WO2011127042A1

WO2011127042A1 - High capacity analyte detection

Info

Publication number: WO2011127042A1
Application number: PCT/US2011/031232
Authority: WO
Inventors: Aravind Subramanian; David D. Peck; Todd R. Golub; Rajiv Narayan; Willis Read-Button
Original assignee: Broad Institute Inc
Current assignee: Broad Institute Inc
Priority date: 2010-04-06
Filing date: 2011-04-05
Publication date: 2011-10-13
Anticipated expiration: 2012-10-06

Abstract

The present invention improves the known compositions and methods for high throughput detection of analytes. Analytes may be derived from samples, including but not limited to biological samples, such that the detection of nucleic acids, proteins, peptides, and/or small organic molecules is facilitated. Different analytes may be attached to solid particles having identical spectral characteristics, wherein the analytes are identified by differential particle count (i.e., for example, subsets of identical particles are assayed in unequal proportions). The simultaneous detection and/or identification all analytes contained within a sample is facilitated by an algorithmic analysis of multimodal distribution patterns.

Description

High Capacity Analyte Detection

Statement Of Governmental Support

This invention was made with government support under U54 6916636 awarded by the National Institutes of Health.. The government has certain rights in the invention.

Field Of Invention

The present invention is related to compositions and methods for the detection of analytes. Analytes capable of detection by this invention include, but are not limited to, nucleic acids, proteins, peptides, and/or small organic molecules (i.e., for example, inorganic and/or organic). Any particular analyte may be detected and/or identified from a sample containing a plurality of other analytes. Further, the invention provides for a capability of simultaneously detecting and/or identifying all of the plurality of analytes contained within a sample (i.e., for example, a biological sample).

Background

The availability of high-performance analyte detection technologies (i.e., for example, for RNA profiling) has been applied to elucidating the mechanisms of action of disease genes and the identification of small molecule therapeutics by molecular signature screening (Lamb et al., Cell 114:323-34 (2003); Stegmaier et al., Nature Genetics 36:257-63 (2004)). For example, detection and quantification of differentially expressed genes in a number of conditions including malignancy, cellular disorders, etc. would be useful in the diagnosis, prognosis and treatment of these pathological conditions. Quantification of gene expression would also be useful in indicating susceptibility to a range of conditions and following up effects of pharmaceuticals or toxins on molecular level. These methods can also be used to screen for molecules that provide a desired gene profile.

The power of being able to simultaneously measure multiple analytes (i.e., for example, transcriptome-wide mRNA species) has been of recent interest. For example, gene expression profiling or microarray analysis has enabled the measurement of thousands of genes in a single sample. Expression profiles have been widely used in both research and clinical settings, for example in classification of cancer subtypes and predicting treatment outcome. However, for nucleic acid analytes, both microarray and tag-sequencing techniques are associated with a number of significant problems. In addition, the array quality is often a problem for cDNA or oligonucleotide microarrays. For example, most researchers cannot confirm the identity of what is immobilized on the surface of a microarray and generally have limited capacity to check and control possible errors in the microarray fabrication.

Additionally, the high costs of microarrays have caused many investigators to perform relatively few control experiments to assess the reliability, validity, and repeatability of their findings. Moreover, given the high costs of microarray fabrication, custom designing arrays to tailor analysis to an individual expression profile is simply impractical in many instances. For the tag-sequencing analysis, a large amount of sequencing effort, generally slow and costly, is needed for tag-based analysis and the sensitivity of tag-based analyses is relatively low and high sensitivity can only be achieved by sequencing a large number of tag sequences.

What is needed in the art is a methodology that avoids the necessity for expensive and laborious microarray systems in favor of solution-based array systems capable of high throughput. Currently available solution-based array systems are still limited in detection capacity (i.e., for example 5-500 analytes per assay). Compositions and methods are described herein that not only allow the detection of a single analyte from a single data bit but expands the current capacity of current solution-based detection methods up to ten-fold by detecting different analytes within a multimodal distribution signal pattern.

