Homogeneous immunoassays for multiple allergens

• US 20070190668A1
• Filed: 04/10/2007
• Published: 08/16/2007
• Est. Priority Date: 12/06/2001
• Status: Active Grant

• Current Assignee: Immunotech SA
• Original Assignee: Immunotech SA
• Inventors: Murai, James, Brown, Christopher
• Granted Patent: US 7,491,553 B2
• Time in Patent Office: Days
• US Class Current: 436/523
• CPC Class Codes: G01N 33/54313 the carrier being characterised by its particulate form
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/6854 Immunoglobulins

Abstract

A homogeneous immunoassay method and system for quantitative determination of total immunoglobulin E and specific antibody levels to a plurality of allergens, in which a relatively small sampling of blood is required. The method utilizes relatively small microparticles in aqueous suspension. The immunoassay procedure is an immunometric sandwich procedure preferably utilizing biotin-streptavidin signal amplification techniques and R-phycoerytherin fluorescent labels.

Claims

1. A method for simultaneously measuring specific immunoglobin antibody levels to from 2 to about 100 allergens in a blood sample, said method comprising:

(a) collecting a blood sample of from about 10 to about 100 μ

L from an individual;

(b) performing a homogeneous immunoassay on the blood sample or a portion thereof, wherein the immunoassay is capable of determining specific immunoglobin antibody levels to from 2 to about 100 allergens; and

(c) determining the said specific immunoglobin antibody levels.

2. A system for simultaneously measuring a plurality of specific immunoglobin antibody levels by use of a homogeneous immunoassay, said system comprising:

(a) a plurality of particles coupled to from 2 to about 100 allergens;

(b) a first conjugate comprising an anti-human IgE or IgG antibody conjugated to a first member of a specific binding pair; and

(c) a second conjugate comprising a second specific binding pair member that binds to the first specific binding pair member;

the system being capable of conducting simultaneous quantitative determination of specific immunoglobin antibody levels to from 2 to about 100 allergens, by a homogeneous immunoassay.

3. A system for quantitative determination of specific immunoglobulin antibody levels to allergens in a blood sample comprising:

(a) a plurality of particles coupled to a plurality of allergens, the particles being in suspension in from about 1 to about 50 μ

L of an aqueous medium, each combination of particles with a specific allergen being distinguishable from combinations of the particles with other allergens;

(b) a first conjugate comprising an anti-human IgE or IgG antibody conjugated to a first member of a specific binding pair, said specific binding pair having the capability of amplifying fluorescent signal output, the first conjugate having from 10 to 30 molecules of the first binding pair member conjugated to one molecule of the anti-human antibody;

(c) a second conjugate comprising a second specific binding pair member that binds to the first specific binding pair member, the second member being coupled to a fluorophore moiety;

(d) the mole ratio of first conjugate to the second conjugate being from about 1;

1 to about 1;

5.

4. A system according to claim 3 in which the plurality of particles contains from about 5 to about 100 allergens.

5. A system according to claim 3 in which the plurality of particles contains from about 10 to 40 allergens.

6. A system according to claim 3 in which the specific antibody is immunoglobulin E.

7. A system according to claim 3 in which the specific antibody comprises immunoglobulin G and subclasses thereof.

8. A system according to claim 3 in which the anti-human antibody is anti-human IgE.

9. A system according to claim 3 in which the anti-human antibody is an anti-human IgE fragment that binds specifically to immunoglobulin E.

10. A system according to claim 3 in which the anti-human antibody is a monoclonal antibody that binds specifically to immunoglobulin E.

11. A system according to claim 3 in which the anti-human antibody is an anti-human IgG.

12. A system according to claim 3 in which the anti-human antibody is an anti-human IgG fragment that binds specifically with immunoglobulin G.

13. A system according to claim 3 in which the anti-human antibody is a monoclonal antibody that binds specifically to immunoglobulin G.

14. A system according to claim 3 in which the fluorophore is a phycobiliprotein.

15. A system according to claim 14 in which the phycobiliprotein is selected from phycoerytherins, phycocyanins, and allophycocyanins.

16. A system according to claim 14 in which the phycobiliprotein is phycoerytherin.

17. A system according to claim 16 in which the phycoerytherin is R-phycoerytherin.

18. A system according to claim 3 in which the second conjugate has a molecular weight of between 400,000 and about 1,000,000 daltons.

19. A system according to claim 3 in which the first specific binding pair member is selected from biotin and digoxin.

20. A system according to claim 3 in which the first specific binding pair member is biotin.

21. A system according to claim 20 in which the biotin is conjugated to the anti-human antibody in a molecular ratio from about 10:

1 to about 30;

1.

22. A system according to claim 20 in which the biotin is conjugated to the anti-human antibody in a molecular ratio from about 15:

1 to 25;

1.

23. A system according to claim 20 in which the biotin is conjugated to the anti-human antibody in a molecular ratio of 20:

1.

24. A system according to claim 3 in which the second specific binding pair member is selected from avidin, streptavidin, and anti-digoxin.

