Methods and Compositions for Detection of Complement Fixing Antibodies

• US 20110281757A1
• Filed: 11/25/2009
• Published: 11/17/2011
• Est. Priority Date: 12/01/2008
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Chen, Ge, Tyan, Dolly B.
• Granted Patent: US 10,338,080 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: G01N 2333/4716 Complement proteins, e.g. anaphylatoxin, C3a, C5a
• CPC Class Codes: G01N 33/543 with an insoluble carrier for immobilising immunochemicals
• CPC Class Codes: G01N 33/6854 Immunoglobulins

Abstract

The invention provides methods for sensitive and specific detection of complement fixing antibodies in a biological sample using complement factor C1q, including autologous complement factor C1q present in the biological sample and a detectably labeled antibody that binds the autologous complement factor C1q, exogenous human complement factor C1q and a detectably labeled antibody that binds the exogenous human complement factor C1q, detectably labeled exogenous human complement factor C1q, or a combination of autologous complement factor C1q and exogenous human complement factor C1q. The invention also features kits, systems, and devices for use in the methods of the invention.

Claims

1. A method for determining the presence or absence of complement fixing antibodies in a biological sample from a subject that bind specifically to an antigen of interest, comprising:

contacting a solid substrate with an antigen of interest (AgI) immobilized thereon with a biological sample from a subject and complement factor C1q, wherein the contacting is for a sufficient time to allow anti-AgI complement fixing antibodies in the sample to bind to the AgI and to allow complement factor C1q to bind the anti-AgI complement fixing antibodies;

incubating the solid substrate with at least one detectably labeled ligand capable of specifically binding the complement factor C1q bound to the anti-AgI complement fixing antibodies bound to the AgI; and

detecting the presence or absence of the detectably labeled ligand bound to the complement factor C1q to determine the presence or absence of complement fixing antibodies that bind specifically to the AgI in the biological sample.

2. The method of claim 1, wherein the complement factor C1q is autologous complement factor C1q.

3. The method of claim 1, wherein the complement factor C1q is exogenous complement factor C1q.

4. The method of claim 1, wherein the complement factor C1q is a combination of autologous complement factor C1q and exogenous complement factor C1q.

5. The method of claim 1, wherein the solid substrate is a multiwell plate.

6. The method of claim 1, wherein the solid substrate is a membrane.

7. The method of claim 1, wherein the solid substrate is a microparticle.

8. The method of claim 7, wherein the microparticle is a polystyrene bead

9. The method of claim 7, wherein the microparticle is a latex bead.

10. The method of claim 7, wherein the microparticle is a magnetic bead.

11. The method of claim 1, wherein the solid substrate is a cell.

12. The method of claim 1, wherein the solid substrate is a cell membrane.

13. The method of claim 1, wherein the biological sample is serum, blood, saliva, plasma, or urine.

14. The method of claim 1, wherein the detectably labeled ligand is a detectably labeled antibody or binding fragment thereof.

15. The method of claim 1, wherein the detectable label is a fluorochrome, a chromophore, an enzyme, a linker molecule, a biotin molecule, an electron donor, an electron acceptor, a dye, a metal, or a radionuclide.

16. A method for determining the presence or absence of complement fixing antibodies in a biological sample from a subject that bind specifically to an antigen of interest, comprising:

contacting a solid substrate with an antigen of interest (AgI) immobilized thereon with a biological sample from a subject and directly labeled exogenous complement factor C1q, wherein the contacting is for a sufficient time to allow anti-AgI complement fixing antibodies in the sample to bind to the AgI and to allow the directly labeled exogenous complement factor C1q to bind to the anti-AgI complement fixing antibodies; and

detecting the presence or absence of the directly labeled exogenous C1q bound to the anti-AgI complement fixing antibodies to determine the presence or absence of complement fixing antibodies in the biological sample that bind specifically to the AgI.

17. The method of claim 16, wherein the solid substrate is a multiwell plate.

18. The method of claim 16, wherein the solid substrate is a membrane.

19. The method of claim 16, wherein the solid substrate is a microparticle.

20. The method of claim 19, wherein the microparticle is a polystyrene bead

21. The method of claim 19, wherein the microparticle is a latex bead.

22. The method of claim 19, wherein the microparticle is a magnetic bead.

23. The method of claim 16, wherein the solid substrate is a cell.

24. The method of claim 16, wherein the solid substrate is a cell membrane.

25. The method of claim 16, wherein the biological sample is serum, blood, saliva, plasma, or urine.

26. The method of claim 16, wherein the directly labeled exogenous C1q is labeled with a fluorochrome, a chromophore, an enzyme, a linker molecule, a biotin molecule, an electron donor, an electron acceptor, a dye, a metal, or a radionuclide.