Summary Of The Invention

In one embodiment, the present invention contemplates a method, comprising: a) providing; i) a sample comprising a plurality of analytes; ii) a plurality of solid substrate populations, wherein each of the solid substrate populations comprise a plurality of subsets, and wherein each subset is present in an unequal proportion from every other subset in the same solid substrate population; iii) a plurality of capture probes capable of attaching to said plurality of analytes, wherein each subset comprises a different capture probe; vi) a means for detecting said plurality of subsets that is capable of creating a multimodal intensity distribution pattern; b) detecting said plurality of subsets with said means, wherein a multimodal intensity distribution pattern is created; c) identifying said plurality of analytes from said multimodal distribution pattern. In one embodiment, the sample may be selected from the group comprising a biological sample, a soil sample, or a water sample. In one embodiment, the plurality of analytes may be selected from the group comprising nucleic acids, proteins, peptides, drugs, small molecules, biological receptors, enzymes, antibodies, polyclonal antibodies, monoclonal antibodies, or Fab fragments. In one embodiment, the solid substrate population comprises a bead-set population. In one embodiment, the unequal proportions comprise two subsets in an approximate ratio of 1.25:0.75. In one embodiment, the unequal proportions comprise three subsets in an approximate ratio of 1.25: 1.00:0.75. In one embodiment, the unequal proportions comprise four subsets in an approximate ratio of 1.25:1.00:0.75:0.50. In one embodiment, the unequal proportions comprise five subsets in an approximate ratio of 1.50:1.25:1.00:0.75:0.50. In one embodiment, the unequal proportions comprise six subsets in an approximate ratio of 1.75: 1 :50:1.25:1.00:0.75:0.50. In one embodiment, the unequal proportions comprise seven subsets in an approximate ratio of 2.00: 1.75:1 :50:1.25:1.00:0.75:0.50. In one embodiment, the unequal proportions comprise eight subsets in an approximate ratio of 2.00:1.75:1 :50:1.25:1:00:0.75:0.50:0.25. In one embodiment, the unequal proportions comprise nine subsets in an approximate ratio of 2.25:2.00:1.75:1 :50:1.25:1.00:0.75:0.50:0.25. In one embodiment, the unequal proportions comprise ten subsets in an approximate ratio of

2.5:2.25:2.00:1.75:1 :50:1.25:1.00:0.75:0.50:0.25.

In one embodiment, the present invention contemplates a method, comprising: a) providing; i) a solid substrate population comprising a first subset and a second subset, wherein the first subset is present in a first proportion and the second subset is present in a second proportion; ii) a first analyte attached to said first subset; iii) a second analyte attached to said second subset; vi) a means for detecting said first subset and second subset that is capable of creating a multimodal intensity distribution pattern; b) detecting said first subset and said second subset with said means, wherein a multimodal intensity distribution pattern is created; and c) identifying said first analyte and said second analyte from said multimodal distribution pattern.

In one embodiment, the solid substrate population comprises a label. In one embodiment, the label comprises a mixture of at least two different fluorophores. In one embodiment, the first proportion is different from the second proportion. In one embodiment, the first analyte is attached to the first subset with a first capture probe. In one embodiment, the second analyte is attached to the second subset with a second capture probe. In one embodiment, the multimodal intensity distribution pattern comprises a first peak corresponding to the first subset. In one embodiment, the multimodal intensity distribution pattern comprises a second peak corresponding to the second subset.

In one embodiment, the present invention contemplates a method, comprising: a) providing; i) a solid substrate population comprising a plurality of subsets; ii) a sample comprising a plurality of analytes, wherein at least one portion of the plurality of analytes comprise related analytes; and iii) a means for detecting said subsets that is capable of creating a multimodal intensity distribution pattern; b) attaching each of the related analyte portions to one of the plurality of subsets; c) detecting said plurality of subsets with said means, wherein a multimodal intensity distribution pattern is created; and d) identifying said related analytes from said multimodal distribution pattern. In one embodiment, the related analytes comprise linked genes.

In one embodiment, the present invention contemplates a method, comprising: a) providing; i) a solid substrate population comprising a plurality of subsets; ii) a sample comprising a plurality of analytes, wherein at least one portion of the plurality of analytes comprise rare event analytes; and iii) a means for detecting said subsets that is capable of creating a multimodal intensity distribution pattern; b) attaching a portion of said plurality of analytes which may contain one or more of the rare event analytes to one of the plurality of subsets; c) detecting said plurality of subsets with said means, wherein a multimodal intensity distribution pattern is created; and d) determining if said rare event analytes occur in said multimodal distribution pattern. In one embodiment, the rare event analyte portion is present in approximately less than 0.01% of said sample. In one embodiment, the rare event analyte comprises a small molecule or drug. In one embodiment, the rare event analyte comprises a nucleic acid mutation. In one embodiment, the rare event analyte comprises a diseased cell. In one embodiment, the rare event analyte comprises an autoimmune antibody. In one embodiment, the rare event analyte comprises a microbe.

In one embodiment, the present invention contemplates a method, comprising: a) providing; i) a solid substrate population comprising a plurality of subsets; ii) a sample comprising a first labeled analyte and a second labeled analyte; and iii) a means for detecting said subsets that is capable of creating a multimodal intensity distribution pattern; b) attaching the first and second labeled analytes in an unequal proportion to one of the plurality of subsets; c) detecting said plurality of subsets with said means, wherein a multimodal intensity distribution pattern is created; and d) identifying said first and second labeled analytes from said multimodal distribution pattern. In one embodiment, the first labeled analyte comprises a normal cell. In one embodiment, the second labeled analyte comprises a tumor cell. In one embodiment, the multimodal intensity distribution pattern comprises a first peak corresponding to the first labeled analyte. In one embodiment, the multimodal intensity distribution pattern comprises a second peak corresponding to the second labeled analyte. In one embodiment, the unequal proportion is equivalent to a ratio of the first and second peaks.