25. A system according to claim 3 in which the second specific binding pair member is streptavidin.

26. A system according to claim 3 in which the suspension of particles has a volume of from about 1 to 10 μ

L.

27. A system according to claim 3 further comprising a blood sample to be tested, having a volume of from about 1 to about 5 μ

L.

28. A homogeneous immunoassay method for simultaneously detecting and quantifying specific immunoglobulin antibody levels to a plurality of allergens in a blood sample, comprising

(a) contacting a blood sample having a volume of from about 1 to about 25 μ

L with a plurality of particles coupled to a plurality of allergens, the particles being in suspension in from about 1 to about 50 μ

L of an aqueous medium, each combination of particles with specific allergen being distinguishable from combinations of the particles with other allergens, under conditions whereby immunoglobulin antibodies present in the blood sample that bind specifically to one or more of the allergens are bound to the particles;

(b) thereafter contacting the materials from step (a) with a first conjugate comprising an anti-human IgE or IgG antibody conjugated to a first member of a specific binding pair, said specific binding pair having the capability of amplifying fluorescent signal output;

(c) thereafter contacting the materials from step (b) with a second conjugate containing a fluorophore moiety bound to a second specific binding pair member that binds to the first specific binding pair member; and

(d) thereafter determining levels of specific immunoglobulin antibody in the materials from step (c) by simultaneously determining the fluorescent emission signals of the particle subsets and measuring the fluorescent emission signals of the fluorophore moiety.

29. A method according to claim 28 in which step (a) is conducted by contacting the sample with a plurality of particles coupled to from about 5 to about 100 specific allergens.

30. A method according to claim 28 in which step (a) is conducted by contacting the sample with a plurality of particles coupled to from about 10 to about 40 specific allergens.

31. A method according to claim 28 in which the antibody comprises specific immunoglobulin E.

32. A method according to claim 28 in which the antibody comprises specific immunoglobulin G and subclasses thereof.

33. A method according to claim 28 in which the first conjugate comprises an anti-human antibody coupled to biotin or digoxin.

34. A method according to claim 33 in which the molecular ratio of anti-human antibody to biotin or digoxin in the first conjugate is from about 1:

10 to about 1;

30.

35. A method according to claim 33 in which the molecular ratio of anti-human antibody to biotin or digoxin in the first conjugate is from about 1:

15 to about 1;

25.

36. A method according to claim 33 in which the molecular ratio of anti-human antibody to biotin or digoxin in the first conjugate is 1:

20.

37. A method according to claim 28 in which the fluorophore moiety of the second conjugate is a phycobiliprotein.

38. A method according to claim 37 in which the phycobiliprotein is selected from phycoerytherins, phycocyanins, and allophycocyanins.

39. a method according to claim 37 in which the phycobiliprotein is phycoerytherin.

40. A method according to claim 37 in which the phycobiliprotein is R-phycoerytherin.

41. A method according to claim 28 in which the second specific binding pair member of the second conjugate is selected from avidin, streptavidin and antidigoxin.

42. A method according to claim 28 in which the second specific binding pair member of the second conjugate is streptavidin.

43. A method according to claim 28 in which the second conjugate has a molecular weight of between 4000,000 and about 1,000,000 daltons.

44. A method according to claim 28 in which the mole ratio of the first conjugate to the second conjugate is from 1:

1 to about 1;

5.

45. A method according to claim 28 in which the plurality of particles has a volume of from about 1 to about 10 μ

L.

46. A method according to claim 28 in which the blood sample has a volume of from about 1 to about 5 μ

L.

47. A method according to claim 28 in which the allergen specific antibodies are determined without diluting the blood sample.

48. A method according to claim 28 in which the blood sample is obtained by providing a blood collection kit so as to enable the providing of a sample of blood for use in the method by pricking the skin to draw blood, collecting a sample of said blood and submitting said sample for use in the method.

49. A system for quantitative determination of total immunoglobulin E levels in a blood sample comprising:

(a) A plurality of particles coupled to anti-human IgE antibody, the particles being in suspension in from about 1 to about 50 μ

L of an aqueous medium;

(b) a first conjugate comprising an anti-human antibody and a first member of a specific binding pair, said specific binding pair having the capability of amplifying fluorescent signal output;

(c) a second conjugate comprising a phycobiliprotein and a second specific binding pair member that binds specifically to the first specific binding pair member and amplifies the fluorescent signal output;

(d) the mole ratio of the first conjugate to the second conjugate being from about 1;

1 to about 1;

5.

50. A system according to claim 49 in which the antibody coupled to particles is selected from polyclonal anti-human IgE.

51. A system according to claim 49 in which the antibody coupled to particles is an anti-human IgE fragment that binds specifically to immunoglobulin E.

52. A system according to claim 49 in which the antibody coupled to particles is a monoclonal antibody that binds specifically to immunoglobulin E.

53. A system according to claim 49 in which the anti-human antibody is anti-human IgE.

54. A system according to claim 49 in which the anti-human antibody is an anti-human IgE fragment that binds specifically to immunoglobulin E.

