Highly multiplexed particle-based assays

• US 8,114,681 B2
• Filed: 10/02/2008
• Issued: 02/14/2012
• 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
• Primary Examiner(s): Gabel, Gail R
• Application Number:
  US12/287,062
• Publication Number:
  US 20090131269A1
• Time in Patent Office: 1,230 Days
• Field of Search: 435/6, 435/7.1, 435/7.92, 435/7.94, 435/973, 435/287.2, 435/288.4, 435/288.3, 436/517, 436/518, 436/523, 436/524, 436/528, 436/172
• US Class Current: 436/523
• 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. A multiplex analyte detection method, which comprises:

providing a first sample comprising or putatively comprising a first group of one or more analytes;

providing a second sample comprising or putatively comprising a second group of one or more analytes;

providing a first population of particles comprising one or more subsets of particles, wherein;

each subset of the one or more subsets of particles in the first population comprises a capture molecule that specifically binds to one of the analytes of the first group or to a molecule that in turn binds to the analyte, which capture molecule is different than that on all other subsets of particles in the first population, wherein a different capture molecule and subset of particles is provided for each different analyte in the first group, and

a plurality of particles in each subset of the one or more subsets of particles in the first population are distinguishable from a plurality of particles in every other subset of particles in the first population;

providing a second population of particles comprising one or more subsets of particles, wherein;

each subset of the one or more subsets of particles in the second population comprises a capture molecule that specifically binds to one of the analytes of the second group or to a molecule that in turn binds to the analyte, which capture molecule is different than that on all other subsets of particles in the second population, wherein a different capture molecule and subset of particles is provided for each different analyte in the second group, and

a plurality of particles in each subset of the one or more subsets of particles in the second population are distinguishable from a plurality of particles in every other subset of particles in the second population and from a plurality of particles in every subset of the first population;

contacting in a first container the first sample and the first population of particles, thereby binding those analytes of the first group that were present in the first sample to the first population of particles;

contacting in a second container the second sample and the second population of particles, thereby binding those analytes of the second group that were present in the second sample to the second population of particles;

combining the first and second populations of particles after contacting them with the samples; and

after combining the first and second populations of particles, detecting which subsets of particles have an analyte captured on the particles, thereby indicating which analytes were present in the samples, by;

providing a detection reagent comprising a label,

contacting the detection reagent with the combined populations of particles, whereby the detection reagent binds directly or indirectly to the analytes captured on the particles, and

identifying at least a portion of the particles from each of the subsets of particles in each of the populations of particles and detecting the presence or absence of the label on those particles, wherein presence of the label on the subset of particles comprising the capture molecule for a particular analyte indicates that that analyte is captured on those particles and therefore indicates that that analyte was present in the sample with which those particles were contacted, thereby detecting multiple analytes.

2. The method according to claim 1, wherein the first sample comprises or putatively comprises a first group of two or more analytes, the second sample comprises or putatively comprises a second group of two or more analytes, the first population of particles comprises two or more subsets of particles, and the second population of particles comprises two or more subsets of particles.

3. The method according to claim 1, wherein the capture molecules on the subsets of the first population of particles are the same as the capture molecules on the subsets of the second population of particles.

4. The method according to claim 1, wherein the analytes are polypeptides or nucleic acids.

5. The method according to claim 1, wherein the analytes of the first group and the analytes of the second group are the same group of target molecules derived from different sources.

6. The method according to claim 1, wherein the detection reagent is a labeled antibody, a labeled polynucleotide, a labeled avidin or a labeled streptavidin.

7. The method according to claim 1, wherein the capture molecules on the first and/or second populations of particles comprise antibodies or polynucleotides.