Definitions

It should be noted that, as used in this specification and the appended claims, the singular forms "a,", "an" and "the" include plural referents unless the content clearly dictates otherwise. It should also be noted that the term "or" is generally employed in its sense including "and/or" unless the content clearly dictates otherwise.

The term, "analyte" as used herein, refers to a variety of biological and chemical molecules including, but not limited to, genes, proteins, ligands (i.e., for example, peptides and/or small organic molecules, mRNA and/or microRNA. For example, the data provided herein utilizes genes as an exemplary analyte though the disclosed embodiments are equally applicable to any other type of analyte. As described herein, an analyte is capable of binding to a capture probe.

The term "related analyte" as used herein, refers to any collection of analytes that are expected to have similar or identical responses to various stimuli, such that they increase or decrease in concentration within a sample in a proportional manner. For example, a particular disease (i.e., for example, cancer) may elevate expression levels of a set of transcripts, such transcripts are then considered to be "related analytes".

The term "rare event analyte" as used herein, refers to any collection of analytes that are detectable within a sample on an infrequent basis (i.e., for example, represented in between approximately 0.1 - 0.00001% of samples)

The term "solid substrate population" as used herein, refers to any collection of particles that are capable of differential labeling such that subsets of solid substrates within the population may be identified. For example, the differential labeling may comprise various mixtures of fluorophores within the particles.

The term, "bead" or "microsphere" as used herein, refers to any solid substrate, often spherically shaped, and having a preferred diameter of 5.6 micrometers, but substrates having larger, or smaller diameters, are contemplated herein. A single bead and/or microsphere has been referred to by others as a "suspended microarray particle". Such solid substrates may be made of a variety of materials including, but not limited to, polystyrene, cross-linked polystyrene, polyacrylate, polylactic acid, polyglycolic acid, poly(lactide coglycolide), polyanhydrides, poly(methyl methacrylate), poly(ethylene-co-vinyl acetate), polysiloxanes, polymeric silica, latexes, dextran polymers and/or epoxies, glass, ceramic, silicon and/or quartz. These materials have a variety of different properties with regard to swelling and porosity, which are well understood in the art. Preferably, the beads are in the size range of approximately 10 nm to 1 mm, and can be manipulated using normal solution techniques when suspended in solution. Beads may be porous or non-porous. In some embodiments, ligands may be attached within the bead as well as on the bead surface. Microspheres and/or beads may be distinguished from one another by characteristics including, but not limited to, color, size, shape, and/or light scattering properties. Finkel et al, Analytical Chemistry 76(19):352A-359A (2004).

The term, "coupled bead" or "tagged bead" refers to any bead attached to a label (i.e., for example, a uniquely colored bead attached to a capture probe).

The term "bead-set", "subset", "microbead region", or "suspended microarray particle category set" as used herein, refers to a solid particle population that are all labeled with the same classifier (i.e., for example, a color and/or bead number set). For example, bead-set may encompass Luminex bead numbers 1-500.

The terms "coupled", "connected", "attached", "linked", or "conjugated" are used interchangeably herein and encompass direct as well as indirect connection, attachment, linkage, or conjugation unless the context clearly dictates otherwise. The attachment of a ligand to a bead may be covalent or non-covalent. Attachment may be reversible or irreversible. Such attachment includes, but is not limited to, covalent bonding, ionic bonding, Van der Waals forces or friction, and the like.

The terms "capture probe", "tag", "label" or "barcode" as used interchangeably herein and encompass any molecule capable of attaching and/or binding to an analyte. For example, a capture probe may be an oligonucleotide, wherein the oligonucleotide is at least partially complementary to an analyte oligonucleotide. Alternatively, a capture probe may include but is not limited to a polyethylene glycol linker, an antibody, a polyclonal antibody, a monoclonal antibody, an Fab fragment, biological receptor complex, an enzyme, a hormone, an antigen, and/or a fragment or portion thereof.

The term, "unequal proportion" as used herein refers to any mixture of multiple components, wherein the multiple components are not present in a 1 :1 ratio. For example, two components may be present in an approximate ratio of 1.25:0.75. Alternatively, three components may be present in an approximate ratio of 1.25: 1.00:0.75. Alternatively, four components maybe present in an approximate ratio of 1.25:1.00:0.75:0.5. Alternatively, five components maybe present in an approximate ratio of 1.50:1.25:1.00:0.75:0.50.

Alternatively, six components may be present in an approximate ratio of

1.75:1:50:1.25:1.00:0.75:0.50. Alternatively, seven components may be present in an approximate ratio of 2.00:1.75:1 :50:1.25:1 :00:0.75:0.50. Alternatively, eight components may be present in an approximate ratio of 2.00: 1.75: 1 :50: 1.25 : 1.00:0.75 :0.50:0.25.