55. A system according to claim 49 in which the anti-human antibody is a monoclonal antibody that binds specifically to immunoglobulin E.

56. A system according to claim 49 in which the phycobiliprotein is selected from phycoerytherins, phycocyanins, and allophycocyanins.

57. A system according to claim 56 in which the phycobiliprotein is phycoerytherin.

58. A system according to claim 57 in which the phycoerytherin is R-phycoerytherin.

59. A system according to claim 49 in which the second conjugate has a molecular weight of between 400,000 and about 1,000,000 daltons.

60. A system according to claim 49 in which the first specific binding pair member is selected from biotin and digoxin.

61. A system according to claim 60 in which the first specific binding pair member is biotin.

62. A system according to claim 61 in which the biotin is conjugated to anti-human antibody in a molecular ratio from about 10:

1 to about 30;

1.

63. A system according to claim 61 in which the biotin is conjugated to anti-human antibody in a molecular ratio from about 15:

1 to 25;

1.

64. A system according to claim 61 in which the biotin is conjugated to anti-human antibody in a molecular ratio of 20:

1.

65. A system according to claim 49 in which the second specific binding pair member is selected from avidin, streptavidin, and anti-digoxin.

66. A system according to claim 49 in which the second specific binding pair member is streptavidin.

67. A system according to claim 49 in which the suspension of particles has a volume of from about 1 to 25 μ

L.

68. A system according to claim 49 further comprising a blood sample to be tested, having a volume of from about 1 to about 2 μ

L.

69. A homogeneous immunoassay method for detecting and quantifying total immunoglobulin E antibodies in a blood sample, comprising

(a) contacting a blood sample having a volume of from about 0.5 to about 10 μ

L with a plurality of particles coupled to anti-human immunoglobulin E, the particles being in suspension in from about 1 to about 50 μ

L of an aqueous medium.

(b) thereafter contacting the materials from step (a) with a first conjugate comprising an anti-human IgE antibody conjugated to a first member of a specific binding pair, said specific binding pair having the capability of amplifying fluorescent signal output;

(c) thereafter contacting the materials from step (b) with a second conjugate containing a fluorophore moiety bound to a second specific binding pair member that binds to the first specific binding pair member; and

(d) thereafter determining levels of specific immunoglobulin antibody in the materials from step (c) by simultaneously determining the fluorescent emission signals of the particles and measuring the fluorescent emission signals of the fluorophore moiety bound to the particles.

70. A method according to claim 69 in which the first conjugate comprises an anti-human IgE antibody coupled to biotin or digoxin.

71. A method according to claim 69 in which the molecular ratio of anti-human IgE antibody to first binding pair member in the first conjugate is from about 1:

10 to about 1;

30.

72. A method according to claim 69 in which the molecular ratio of anti-human antibody to first binding pair member in the first conjugate is from about 1:

15 to about 1;

25.

73. A method according to claim 69 in which the molecular ratio of anti-human antibody to first binding pair member in the first conjugate is 1:

20.

74. A method according to claim 69 in which the fluorophore of the second conjugate is a phycobiliprotein.

75. A method according to claim 74 in which the phycobiliprotein is selected from phycoerytherins, phycocyanins, and allophycocyanins.

76. a method according to claim 75 in which the phycobiliprotein is phycoerytherin.

77. A method according to claim 75 in which the phycobiliprotein is R-phycoerytherin.

78. A method according to claim 69 in which the second specific binding pair member of the second conjugate comprises avidin, streptavidin, or antidigoxin.

79. A method according to claim 78 in which the second specific binding pair member of the second conjugate is streptavidin.

80. A method according to claim 69 in which the second conjugate has a molecular weight of between 4000,000 and about 1,000,000 daltons.

81. A method according to claim 69 in which the mole ratio of the first conjugate to the second conjugate is from 1:

1 to about 1;

5.

82. A method according to claim 69 in which the particles have a volume of from about 1 to about 25.

83. A method according to claim 69 in which the blood sample has a volume of from about 1 to about 5 μ

L.

84. A method according to claim 69 in which the total IgE concentration is determined without diluting the sample.

85. A method according to claim 69 in which the blood sample is obtained by providing a blood collection kit so as to enable the providing of a sample of blood for use in the method by pricking the skin to draw blood, collecting a sample of said blood and submitting said sample for use in the method.

86. A method for conducting and providing tests for the presence of specific antibodies to allergens or for total IgE in a sample, comprising:

(a) providing a blood collection kit so as to enable an individual to provide a sample of blood for use in the method by pricking the skin to draw blood, collecting a sample of said blood and submitting said sample for use in the method;

(b) conducting a homogeneous immunoassay to detect the presence of antibodies to specific allergens or of total IgE in the sample from step (a);

(c) entering results of the immunoassay from step (b) into a database; and

(d) providing the individual access to the results of the immunoassay in the database of step (c) via a Web site or Web page of a global computer system.