8. The method according to claim 1,

wherein the one or more analytes are nucleic acids and the capture molecules are polynucleotide capture probes;

wherein the contacting steps comprise;

providing a number of subsets of capture extenders equivalent to the number of subsets of particles in the first and second populations of particles, each subset of capture extenders being configured to hybridize simultaneously to one of the analytes and to one of the capture probes,

contacting the first sample with the corresponding one or more subsets of capture extenders and with the first population of particles, whereby the one or more analytes are bound to the one or more subsets of particles, and

contacting the second sample with the corresponding one or more subsets of capture extenders and with the second population of particles, whereby the one or more analytes are bound to the one or more subsets of particles; and

wherein detecting which subsets of particles have an analyte captured on the particles comprises;

hybridizing one or more label extenders and a label probe system comprising a label to the nucleic acid analytes captured on the particles, and

identifying at least a portion of the particles from each of the subsets of particles in each of the populations of particles and detecting the presence or absence of the label on those particles, wherein presence of the label on the subset of particles comprising the capture molecule for a particular analyte indicates that that analyte is captured on those particles and therefore indicates that that analyte was present in the sample with which those particles were contacted, thereby detecting multiple analytes.

9. The method according to claim 1,

wherein the one or more analytes are biotinylated nucleic acids;

wherein the capture molecules are polynucleotides, each of which is complementary to one of the nucleic acid analytes;

wherein providing a first sample comprising or putatively comprising a first group of one or more analytes comprises isolating a first group of one or more biotinylated nucleic acids bound by one or more transcription factors;

wherein providing a second sample comprising or putatively comprising a second group of one or more analytes comprises isolating a second group of one or more biotinylated nucleic acids bound by one or more transcription factors;

wherein binding those analytes of the first or second group that were present in the first or second sample to the first or second population of particles comprises hybridizing each of the biotinylated nucleic acid analytes present in the first or second sample to its complementary polynucleotide capture molecule;

wherein detecting which subsets of particles have an analyte captured on the particles comprises;

binding labeled streptavidin or labeled avidin to the biotinylated nucleic acid analytes, and

identifying at least a portion of the particles from each of the subsets of particles in each of the populations of particles and detecting the presence or absence of the label on those particles, wherein presence of the label on the subset of particles comprising the capture molecule for a particular analyte indicates that that analyte is captured on those particles and therefore indicates that that analyte was present in the sample with which those particles were contacted, thereby detecting multiple analytes.

10. The method according to claim 1,

wherein the one or more analytes are tyrosine-phosphorylated polypeptides;

wherein the capture molecules comprise SH2 peptide domains; and

wherein detecting which subsets of particles have an analyte captured on the particles comprises;

contacting, and thereby binding, a biotinylated anti-phosphotyrosine antibody with the tyrosine-phosphorylated polypeptide analytes bound to the particles,

contacting, and thereby binding, labeled streptavidin or labeled avidin to the biotinylated anti-phosphotyrosine antibody, and

identifying at least a portion of the particles from each of the subsets of particles in each of the populations of particles and detecting the presence or absence of the label on those particles, wherein presence of the label on the subset of particles comprising the capture molecule for a particular analyte indicates that that analyte is captured on those particles and therefore indicates that that analyte was present in the sample with which those particles were contacted, thereby detecting multiple analytes.

11. The method according to claim 1, further comprising:

providing a third sample comprising or putatively comprising a third group of one or more analytes;

providing a third population of particles comprising one or more subsets of particles, wherein,

a plurality of particles in each subset of the one or more subsets of particles in the third population are distinguishable from a plurality of particles in every other subset of particles in the third population and from a plurality of particles in every subset of particles in the first and second populations; and

each subset of the one or more subsets of particles in the third population comprises a capture molecule that specifically binds to one of the analytes in the third group or to a molecule that in turn binds to the analyte, which capture molecule is different than that on all other subsets of particles in the third population of particles, wherein a different capture molecule and subset of particles is provided for each different analyte in the third group;

wherein the contacting steps further comprise contacting the third sample and the third population of particles in a third container, thereby binding those analytes of the third group that were present in the third sample to the third population of particles;

wherein combining the first and second populations of particles comprises combining the first, second, and third populations of particles.