Novel microarrays and methods of use thereof

• US 20040253634A1
• Filed: 05/13/2004
• Published: 12/16/2004
• Est. Priority Date: 04/10/2001
• Status: Abandoned Application

• Current Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Original Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Inventors: Wang, Denong
• Application Number:
  US10/474,449
• Publication Number:
  US 20040253634A1
• Time in Patent Office: Days
• US Class Current: 435/7.1
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00617 by chemical means
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00641 the porous medium being continuous, e.g. porous oxide substrates
• CPC Class Codes: B01J 2219/00644 the porous medium being present in discrete locations, e.g. gel pads
• CPC Class Codes: B01J 2219/00725 Peptides
• CPC Class Codes: B01J 2219/00727 Glycopeptides
• CPC Class Codes: B01J 2219/0074 Biological products
• CPC Class Codes: B01L 3/5085 for multiple samples, e.g. microtitration plates
• CPC Class Codes: C40B 40/10 Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: G01N 2400/00 Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: G01N 33/548 Carbohydrates, e.g. dextran

Abstract

This invention provides novel nitrocellulose-based or llydrogel-based microarrays and methods of making and using them (1) to detect the presence of one or more agents in a sample, (2) to determine the amount of one or more agents in a sample, (3) to determine whether a subject is afflicted with a disorder, and (4) to determine whether an agent known to specifically bind to a first compound also specifically binds to a second compound. This invention also provides kits which comprise the instant micoarrays. This invention further provides antibodies capable of specifically binding to a glycomer present both on the surface of a mammalian macrophage or intestinal epithelial cell, and on a bacterial cell. Finally, this invention provides diagnostic methods using the instant antibodies.

Claims

1. A microarray comprising a nitrocellulose or Hydrogel support having affixed to its surface at discrete loci a plurality of compounds, wherein (a) at at least one discrete locus is affixed a compound selected from the group consisting of a glycomer, an insoluble protein, a lectin and an antibody, and (b) the composition of compounds at each discrete locus differs from the composition of compounds at at least one other discrete locus.

2. The microarray of claim 1, wherein the nitrocellulose or Hydrogel support is selected from the group consisting of a chip, a slide, a filter, and a plate.

5. The microarray of claim 1 or 3, wherein the number of discrete loci is at least 100.

6. The microarray of claim 1 or 3, wherein the number of discrete loci is at least 1000.

7. The microarray of claim 1 or 3, wherein the number of discrete loci is at least 10,000.

8. The microarray of claim 1 or 3, wherein a glycomer is affixed at at least one locus.

9. The microarray of claim 1 or 3, wherein an insoluble protein is affixed at at least one locus.

10. The microarray of claim 1 or 3, wherein a lectin is affixed at at leastone locus.

11. The microarray of claim 1 or 3, wherein an antibody is affixed at at least one locus.

12. The microarray of claim 1 or 3, wherein at each locus is affixed only one compound.

13. The microarray of claim 1 or 3, wherein at at least one locus is affixed a plurality of compounds.

14. The microarray of claim 1 or 3, wherein the microarray has affixed to its surface two or more compounds selected from the group consisting of a glycomer, an insoluble protein, a lectin and an antibody.

15. The microarray of claim 1 or 3, wherein the microarray has further affixed to its surface a compound selected from the group consisting of a soluble protein, a nucleic acid and a small molecule.

35. A method of detecting in a sample the presence of one or more agents which specifically bind to one or mote known glycomers, which method comprises:

(a) contacting the sample with the microarray of claim 1 or 3, wherein each known glycomer is affixed at at least one discrete locus and wherein the contacting is performed under conditions which would permit an agent, if present in the sample, to specifically bind to its corresponding glycomer in the microarray; and

(b) determining whether any known glycomer in the microarray has an agent specifically bound thereto,

thereby detecting the presence of the one or more agents in the sample.

36. The method of claim 35, wherein the agent is an antibody which correlates with an inflammatory disease.

37. The method of claim 35, wherein the agent is an antibody which correlates with an infection.

38. The method of claim 35, wherein the agent is an antibody which correlates with the presence of a tumor.

39. The method of claim 35, wherein the method comprises detecting the presence of a plurality of agents in the sample, each of which binds to a plurality of glycomers.

40. The method of claim 35, wherein the method comprises determining the amount of a plurality of agents in the sample, each of which binds to one glycomer.

