Highly Multiplexed Particle-Based Assays

• US 20120202293A1
• Filed: 01/10/2012
• Published: 08/09/2012
• Est. Priority Date: 10/05/2007
• Status: Active Grant

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Martin, Jason, Nguyen, Quan Ngoc
• Granted Patent: US 8,685,753 B2
• Time in Patent Office: Days
• US Class Current: 436/94
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 2525/313 Branched oligonucleotides
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: G01N 33/54333 Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
• CPC Class Codes: Y10S 435/973 Simultaneous determination of more than one analyte
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Methods are provided for detecting and optionally quantitating multiple analytes, including nucleic acid and/or polypeptide analytes, in particle-based assays that can be highly multiplexed. Compositions, systems, and kits related to the methods are also featured.

Claims

1-32. -32. (canceled)

33. A method of detecting multiple analytes, which comprises:

providing a first population of particles comprising two or more subsets of particles, wherein each subset of particles comprises a capture molecule that specifically binds directly or indirectly to an analyte;

providing a second population of particles comprising two or more subsets of particles, wherein each subset of particles comprises a capture molecule that specifically binds directly or indirectly to an analyte;

incubating a first sample with the first population of particles, and separately incubating a second sample with the second population of particles, wherein each sample comprises or putatively comprises, together or individually, multiple analytes;

then combining the first and second populations of particles; and

then detecting whether one or more analytes are associated with at least a portion of the particles from each subset of particles, wherein each subset of particles is distinguishable from all other subsets of particles.

34. The method according to claim 33, wherein the capture molecule is DNA.

35. The method according to claim 33, wherein the analyte is nucleic acid.

36. The method according to claim 35, wherein the nucleic acid is DNA.

37. The method according to claim 33, wherein the analyte is labeled.

38. The method according to claim 37, wherein the label is fluorescent.

39. The method according to claim 33, wherein the particles are distinguishable by the color of the particles.

40. The method according to claim 33, wherein the first sample is derived from a human source.

41. The method according to claim 33, wherein both the first and second samples are derived from the same human subject.

42. The method according to claim 33, wherein each sample contains or putatively contains at least five different analytes.

43. The method according to claim 42, wherein the first population of particles comprises at least five subsets of particles and wherein the second population of particles comprises at least five subsets of particles.

44. The method according to claim 33, wherein each sample contains or putatively contains at least ten different analytes.

45. The method according to claim 44, wherein the first population of particles comprises at least ten subsets of particles and wherein the second population of particles comprises at least ten subsets of particles.

46. The method according to claim 33, wherein the capture molecule is an antibody.

47. The method according to claim 33, wherein the analyte is a polypeptide.

48. The method according to claim 33, wherein the particles with bound analytes are detected by bDNA assay.

49. The method according to claim 33, wherein only a representative portion of the particles are combined and detected.

50. The method according to claim 33, comprising:

providing one or more additional populations of particles each comprising two or more subsets of particles, wherein each subset of particles comprises a capture molecule that specifically binds directly or indirectly to an analyte,

separately incubating the one or more additional populations of particles with one or more additional samples;

then combining the populations of particles; and

then detecting whether one or more analytes are associated with at least a portion of the particles from each subset of particles, wherein each subset of particles is distinguishable from all other subsets of particles.

51. The method according to claim 50, wherein the particles with bound analytes are detected by bDNA assay.

52. The method according to claim 50, wherein each sample comprises or putatively comprises at least five different analytes, and wherein each population of particles comprises at least five subsets of particles.

53. A method of detecting multiple analytes, which comprises:

providing a first population of particles comprising five or more subsets of particles, wherein each subset of particles comprises a capture molecule that specifically binds directly or indirectly to an analyte;

providing a second population of particles comprising five or more subsets of particles, wherein each subset of particles comprises a capture molecule that specifically binds directly or indirectly to an analyte;

providing a third population of particles comprising five or more subsets of particles, wherein each subset of particles comprises a capture molecule that specifically binds directly or indirectly to an analyte;

separately incubating a first sample with the first population of particles, a second sample with the second population of particles, and a third sample with the third population of particles, wherein each sample individually comprises or putatively comprises at least five different analytes;

then combining the first, second, and third populations of particles; and

then detecting whether one or more analytes are associated with at least a portion of the particles from each subset of particles, wherein each subset of particles is distinguishable from all other subsets of particles.

54. The method according to claim 53, wherein the particles with bound analytes are detected by bDNA assay.

55. A method of detecting multiple nucleic acid analytes, which comprises:

providing a first population of particles comprising two or more subsets of particles, wherein each subset of particles comprises a DNA molecule that specifically binds directly or indirectly to a nucleic acid analyte;

providing a second population of particles comprising two or more subsets of particles, wherein each subset of particles comprises a DNA molecule that specifically binds directly or indirectly to a nucleic acid analyte;

incubating a first sample with the first population of particles, and separately incubating a second sample with the second population of particles, wherein each sample comprises or putatively comprises, together or individually, multiple fluorescently labeled nucleic acid analytes;

then combining the first and second populations of particles; and

then detecting whether one or more nucleic acid analytes are associated with at least a portion of the particles from each subset of particles, wherein each subset of particles is distinguishable from all other subsets of particles by the color of the particles.

56. The method according to claim 55, wherein the particles with bound analytes are detected by bDNA assay.

57. The method according to claim 55, wherein each sample comprises or putatively comprises at least five different analytes, and wherein each population of particles comprises at least five subsets of particles.