Alternatively, nine components may be present in an approximate ratio of

2.25:2.00:1.75:1 :50:1.25:1.00:0.75:0.50:0.25. Alternatively, ten components may be present in an approximate ratio of 2.50:2.25:2.00: 1.75: 1 :50: 1.25: 1.00:0.75:0.50:0.25.

The term "counts" as used herein, refers to the number of measured coupled beads that are attached or bound to at least one analyte.

The term "multimodal distribution pattern' refers to any data set of at least two tagged-beads plotted as bead count versus label intensity. For example, a first tagged-bead is conjugated to a first analyte while the second tagged bead is conjugated to a second analyte. Any specific intensity peak within the multimodal distribution pattern may represent data collected from a single tagged-bead.

The term "luminescence" as used herein, refers to any spontaneous emission of photons in the range from ultraviolet to infrared, after optical or other than optical excitation, such

as electrical or chemical or biochemical or thermal excitation. For example,

chemiluminescence, bioluminescence, electroluminescence, and especially fluorescence and phosphorescence are included commonly under the term "luminescence".

The term "high throughput" as used herein, refers to the detection or analysis of more than one reaction in a single process, where each reaction is itself a multiplex reaction, detecting more than one analytes simultaneously. For example, 2-10,000 multiplex reactions can be processed simultaneously.

The term "a means for detecting" as used herein, refers to any method and/or device that is capable of individually sensing each subset of a solid particle population, even if the subset comprises a single solid particle. For example, the means may be a flow cytometer that detects the solid particles using laser scanning.

The term "disease", as used herein, refers to any impairment of the normal state of the living animal or plant body or one of its parts that interrupts or modifies the performance of the vital functions. Typically manifested by distinguishing signs and symptoms, it is usually a response to: i) environmental factors (as malnutrition, industrial hazards, or climate); ii) specific infective agents (as worms, bacteria, or viruses); iii) inherent defects of the organism (as genetic anomalies); and/or iv) combinations of these factors.

The term "affinity" as used herein, refers to any attractive force between substances or particles that causes them to enter into and remain in chemical combination. For example, an inhibitor compound that has a high affinity for a receptor will provide greater efficacy in preventing the receptor from interacting with its natural ligands, than an inhibitor with a low affinity.

The term "derived from" as used herein, refers to the source of a compound or sequence. In one respect, a compound or sequence may be derived from an organism or particular species. In another respect, a compound or sequence may be derived from a larger complex or sequence.

The term "protein" as used herein, refers to any of numerous naturally occurring extremely complex substances (as an enzyme or antibody) that consist of amino acid residues joined by peptide bonds, contain the elements carbon, hydrogen, nitrogen, oxygen, usually sulfur. In general, a protein comprises amino acids having an order of magnitude within the hundreds.

The term "peptide" as used herein, refers to any of various amides that are derived from two or more amino acids by combination of the amino group of one acid with the carboxyl group of another and are usually obtained by partial hydrolysis of proteins. In general, a peptide comprises amino acids having an order of magnitude with the tens.

The term, "purified" or "isolated", as used herein, may refer to a peptide composition that has been subjected to treatment (i.e., for example, fractionation) to remove various other components, and which composition substantially retains its expressed biological activity. Where the term "substantially purified" is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the composition (i.e., for example, weight/weight and/or weight/volume). The term "purified to homogeneity" is used to include compositions that have been purified to

'apparent homogeneity" such that there is single protein species (i.e., for example, based upon SDS-PAGE or HPLC analysis). A purified composition is not intended to mean that some trace impurities may remain.

As used herein, the term "substantially purified" refers to molecules, either nucleic or amino acid sequences, that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and more preferably 90% free from other components with which they are naturally associated. An "isolated polynucleotide" is therefore a substantially purified polynucleotide.

"Nucleic acid sequence" and "nucleotide sequence" as used herein refer to an oligonucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense or antisense strand.

The term "an isolated nucleic acid", as used herein, refers to any nucleic acid molecule that has been removed from its natural state (e.g., removed from a cell and is, in a preferred embodiment, free of other genomic nucleic acid).

The terms "amino acid sequence" and "polypeptide sequence" as used herein, are interchangeable and to refer to a sequence of amino acids.

As used herein the term "portion" when in reference to a protein (as in "a portion of a given protein") refers to fragments of that protein. The fragments may range in size from four amino acid residues to the entire amino acid sequence minus one amino acid.

The term "portion" when used in reference to a nucleotide sequence refers to fragments of that nucleotide sequence. The fragments may range in size from 5 nucleotide residues to the entire nucleotide sequence minus one nucleic acid residue.

The term "antibody" refers to immunoglobulin evoked in animals by an immunogen (antigen). It is desired that the antibody demonstrates specificity to epitopes contained in the immunogen. The term "polyclonal antibody" refers to immunoglobulin produced from more than a single clone of plasma cells; in contrast "monoclonal antibody" refers to

immunoglobulin produced from a single clone of plasma cells.