41. A method of detecting in a sample the presence of one or more agents which specifically bind to one or more known insoluble proteins, which method comprises:

(a) contacting the sample with the microarray of claim 1 or 3, wherein each known insoluble protein is affixed at at least one discrete locus and wherein the contacting is performed under conditions which would permit an agent, if present in the sample, to specifically bind to its corresponding insoluble protein in the microarray; and

(b) determining whether any known insoluble protein in the microarray has an agent specifically bound thereto,

thereby detecting the presence of the one or more agents in the sample.

42. The method of claim 41, wherein the agent is an antibody which correlates with a disease.

43. The method of claim 41, wherein the agent is an antibody which correlates with an infection.

44. The method of claim 41, wherein the agent is an antibody which correlates with the presence of a tumor.

45. The method of claim 41, wherein the method comprises detecting the presence of a plurality of agents in the sample, each of which binds to a plurality of insoluble proteins.

46. The method of claim 41, wherein the method comprises determining the amount of a plurality of agents in the sample, each of which binds to one insoluble protein.

47. A method of detecting in a sample the presence of one or more agents which specifically bind to one or more known antibodies or lectins, which method comprises:

(a) contacting the sample with the microarray of claim 1 or 3, wherein each known antibody or lectin is affixed at at least one discrete locus and wherein the contacting is performed under conditions which would permit an agent, if present in the sample, to specifically bind to its corresponding antibody or lectin in the microarray; and

(b) determining whether any known antibody or lectin in the microarray has an agent specifically bound thereto,

thereby detecting the presence of the one or more agents in the sample.

48. The method of claim 47, wherein the agent is an antibody which correlates with a disease.

49. The method of claim 47, wherein the agent is an antibody which corresponds to an infection.

50. The method of claim 47, wherein the agent is an antibody which correlates with the presence of a tumor.

51. The method of claim 47, wherein the method comprises detecting the presence of a plurality of agents in the sample, each of which binds to a plurality of lectins or antibodies.

52. The method of claim 47, wherein the method comprises determining the amount of a plurality of agents in the sample, each of which binds to one lectin or antibody.

53. A method of determining the amount of one or more agents in a sample, each of which specifically binds to one or more known glycomers, which method comprises:

(a) contacting the sample with the microarray of claim 1 or 3, wherein each known glycomer is affixed at at least one discrete locus, and wherein the contacting is performed under conditions which would permit an agent, if present in the sample, to specifically bind to its corresponding glycomer in the microarray;

(b) for each known glycomer in the microarray, determining the amount of agent specifically bound thereto; and

(c) comparing the amounts so determined to a known standard,

thereby determining the amount of the one or more agents in the sample.

54. The method of claim 53, wherein the agent is an antibody which correlates with an inflammatory disease.

55. The method of claim 53, wherein the agent is an antibody which correlates with an infection.

56. The method of claim 53, wherein the agent is an antibody which correlates with the presence of a tumor.

57. The method of claim 53, wherein the method comprises determining the amount of a plurality of agents in the sample, each of which binds to a plurality of glycomers.

58. The method of claim 53, wherein the method comprises determining the amount of a plurality of agents in the sample, each of which binds to one glycomer.

59. A method of determining the amount of one or more agents in a sample, each of which specifically binds to one or more known insoluble proteins, which method comprises:

(a) contacting the sample with the microarray of claim 1 or 3, wherein each known insoluble protein is affixed at at least one discrete locus, and wherein the contacting is performed under conditions which would permit an agent, if present in the sample, to specifically bind to its corresponding insoluble protein in the microarray;

(b) for each known insoluble protein in the microarray, determining the amount of agent specifically bound thereto; and

(c) comparing the amounts so determined to a known standard,

thereby determining the amount of the one or more agents in the sample.

60. The method of claim 59, wherein the agent is an antibody which correlates with an inflammatory disease.

61. The method of claim 59, wherein the agent is an antibody which correlates with an infection.

62. The method of claim 59, wherein the agent is an antibody which correlates with the presence of a tumor.

63. The method of claim 59, wherein the method comprises determining the amount of a plurality of agents in the sample, each of which binds to a plurality of insoluble proteins.

64. The method of claim 59, wherein the method comprises determining the amount of a plurality of agents in the sample, each of which binds to one insoluble protein.

