymer, the at least one first secondary goat antibody comprising an antigen binding domain which specifically binds to a mouse antibody;

c. at least one second secondary goat antibody linked to a second dextran polymer, the at least one second secondary goat antibody comprising an antigen binding domain which specifically binds to a rabbit antibody;

d. at least one tertiary rabbit antibody linked to a third dextran polymer, the at least one tertiary rabbit antibody comprising an antigen binding domain which specifically binds to a goat antibody wherein at least one horse radish peroxidase molecule is linked to at least one of

i. the at least one tertiary rabbit antibody;

ii. the third dextran polymer; and

iii. both the at least one tertiary rabbit antibody and the third dextran polymer.

79. A composition comprising

a. at least one primary mouse antibody;

b. at least one secondary rabbit antibody comprising an antigen binding domain which specifically binds to a mouse antibody;

c. at least one tertiary goat antibody linked to a dextran polymer, the at least one tertiary goat antibody comprising an antigen binding domain which specifically binds to a rabbit antibody wherein at least one horse radish peroxidase molecule is linked to at least one of

i. the least one tertiary goat antibody;

ii. the dextran polymer; and

iii. both the at least one tertiary goat antibody and the first dextran polymer.

80. A composition comprising

a. at least one primary mouse antibody;

b. at least one secondary rabbit antibody linked to a first dextran polymer, the at least one secondary rabbit antibody comprising an antigen binding domain which specifically binds to a mouse antibody;

c. at least one first tertiary goat antibody linked to a second dextran polymer, the at least one first tertiary goat antibody comprising an antigen binding domain which specifically binds to a rabbit antibody;

d. at least one second tertiary goat antibody linked to a third dextran polymer, the at least one second tertiary goat antibody comprising an antigen binding domain which specifically binds to a mouse antibody;

wherein at least one horse radish peroxidase molecule is linked to at least one of

i. any at least one tertiary antibody;

ii. at least one second dextran polymer;

iii. at least one third dextran polymer; and

iv. both at least one second dextran molecule and at least one third dextran molecule.

82. A composition comprising

a. at least one primary antibody chosen from a mouse antibody and a rabbit antibody;

b. at least one first secondary goat antibody linked to a first dextran polymer, the first secondary goat antibody comprising an antigen binding domain which specifically binds to a mouse antibody;

c. at least one second secondary goat antibody linked to a second dextran polymer, the second secondary goat antibody comprising an antigen binding domain which specifically binds to a rabbit antibody; and

d. at least one hapten molecule linked to at least one of

i. the at least one first secondary goat antibody;

ii. the first dextran polymer; and

iii. both the at least one first secondary goat antibody and the first dextran polymer; and

e. at least one hapten molecule linked to at least one of

i. the at least one second secondary goat antibody;

ii. the second dextran polymer; and

iii. both the at least one second secondary goat antibody and the second dextran polymer; and

f. at least one tertiary antibody, chosen from a rabbit antibody, a mouse antibody, a goat antibody, a swine antibody, and a rat antibody, linked to a third dextran polymer, the at least one tertiary antibody comprising an antigen binding domain which specifically reacts with at least one hapten molecule wherein at least one horse radish peroxidase molecule is linked to at least one of

i. the at least one tertiary antibody;

ii. the third dextran polymer; and

iii. both the at least one tertiary antibody and the third dextran polymer.

83. A kit for detecting a biological marker in a sample comprising;

a. optionally, a first binding agent which specifically binds to a biological marker in a sample;

b. a second binding agent which specifically binds to the first binding agent, where the second binding agent is optionally conjugated to a polymer, or is polymerizable without the addition of a polymer backbone;

c. a third binding agent which specifically binds to the second binding agent, where the third binding agent is conjugated to a polymer, or is polymerizable without the addition of a polymer backbone;

d. at least one detectable substance linked to at least one of a) b) and c) and

e. at least one container.

84. A kit for performing immuno-histochemistry on a sample comprising;

a. at least one secondary antibody optionally linked to a first polymer or polymerizable without the addition of a polymer backbone;

b. at least one tertiary antibody linked to a second polymer, or polymerizable without the addition of a polymer backbone and wherein a first detectable agent is linked to at least one of

i. the at least one tertiary antibody;

ii. the first polymer; and

iii. both the first polymer and the tertiary antibody;

c. optionally a primary antibody;

d. at least one container.