The terms "specific binding" or "specifically binding" when used in reference to the interaction of an antibody and a protein or peptide means that the interaction is dependent upon the presence of a particular structure (i.e., for example, an antigenic determinant or epitope) on a protein; in other words an antibody is recognizing and binding to a specific protein structure rather than to proteins in general. For example, if an antibody is specific for epitope "A", the presence of a protein containing epitope A (or free, unlabelled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A bound to the antibody.

The term "small organic molecule" as used herein, refers to any molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size from approximately 10 Da up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.

As used herein, the term "antisense" is used in reference to RNA sequences which are complementary to a specific RNA sequence (e.g., mRNA). Antisense RNA may be produced by any method, including synthesis by splicing the gene(s) of interest in a reverse orientation to a viral promoter which permits the synthesis of a coding strand. Once introduced into a cell, this transcribed strand combines with natural mRNA produced by the cell to form duplexes. These duplexes then block either the further transcription of the mRNA or its translation. In this manner, mutant phenotypes may be generated. The term "antisense strand" is used in reference to a nucleic acid strand that is complementary to the "sense" strand. The designation (-) (i.e., "negative") is sometimes used in reference to the antisense strand, with the designation (+) sometimes used in reference to the sense (i.e., "positive") strand.

As used herein, the terms "siRNA" refers to either small interfering RNA, short interfering RNA, or silencing RNA. Generally, siRNA comprises a class of double-stranded RNA molecules, approximately 20-25 nucleotides in length. Most notably, siRNA is involved in RNA interference (RNAi) pathways and/or RNAi-related pathways, wherein the compounds interfere with gene expression.

As used herein, the term "shRNA" refers to any small hairpin RNA or short hairpin RNA. Although it is not necessary to understand the mechanism of an invention, it is believed that any sequence of RNA that makes a tight hairpin turn can be used to silence gene expression via RNA interference. Typically, shRNA uses a vector stably introduced into a cell genome and is constitutively expressed by a compatible promoter. The shRNA hairpin structure may also cleaved into siRNA, which may then become bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs which match the siRNA that is bound to it.

As used herein, the term "microRNA", "miRNA", or "μΚΝΑ" refers to any single-stranded RNA molecules of approximately 21-23 nucleotides in length, which regulate gene expression. miRNAs may be encoded by genes from whose DNA they are transcribed but miRNAs are not translated into protein (i.e. they are non-coding RNAs). Each primary transcript (a pri-miRNA) is processed into a short stem-loop structure called a pre-miRNA and finally into a functional miRNA. Mature miRNA molecules are partially complementary to one or more messenger RNA (mRNA) molecules, and their main function is to down- regulate gene expression. The term "sample" as used herein is used in its broadest sense and includes environmental and biological samples. Environmental samples include material from the environment such as soil and water. Biological samples may be animal, including, human, fluid (e.g., blood, plasma and serum), solid (e.g., stool), tissue, liquid foods (e.g., milk), and solid foods (e.g., vegetables). For example, a pulmonary sample may be collected by bronchoalveolar lavage (BAL) which comprises fluid and cells derived from lung tissues. A biological sample may comprise a cell, tissue extract, body fluid, chromosomes or extrachromosomal elements isolated from a cell, genomic DNA (in solution or bound to a solid support such as for Southern blot analysis), RNA (in solution or bound to a solid support such as for Northern blot analysis), cDNA (in solution or bound to a solid support) and the like.

As used herein, the terms "complementary" or "complementarity" are used in reference to "polynucleotides" and "oligonucleotides" (which are interchangeable terms that refer to a sequence of nucleotides) related by the base-pairing rules. For example, the sequence "C-A-G-T," is complementary to the sequence "G-T-C-A." Complementarity can be "partial" or "total." "Partial" complementarity is where one or more nucleic acid bases is not matched according to the base pairing rules. "Total" or "complete" complementarity between nucleic acids is where each and every nucleic acid base is matched with another base under the base pairing rules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods which depend upon binding between nucleic acids.

The terms "homology" and "homologous" as used herein in reference to nucleotide sequences refer to a degree of complementarity with other nucleotide sequences. There may be partial homology or complete homology (i.e., identity). A nucleotide sequence which is partially complementary, i.e., "substantially homologous," to a nucleic acid sequence is one that at least partially inhibits a completely complementary sequence from hybridizing to a target nucleic acid sequence. The inhibition of hybridization of the completely

complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a completely homologous sequence to a target sequence under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction. The absence of nonspecific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of nonspecific binding the probe will not hybridize to the second non-complementary target.

The terms "homology" and "homologous" as used herein in reference to amino acid sequences refer to the degree of identity of the primary structure between two amino acid sequences. Such a degree of identity may be directed to a portion of each amino acid sequence, or to the entire length of the amino acid sequence. Two or more amino acid sequences that are "substantially homologous" may have at least 50% identity, preferably at least 75% identity, more preferably at least 85% identity, most preferably at least 95%, or 100% identity.