65. A method of determining the amount of one or more agents in a sample, each of which specifically binds to one or more known antibodies or lectins, which method comprises:

(a) contacting the sample with the microarray of claim 1 or 3, wherein each known antibody or lectin is affixed at at least one discrete locus, and wherein the contacting is performed under conditions which would permit an agent, if present in the sample, to specifically bind to its corresponding antibody or lectin in the microarray;

(b) for each known antibody or lectin in the microarray, determining the amount of agent specifically bound thereto; and

(c) comparing the amounts so determined to a known standard,

thereby determining the amount of the one or more agents in the sample.

66. The method of claim 65, wherein the agent is an antibody which correlates with an inflammatory disease.

67. The method of claim 65, wherein the agent is an antibody which correlates with an infection.

68. The method of claim 65, wherein the agent is an antibody which correlates with the presence of a tumor.

69. The method of claim 65, which method comprises determining the amount of a plurality of agents in the sample, each of which binds to a plurality of lectins or antibodies.

70. The method of claim 65, wherein the method comprises determining the amount of a plurality of agents in the sample, each of which binds to one lectin or antibody.

71. A method of determining whether a subject is afflicted with a disorder characterized by the presence or absence in an afflicted subject of an agent which specifically binds to a known glycomer, which method comprises:

(a) contacting a suitable sample from the subject with the microarray of claim 1 or 3, wherein the known glycomer is affixed at at least one discrete locus and wherein the contacting is performed under conditions which would permit the agent, if present in the sample, to specifically bind to the known glycomer in the microarray; and

(b) determining whether the known glycomer in the microarray has the agent specifically bound thereto,

thereby determining whether the subject is afflicted with the disorder.

72. The method of claim 71, wherein the subject is human.

73. The method of claim 71, wherein the disorder is an inflammatory disorder.

74. The method of claim 73, wherein the inflammatory disorder is celiac disease.

75. A method of determining whether a subject is afflicted with a disorder characterized by the presence or absence in an afflicted subject of an agent which specifically binds to a known insoluble protein, which method comprises:

(a) contacting a suitable sample from the subject with the microarray of claim 1 or 3, wherein the known insoluble protein is affixed at at least one discrete locus and wherein the contacting is performed under conditions which would permit the agent, if present in the sample, to specifically bind to the known insoluble protein in the microarray; and

(b) determining whether the known insoluble protein in the microarray has the agent specifically bound thereto,

thereby determining whether the subject is afflicted with the disorder.

76. The method of claim 75, wherein the subject is human.

77. A method of determining whether a subject is afflicted with a disorder characterized by the presence or absence in an afflicted subject of an agent which specifically binds to a known antibody or lectin, which method comprises:

(a) contacting a suitable sample from the subject with the microarray of claim 1 or 3, wherein the known antibody or lectin is affixed at at least one discrete locus and wherein the contacting is performed under conditions which would permit the agent, if present in the sample, to specifically bind to the known antibody or lectin in the microarray; and

(b) determining whether the known antibody or lectin in the microarray has the agent specifically bound thereto,

thereby determining whether the subject is afflicted with the disorder.

78. The method of claim 77, wherein the subject is human.

79. The method of claim 77, wherein the disorder is HIV-1 infection.

80. A method of determining whether an antibody known to specifically bind to a first glycomer also specifically binds to a second glycomer, which method comprises:

(a) contacting the antibody with the microarray of claim 1 or 3, wherein a plurality of glycomers, other than the first glycomer, are affixed at discrete loci in the microarray, and wherein the contacting is performed under conditions which would permit the antibody to specifically bind to the first glycomer if it were present in the microarray; and

(b) determining whether any of the glycomers in the microarray, other than the first glycomer, has the antibody specifically bound thereto,

thereby determining whether the antibody also specifically binds to a second glycomer.

81. A method of determining whether an antibody known to specifically bind to a first insoluble protein also specifically binds to a second insoluble protein, which method comprises:

(a) contacting the antibody with the microarray of claim 1 or 3, wherein a plurality of insoluble proteins, other than the first insoluble protein, are affixed at discrete loci in the microarray, and wherein the contacting is performed under conditions which would permit the antibody to specifically bind to the first insoluble protein if it were present in the microarray; and

(b) determining whether any of the insoluble proteins in the microarray, other than the first insoluble protein, has the antibody specifically bound thereto,

thereby determining whether the antibody also specifically binds to a second insoluble protein.