27. A method for determining the presence or absence of complement fixing antibodies in a biological sample from a subject that bind specifically to human leukocyte antigens (HLAs), the method comprising:

incubating a biological sample from a subject with a collection of microparticles of different subtypes and complement factor C1q, wherein each microparticle is coated with a different purified HLA subtype, is derived from a cell population presenting the same HLAs, and wherein the incubating is for a sufficient time to allow anti-HLA complement fixing antibodies in the biological sample to bind to the HLAs and to allow the complement factor C1q to bind the anti-HLA complement fixing antibodies in the biological sample;

incubating the microparticles with at least one detectably labeled ligand capable of specifically binding with the complement factor C1q bound to the anti-HLA complement fixing antibodies bound to the HLAs; and

detecting the presence or absence of the detectably labeled ligand bound to the complement factor C1q to determine the presence or absence of complement fixing antibodies.

28. The method of claim 27, wherein the complement factor C1q is autologous complement factor C1q.

29. The method of claim 27, wherein the complement factor C1q is exogenous complement factor C1q.

30. The method of claim 27, wherein the complement factor C1q is a combination of autologous complement factor C1q and exogenous complement factor C1q.

31. The method of claim 27, wherein the microparticle is an agarose bead.

32. The method of claim 27, wherein the microparticle is a latex bead.

33. The method of claim 27, wherein the microparticle is a magnetic bead.

34. The method of claim 27, wherein the biological sample is serum, blood, saliva, plasma, or urine.

35. The method of claim 27, wherein the detectably labeled ligand is a detectably labeled antibody or binding fragment thereof.

36. The method of claim 27, wherein the detectable label is a fluorochrome, a chromophore, an enzyme, a linker molecule, a biotin molecule, an electron donor, an electron acceptor, a dye, a metal, or a radionuclide.

37. The method of claim 27, wherein the detecting is by flow cytometry.

38. A method for determining the presence or absence of complement fixing antibodies in a biological sample from a subject that bind specifically to human leukocyte antigens (HLAs), the method comprising:

incubating a biological sample from a subject with a collection of microparticles of different subtypes and directly labeled exogenous complement factor C1q, wherein each microparticle is coated with a different purified HLA subtype, is derived from a cell population presenting the same HLAs, and wherein the incubating is for a sufficient time to allow anti-HLA complement fixing antibodies in the biological sample to bind to the HLAs and to allow the complement factor C1q to bind the anti-HLA complement fixing antibodies in the biological sample; and

detecting the presence or absence of the directly labeled exogenous complement factor C1q bound to the anti-HLA complement fixing antibodies to determine the presence or absence of complement fixing antibodies in the biological sample that bind specifically to HLAs.

39. The method of claim 38, wherein the microparticle is an agarose bead.

40. The method of claim 38, wherein the microparticle is a latex bead.

41. The method of claim 38, wherein the microparticle is a magnetic bead.

42. The method of claim 38, wherein the biological sample is serum, blood, saliva, plasma, or urine.

43. The method of claim 38, wherein the directly labeled exogenous C1q is labeled with a fluorochrome, a chromophore, an enzyme, a linker molecule, a biotin molecule, an electron donor, an electron acceptor, a dye, a metal, or a radionuclide.

44. The method of claim 38, wherein the detecting is by flow cytometry.

45. A kit for determining the presence or absence of complement fixing antibodies in a biological sample from a subject that bind specifically to an antigen of interest, comprising:

a solid substrate with an antigen of interest (AgI) immobilized thereon;

a detectably labeled ligand capable of specifically binding to complement factor C1q; and

instructions for determining the presence or absence of complement fixing antibodies in a biological sample from a subject against the antigen of interest.