An oligonucleotide sequence which is a "homolog" is defined herein as an oligonucleotide sequence which exhibits greater than or equal to 50% identity to a sequence, when sequences having a length of 100 bp or larger are compared.

Low stringency conditions comprise conditions equivalent to binding or hybridization at 42°C in a solution consisting of 5 x SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH2P04-H20 and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5x Denhardfs reagent {50x

Denhardt's contains per 500 ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)} and 100 μg/ml denatured salmon sperm DNA followed by washing in a solution comprising 5x SSPE, 0.1% SDS at 42°C when a probe of about 500 nucleotides in length, is employed. Numerous eqtiivalent conditions may also be employed to comprise low stringency conditions; factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target ( DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol), as well as components of the hybridization solution may be varied to generate conditions of low stringency hybridization different from, but equivalent to, the above listed conditions. In addition, conditions which promote hybridization under conditions of high stringency (e.g., increasing the temperature of the hybridization and/or wash steps, the use of formamide in the hybridization solution, etc.) may also be used.

As used herein, the term "hybridization" is used in reference to the pairing of complementary nucleic acids using any process by which a strand of nucleic acid joins with a complementary strand through base pairing to form a hybridization complex. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementarity between the nucleic acids, stringency of the conditions involved, the Tm of the formed hybrid, and the G:C ratio within the nucleic acids.

As used herein the term "hybridization complex" refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bounds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds maybe further stabilized by base stacking interactions. The two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration. A hybridization complex maybe formed in solution (e.g., CO t or R0 1 analysis) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized to a solid support (e.g., a nylon membrane or a nitrocellulose filter as employed in Southern and Northern blotting, dot blotting or a glass slide as employed in in situ hybridization, including FISH (fluorescent in situ hybridization)).

As used herein, the term "Tm " is used in reference to the "melting temperature." The melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands. As indicated by standard references, a simple estimate of the Tm value may be calculated by the equation: Tm = 81.5 + 0.41 (% G+C), when a nucleic acid is in aqueous solution at 1M NaCl. Anderson et al, "Quantitative Filter Hybridization" In: Nucleic Acid Hybridization (1985). More sophisticated

computations take structural, as well as sequence characteristics, into account for the calculation of Tm.

As used herein the term "stringency" is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted. "Stringency" typically occurs in a range from about Tm to about 20°C to 25°C below Tm. A "stringent hybridization" can be used to identify or detect identical polynucleotide sequences or to identify or detect similar or related polynucleotide sequences. For example, when fragments are employed in

hybridization reactions under stringent conditions the hybridization of fragments which contain unique sequences (i.e., regions which are either non-homologous to or which contain less than about 50% homology or complementarity) are favored. Alternatively, when conditions of "weak" or "low" stringency are used hybridization may occur with nucleic acids that are derived from organisms that are genetically diverse (i.e., for example, the frequency of complementary sequences is usually low between such organisms). As used herein, the term "amplifiable nucleic acid" is used in reference to nucleic acids which maybe amplified by any amplification method. It is contemplated that

"amplifiable nucleic acid" will usually comprise "sample template."

As used herein, the term "sample template" refers to nucleic acid originating from a sample which is analyzed for the presence of a target sequence of interest. In contrast,

"background template" is used in reference to nucleic acid other than sample template which may or may not be present in a sample. Background template is most often inadvertent. It may be the result of carryover, or it may be due to the presence of nucleic acid contaminants sought to be purified away from the sample. For example, nucleic acids from organisms other than those to be detected may be present as background in a test sample.

"Amplification" is defined as the production of additional copies of a nucleic acid sequence and is generally carried out using polymerase chain reaction. Dieffenbach C. W. and G. S. Dveksler (1995) In: PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.

As used herein, the term "polymerase chain reaction" ("PCR") refers to the method of

K. B. Mullis U.S. Pat. Nos. 4,683,195 and 4,683,202, herein incorporated by reference, which describe a method for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. The length of the amplified segment of the desired target sequence is determined by the relative positions of two oligonucleotide primers with respect to each other, and therefore, this length is a controllable parameter. By virtue of the repeating aspect of the process, the method is referred to as the "polymerase chain reaction" (hereinafter "PCR"). Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be "PCR amplified". With PCR, it is possible to amplify a single copy of a specific target sequence in genomic DNA to a level detectable by several different methodologies (e.g., hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of 32P-labeled

deoxynucleotide triphosphates, such as dCTP or dATP, into the amplified segment). In addition to genomic DNA, any oligonucleotide sequence can be amplified with the appropriate set of primer molecules. In particular, the amplified segments created by the PCR process itself are, themselves, efficient templates for subsequent PCR amplifications.

As used herein, the term "primer" refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, (i.e., in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH). The primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxy-ribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method.

