Methods and kits for detecting a target cell

• US 20070134713A1
• Filed: 01/18/2007
• Published: 06/14/2007
• Est. Priority Date: 04/25/2002
• Status: Abandoned Application

• Current Assignee: Semibio Holdings Limited
• Original Assignee: Semibio Holdings Limited
• Inventors: Cao, Bo
• Application Number:
  US11/655,607
• Publication Number:
  US 20070134713A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: G01N 33/54353 with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393
• CPC Class Codes: G01N 33/56966 Animal cells
• CPC Class Codes: Y10T 428/315 Surface modified glass [e.g., tempered, strengthened, etc.]

Abstract

The present invention concerns the identification of specific target cells in whole blood. The present invention discloses methods for detecting and optionally quantifying a target cell in untreated or substantially untreated whole blood. The present invention further discloses kits for detecting and optionally quantifying a target cell in untreated or substantially untreated whole blood.

Claims

1-46. -46. (canceled)

47. A kit for detecting a target cell in a sample of untreated or substantially untreated whole blood, comprising a solid substrate, wherein

said target cell is selected from the group consisting of erythrocytes, lymphocytes, monocytes, megakaryocytes, granulocytes, hematopoietic stem cells, hematopoietic progenitor cells, dendritic cells, Langerhans cells, epithelial cells, fibroblasts, metastatic cancer cells, and circulating fetal cells;

said untreated or substantially untreated whole blood has not been treated for erythrocyte lysis;

said untreated or substantially untreated whole blood has not been subjected to cell concentration, centrifugation, filtration, buffy coat preparation, plasma removal, serum removal, or isolation of a group of cells;

said solid substrate comprises;

a multi-layered composition comprising;

polyanion layers and polycation layers in an alternating arrangement substantially parallel to the surface of said solid substrate;

a high affinity binding pair, said high affinity binding pair selected from the group consisting of avidin and biotin, protein A and

immunoglobulin, protein G and immunoglobulin, protein L and immunoglobulin, and a ligan-receptor pair; and

a specific binding agent that specifically binds said target cell, wherein said specific binding agent is bound to said solid substrate by a non-covalent binding reaction, and wherein said specific binding agent is selected from the group consisting of antibodies, antibody fragments, antigen-binding sites of antibodies, antigens, ligands, and receptors; and

said detecting is by optical methods.

48. The kit of claim 48, wherein said detecting permits quantifying said target cells bound to said solid substrate.

49. The kit of claim 48, further comprising means for providing a sample of untreated or substantially untreated whole blood.

50. The kit of claim 48, further comprising means for contacting said sample to said solid substrate.

51. The kit of claim 48, further comprising means for washing said solid substrate.

52. The kit of claim 48, further comprising means for staining said bound target cells.

53. The kit of claim 48, further comprising instructions for use.

54. The kit of claim 48, wherein said target cell is a lymphocyte.

55. The kit of claim 48, wherein said target cell is selected from the group consisting of a CD4⁺ T-lymphocyte, a CD8⁺ T-lymphocyte, a CD3⁺ T-lymphocyte, a CD19⁺ B-lymphocyte, a CD23⁺ B-lymphocyte, a CD25⁺ T-lymphocyte, a CD56⁺ natural killer lymphocyte, a CD65⁺ lymphocyte, and a CD34⁺ hematopoietic progenitor cell.

56. The kit of claim 48, wherein said solid substrate is selected from the group consisting of glass, quartz, silicon, silica oxides, ceramics, polymeric plastics, cycloolefins, cellulose polymers, metals, and composites.

57. The kit of claim 48, wherein said solid substrate is substantially optically transparent.

58. The kit of claim 48, wherein said solid surface comprises a glass slide.

59. The kit of claim 48, wherein said solid surface comprises a substantially planar surface.

60. The kit of claim 48, wherein said solid substrate comprises a chamber.

63. The kit of claim 48, wherein a pattern of known dimensions aids in said detecting of said target cells bound to said solid substrate.

64. The kit of claim 48, wherein the binding between each polyanion layer and each adjacent polycation layer is by means of electrostatic forces.

65. The kit of claim 48, wherein the polyanions are selected from the group consisting of polyphosphorus acids, polysulphur acids, polyboric acids, polysilicic acids, polycarboxylic cids, anionic polyaminoacids, anionic polypeptides, anionic polyols, anionic polythiols, anionic polyimides, and combinations thereof.

66. The kit of claim 48, wherein the polycations are selected from the group consisting of polyamines, polyaminiums, polyphosphoniums, polyyliums, polyoxoniums, cationic polyols, cationic polythiols, cationic polyaminoacids, cationic polyaldehydes, and combinations thereof.

67. The kit of claim 48, wherein said multi-layered composition further comprises the first member of a high affinity binding pair, and said specific binding agent is labeled with the second member of said high affinity binding pair, thus attaching said specific binding agent to said solid substrate by a non-covalent binding reaction.

69. The kit of claim 48, wherein said multi-layered composition further comprises one unit of the first member of a high affinity binding pair, and said specific binding agent is labeled with another unit of the first member of said high affinity binding pair, and said two units of the first member of said high affinity binding pair are both bound to the second member of the high affinity binding pair, thus attaching said specific binding agent to said solid substrate by a non-covalent binding reaction.

61. The kit of claim 61, wherein said chamber is of known dimensions.

62. The kit of claim 61, wherein said solid substrate further comprises a pattern of known dimensions.

68. The kit of claim 68, wherein the top polyionic layer of said multi-layered composition is covalently bound to said first member of said high affinity binding pair.

70. The kit of claim 70 wherein the top polyionic layer of said multi-layered composition is covalently bound to said one unit of the first member of said high affinity binding pair.