88. A kit comprising the microarray of claim 1, 3, 18 or 22 and instructions for use.

89. A kit comprising the microarray of claim 1, 3, 18 or 22 and a desiccant.

90. A kit comprising the microarray of claim 1, 3, 18 or 22 immersed in an aqueous solution.

91. A kit for practicing the method of claim 71, which comprises:

(a) a microarray comprising a nitrocellulose or Hydrogel support having affixed to its surface at discrete loci a plurality of compounds, wherein (i) at at least one discrete locus is affixed the glycomer to which the agent present or absent in an afflicted subject specifically binds, and (ii) the composition of compounds at each discrete locus differs from the composition of compounds at at least one other discrete locus; and

(b) instructions for use.

92. A kit for practicing the method of claim 75, which comprises:

(a) a microarray comprising a nitrocellulose or Hydrogel support having affixed to its surface at discrete loci a plurality of compounds, wherein (i) at at least one discrete locus is affixed the insoluble protein to which the agent present or absent in an afflicted subject specifically binds, and (ii) the composition of compounds at each discrete locus differs from the composition of compounds at at least one other discrete locus; and

(b) instructions for use.

93. A kit for practicing the method of claim 77, which comprises:

(a) a microarray comprising a nitrocellulose or Hydrogel support having affixed to its surface at discrete loci a plurality of compounds, wherein (i) at at least one discrete locus is affixed the antibody or lectin to which the agent present or absent in an afflicted subject specifically binds, and (ii) the composition of compounds at each discrete locus differs from the composition of compounds at at least one other discrete locus; and

(b) instructions for use.

3. A microarray comprising a plurality of nitrocellulose or Hydrogel or Hydrogel supports, each support having one or a plurality of compounds affixed to its surface at a single discrete locus or a plurality of compounds affixed to its surface at discrete loci, wherein (a) at at least one discrete locus is affixed a compound selected from the group consisting of a glycomer, an insoluble protein, a lectin and an antibody, and (b) the composition of compounds at each discrete locus differs from the composition of compounds at at least one other discrete locus.

4. The microarray of claim 3, wherein the nitrocellulose or Hydrogel or Hydrogel support is selected from the group consisting of a chip, a slide, a filter, a plate, and a bead.

16. An article comprising a nitrocellulose or Hydrogel support having dextran affixed to its surface at discrete loci.

17. The article of claim 16, wherein the dextran is α

(1,6)dextran.

18. A microarray comprising the article of claim 16, wherein at least one compound is affixed to the dextran at each discrete locus, the composition of compounds at each discrete locus differing from the composition of compounds at at least one other discrete locus.

19. The microarray of claim 18, wherein the nitrocellulose or Hydrogel support is selected from the group consisting of a chip, a slide, a filter, and a plate.

24. The microarray of claim 18 or 22, wherein the number of discrete loci is at least 100.

25. The microarray of claim 18 or 22, wherein the number of discrete loci is at least 1000.

26. The microarray of claim 18 or 22, wherein the number of discrete loci is at least 10,000.

27. The microarray of claim 18 or 22, wherein a glycomer is affixed to the dextran at at least one locus.

28. The microarray of claim 18 or 22, wherein an insoluble protein is affixed to the dextran at at least one locus.

29. The microarray of claim 18 or 22, wherein a lectin is affixed to the dextran at at least one locus.

30. The microarray of claim 18 or 22, wherein an antibody is affixed to the dextran at at least one locus.

31. The microarray of claim 18 or 22, wherein at each locus is affixed only one compound.

32. The microarray of claim 18 or 22, wherein at at least one locus is affixed a plurality of compounds.

33. The microarray of claim 18 or 22, wherein the microarray has affixed to the dextran two or more compounds selected from the group consisting of a glycomer, an insoluble protein, a lectin and an antibody.

34. The microarray of claim 18 or 22, wherein the microarray has affixed to its surface a compound selected from the group consisting of a soluble protein, a nucleic acid and a small molecule.

84. A method of making the article of claim 16 comprising contacting a nitrocellulose or Hydrogel support with dextran at discrete loci under suitable conditions.