As used herein, the term "probe" refers; to an oligonucleotide (i.e., a sequence of nucleotides), whether occurring naturally as in a purified restriction digest or produced synthetically, recombinantly or by PCR amplification, which is capable of hybridizing to another oligonucleotide of interest. A probe may be single-stranded or double-stranded. Probes are useful in the detection, identification and isolation of particular gene sequences. It is contemplated that any probe used in the present invention will be labeled with any

"reporter molecule," so that is detectable in any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the present invention be limited to any particular detection system or label.

As used herein, the terms "restriction endonucleases" and "restriction enzymes" refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.

DNA molecules are said to have "5' ends" and "3' ends" because mononucleotides are reacted to make oligonucleotides in a manner such that the 5' phosphate of one

mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage. Therefore, an end of an oligonucleotide is referred to as the "5' end" if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring. An end of an oligonucleotide is referred to as the "3' end" if its 3' oxygen is not linked to a 5' phosphate of another mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5' and 3' ends. In either a linear or circular DNA molecule, discrete elements are referred to as being "upstream" or 5' of the "downstream" or 3' elements. This terminology reflects the fact that transcription proceeds in a 5' to 3' fashion along the DNA strand. The promoter and enhancer elements which direct transcription of a linked gene are generally located 5' or upstream of the coding region. However, enhancer elements can exert their effect even when located 3' of the promoter element and the coding region. Transcription termination and polyadenylation signals are located 3' or downstream of the coding region.

As used herein, the term "an oligonucleotide having a nucleotide sequence encoding a gene" means a nucleic acid sequence comprising the coding region of a gene, i.e. the nucleic acid sequence which encodes a gene product. The coding region may be present in a cDNA, genomic DNA or RNA form. When present in a DNA form, the oligonucleotide may be single-stranded (i.e., the sense strand) or double-stranded. Suitable control elements such as enhancers/promoters, splice junctions, polyadenylation signals, etc. maybe placed in close proximity to the coding region of the gene if needed to permit proper initiation of transcription and/or correct processing of the primary RNA transcript. Alternatively, the coding region utilized in the expression vectors of the present invention may contain endogenous enhancers/promoters, splice junctions, intervening sequences, polyadenylation signals, etc. or a combination of both endogenous and exogenous control elements.

As used herein, the term "regulatory element" refers to a genetic element which controls some aspect of the expression of nucleic acid sequences. For example, a promoter is a regulatory element which facilitates the initiation of transcription of an operably linked coding region. Other regulatory elements are splicing signals, polyadenylation signals, termination signals, etc.

The term "in operable combination" as used herein, refers to any linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced. Regulatory sequences may be operably combined to an open reading frame including but not limited to initiation signals such as start (i.e., ATG) and stop codons, promoters which maybe constitutive (i.e., continuously active) or inducible, as well as enhancers to increase the efficiency of expression, and transcription termination signals.

Transcriptional control signals in eukaryotes comprise "promoter" and "enhancer" elements. Promoters and enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription. Maniatis, T. et al., Science 236:1237 (1987). Promoter and enhancer elements have been isolated from a variety of eukaryotic sources including genes in plant, yeast, insect and mammalian cells and viruses (analogous control elements, i.e., promoters, are also found in prokaryotes). The selection of a particular promoter and enhancer depends on what cell type is to be used to express the protein of interest. The presence of "splicing signals" on an expression vector often results in higher levels of expression of the recombinant transcript. Splicing signals mediate the removal of introns from the primary RNA transcript and consist of a splice donor and acceptor site. Sambrook, J. et al., In: Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor laboratory Press, New York (1989) pp. 16.7-16.8. A commonly used splice donor and acceptor site is the splice junction from the 16S RNA of SV40.

The term "poly A site" or "poly A sequence" as used herein denotes a DN A sequence which directs both the termination and polyadenylation of the nascent RNA transcript.

Efficient polyadenylation of the recombinant transcript is desirable as transcripts lacking a poly A tail are unstable and are rapidly degraded. The poly A signal utilized in an expression vector may be "heterologous" or "endogenous." An endogenous poly A signal is one that is found naturally at the 3' end of the coding region of a given gene in the genome. A

heterologous poly A signal is one which is isolated from one gene and placed 3' of another gene. Efficient expression of recombinant DNA sequences in eukaryotic cells involves expression of signals directing the efficient termination and polyadenylation of the resulting transcript. Transcription termination signals are generally found downstream of the polyadenylation signal and are a few hundred nucleotides in length.

As used herein, the terms "nucleic acid molecule encoding", "DNA sequence encoding," and "DNA encoding" refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus codes for the amino acid sequence.

The term "Southern blot" refers to the analysis of DNA on agarose or acrylamide gels to fractionate the DNA according to size, followed by transfer and immobilization of the DNA from the gel to a solid support, such as nitrocellulose or a nylon membrane. The immobilized DNA is then probed with a labeled oligodeoxyribonucleotide probe or DNA probe to detect DNA species complementary to the probe used. The DNA may be cleaved with restriction enzymes prior to electrophoresis. Following electrophoresis, the DNA may be partially depurinated and denatured prior to or during transfer to the solid support.