46. The kit of claim 45, wherein the solid substrate is a multiwell plate.

47. The kit of claim 45, wherein the solid substrate is a membrane.

48. The kit of claim 45, wherein the solid substrate is a microparticle.

49. The kit of claim 48, wherein the microparticle is an agarose bead.

50. The kit of claim 48, wherein the microparticle is a latex bead.

51. The kit of claim 48, wherein the microparticle is a magnetic bead.

52. The kit of claim 45, wherein the biological sample is serum, blood, saliva, plasma, or urine.

53. The kit of claim 45, wherein the detectably labeled ligand is a detectably labeled antibody or binding fragment thereof.

54. The kit of claim 45, wherein the detectable label is a fluorochrome, a chromophore, an enzyme, a linker molecule, a biotin molecule, an electron donor, an electron acceptor, a dye, a metal, or a radionuclide.

55. A kit for determining the presence or absence of complement fixing antibodies in a biological sample from a subject that bind specifically to an antigen of interest, comprising:

a solid substrate with an antigen of interest (AgI) immobilized thereon;

a directly labeled exogenous C1q capable of binding to complement fixing antibodies; and

instructions for determining the presence or absence of complement fixing antibodies in a biological sample from a subject against the antigen of interest.

56. The kit of claim 55, wherein the solid substrate is a multiwell plate.

57. The kit of claim 55, wherein the solid substrate is a membrane.

58. The kit of claim 55, wherein the solid substrate is a cell.

59. The kit of claim 55, wherein the solid substrate is a microparticle.

60. The kit of claim 59, wherein the microparticle is an agarose bead.

61. The kit of claim 59, wherein the microparticle is a latex bead.

62. The kit of claim 59, wherein the microparticle is a magnetic bead.

63. The kit of claim 58, wherein the microparticle is a polystyrene bead

64. The kit of claim 55, wherein the biological sample is serum, blood, saliva, plasma, or urine.

65. The kit of claim 55, wherein the directly labeled exogenous C1q is labeled with a fluorochrome, a chromophore, an enzyme, a linker molecule, a biotin molecule, an electron donor, an electron acceptor, a dye, a metal, or a radionuclide.

66. A kit for determining the presence or absence of complement fixing antibodies in a biological sample from a subject against human leukocyte antigens (HLAs), comprising:

a collection of microparticle subtypes wherein each microparticle subtype is coated with different purified HLAs to represent the HLA antigen population of a single cell line or multiple cell lines such that the collection simulates the distribution of HLAs in a normal human population;

a detectably labeled ligand capable of specifically binding with a complement factor C1q; and

instructions for determining the presence or absence of complement fixing antibodies in a sample from a subject against HLAs.

67. The kit of claim 66, wherein the microparticle is an agarose bead.

68. The kit of claim 66, wherein the microparticle is a latex bead.

69. The kit of claim 66, wherein the microparticle is a magnetic bead.

70. The kit of claim 66, wherein the microparticle is a polystyrene bead.

71. The kit of claim 66, wherein the biological sample is serum, blood, saliva, plasma, or urine.

72. The kit of claim 66, wherein the detectably labeled ligand is a detectably labeled antibody or binding fragment thereof.

73. The kit of claim 66, wherein the detectable label is a fluorochrome, a chromophore, an enzyme, a linker molecule, a biotin, an electron donor, an electron acceptor, a dye, a metal, or a radionuclide.

74. A kit for determining the presence or absence of complement fixing antibodies in a biological sample from a subject against human leukocyte antigens (HLAs), comprising:

a collection of microparticle subtypes wherein each microparticle subtype is coated with different purified HLAs to represent the HLA antigen population of a single cell line or multiple cell lines such that the collection simulates the distribution of HLAs in a normal human population;

a directly labeled exogenous C1q capable of binding to complement fixing antibodies; and

instructions for determining the presence or absence of complement fixing antibodies in a sample from a subject against HLAs.

75. The kit of claim 74, wherein the microparticle is an agarose bead.

76. The kit of claim 74, wherein the microparticle is a latex bead.

77. The kit of claim 74, wherein the microparticle is a magnetic bead.

78. The kit of claim 74, wherein the microparticle is a polystyrene bead.

79. The kit of claim 74, wherein the biological sample is serum, blood, saliva, plasma, or urine.

80. The kit of claim 74, wherein the directly labeled exogenous C1q is labeled with a fluorochrome, a chromophore, an enzyme, a linker molecule, a biotin, an electron donor, an electron acceptor, a dye, a metal, or a radionuclide.