85. The method of claim 84, further comprising the step of affixing at least one compound to the dextran at each discrete locus, whereby the composition of compounds at each discrete locus differs from the composition of compounds at at least one other discrete locus.

20. An article comprising a plurality of nitrocellulose or Hydrogel supports, each support having dextran affixed to its surface at one or more discrete loci.

21. The article of claim 20, wherein the dextran is α

(1,6)dextran.

22. A microarray comprising the article of claim 20, wherein at least one compound is affixed to the dextran at each discrete locus, the composition of compounds at each discrete locus differing from the composition of compounds at at least one other discrete locus.

23. The microarray of claim 22, wherein the nitrocellulose or Hydrogel support is selected from the group consisting of a chip, a slide, a filter, a plate, and a bead.

86. A method of making the article of claim 20 comprising contacting a plurality of nitrocellulose or Hydrogel supports with dextran, whereby each support has dextran affixed to its surface at one or more discrete loci.

87. The method of claim 86, further comprising the step of affixing at least one compound to the dextran at each discrete locus, whereby the composition of compounds at each discrete locus differs from the composition of compounds at at least one other discrete locus.

82. A method of making a microarray comprising a nitrocellulose or Hydrogel support having affixed to its surface at discrete loci a plurality of compounds, which method comprises contacting the nitrocellulose or Hydrogel support with the compounds under suitable conditions, whereby (a) at at least one discrete locus is affixed a compound selected from the group consisting of a glycomer, an insoluble protein, a lectin and an antibody, and (b) the composition of compounds at each discrete locus differs from the composition of compounds at at least one other discrete locus.

83. A method of making a microarray comprising a plurality of nitrocellulose or Hydrogel supports, each support having one or a plurality of compounds affixed to its surface at a single discrete locus or a plurality of compounds affixed to its surface at discrete loci, which method comprises contacting the nitrocellulose or Hydrogel supports with the compounds under suitable conditions, whereby (a) at at least one discrete locus is affixed a compound selected from the group consisting of a glycomer, an insoluble protein, a lectin and an antibody, and (b) the composition of compounds at each discrete locus differs from the composition of compounds at at least one other discrete locus.

94. An antibody capable of specifically binding to a glycomer present on the surface of a mammalian macrophage, which glycomer, or structural mimic thereof, is also endogenous to, and present on the surface of, a bacterial cell.

95. The antibody of claim 94, wherein the antibody is a groove-type antibody.

96. The antibody of claim 94, wherein the antibody is designated 4.3. F1 (ATCC Accession No. PTA-3259).

97. The antibody of claim 94, wherein the antibody is designated 45.21.1 (ATCC Accession No. PTA-3260).

101. A method of determining whether a subject is afflicted with a disorder characterized by the presence of a glycomer on the surface of macrophages in an afflicted subject, which glycomer, or structural mimic thereof, is also endogenous to, and present on the surface of, a bacterial cell, comprising:

(a) contacting a sample of the subject'"'"'s macrophages with the antibody of claim 94;

and

(b) determining whether the antibody specifically binds to the macrophages in the sample,

such binding indicating that the subject is afflicted with the disorder.

102. The method of claim 101, wherein the subject is human.

103. The method of claim 101, wherein the disorder is an immune disorder or an inflammatory disorder.

98. An antibody capable of specifically binding to a glycomer present on the surface of a mammalian intestinal epithelial cell, which glycomer, or structural mimic thereof, is also endogenous to, and present on the surface of, a bacterial cell.

99. The antibody of claim 98, wherein the antibody is a cavity-type antibody.

100. The antibody of claim 98, wherein the antibody is designated 16.4.12E (ATCC Accession No. PTA-3261).

104. A method of determining whether a subject is afflicted with a disorder characterized by the presence of a glycomer on the surface of intestinal epithelial cells in an afflicted subject, which glycomer, or structural mimic thereof, is also endogenous -to, and present on the surface of, a bacterial cell, comprising:

(a) contacting a sample of the subject'"'"'s intestinal epithelial cells with the antibody of claim 98;

and

(b) determining whether the antibody specifically binds to the intestinal epithelial cells in the sample,

such binding indicating that the subject is afflicted with the disorder.

105. The method of claim 104, wherein the subject is human.

106. The method of claim 104, wherein the disorder is an immune disorder or an inflammatory disorder.

107. The method of claim 106, wherein the disorder is celiac disease.