Southern blots are a standard tool of molecular biologists. J. Sambrook et al. (1989) In:

Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, pp 9.31-9.58.

The term "Northern blot" as used herein refers to the analysis of RNA by

electrophoresis of RNA on agarose gels to fractionate the RNA according to size followed by transfer of the RNA from the gel to a solid support, such as nitrocellulose or a nylon membrane. The immobilized RNA is then probed with a labeled oligodeoxyribonucleotide probe or DNA probe to detect RNA species complementary to the probe used. Northern blots are a standard tool of molecular biologists. J. Sambrook, J. et al. (1989) supra, pp 7.39-7.52.

The term "reverse Northern blot" as used herein refers to the analysis of DNA by electrophoresis of DNA on agarose gels to fractionate the DNA on the basis of size followed by transfer of the fractionated DNA from the gel to a solid support, such as nitrocellulose or a nylon membrane. The immobilized DNA is then probed with a labeled oligoribomiclotide probe or RNA probe to detect DNA species complementary to the ribo probe used.

As used herein the term "coding region" when used in reference to a structural gene refers to the nucleotide sequences which encode the amino acids found in the nascent polypeptide as a result of translation of a mRNA molecule. The coding region is bounded, in eukaryotes, on the 5' side by the nucleotide triplet "ATG" which encodes the initiator methionine and on the 3' side by one of the three triplets which specify stop codons (i.e., TAA, TAG, TGA).

As used herein, the term "structural gene" refers to a DNA sequence coding for RNA or a protein. In contrast, "regulatory genes" are structural genes which encode products which control the expression of other genes (e.g., transcription factors).

As used herein, the term "gene" means the deoxyribonucleotide sequences comprising the coding region of a structural gene and including sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb on either end such that the gene corresponds to the length of the full-length mRNA. The sequences which are located 5' of the coding region and which are present on the mRNA are referred to as 5' non-translated sequences. The sequences which are located 3' or downstream of the coding region and which are present on the mRNA are referred to as 3' non-translated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed "introns" or "intervening regions" or "intervening sequences." Introns are segments of a gene which are transcribed into heterogeneous nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or "spliced out" from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

In addition to containing introns, genomic forms of a gene may also include sequences located on both the 5' and 3' end of the sequences which are present on the RNA transcript. These sequences are referred to as "flanking" sequences or regions (these flanking sequences are located 5' or 3' to the non-translated sequences present on the mRNA transcript). The 5' flanking region may contain regulatory sequences such as promoters and enhancers which control or influence the transcription of the gene. The 3' flanking region may contain sequences which direct the termination of transcription, posttranscriptional cleavage and polyadenylation.

The term "label" or "detectable label" are used herein, to refer to any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Such labels include biotin for staining with labeled streptavidin conjugate, magnetic beads (e.g., Dynabeads®), fluorescent dyes (e.g., fluorescein, texas red, rhodamine, green fluorescent protein, and the like), radiolabels (e.g., 3H, 1251, 35S, 14C, or 32P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Patents teaching the use of such labels include, but are not limited to, U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241 (all herein incorporated by reference). The labels contemplated in the present invention may be detected by many methods. For example, radiolabels maybe detected using photographic film or scintillation counters, fluorescent markers may be detected using a photodetector to detect emitted light. Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting, the reaction product produced by the action of the enzyme on the substrate, and calorimetric labels are detected by simply visualizing the colored label.

Brief Description Of The Figures

The file of this patent contains at least one drawing executed in color. Copies of this patent with color drawings will be provided by the Patent and Trademark Office upon request and payment of the necessary fee. Figure 1 presents one embodiment of a commercially available Luminex bead region. For example, a three-dimensional plot is presented showing the respective coordinates of each of the 500 bead color variation regions recognized by the Luminex system

Figure 2 presents a schematic showing one embodiment for detecting two

independent collections of tagged-beads, each detecting 500 analytes in separate wells.

Figure 3 presents a schematic showing embodiments for detecting two mixed collections of tagged-beads, each detecting 500 analytes in a single well. The left hand panel shows previously reported "ligand mediated amplification" (LMA) and indicates several problems with this approach. The right hand panel shows improvements contemplated by some embodiments of the present invention.

Figure 4 presents exemplary data comparing a median based peak algorithm summarization with kernel based peak algorithm summarization for analyte 179 and analyte 289. Dashed green line = MFI value. Red line = smoothed kernel density estimate. White circle = kernel-based summary value.

Figure 5 presents exemplary data from the output of a kernel density peak detection algorithm comparing a combined collection of two bead-sets of unequal proportion providing a bimodal distribution (center plot) of Gene 1 and Gene 2. Their respective unimodal distribution patterns are also presented (right and left plots). A bimodal distribution is presented a bead first color variation attached to Gene 1 in a first unequal proportion (Peak 1) and a bead first color variation attached to Gene 2 in a second unequal proportion (Peak 2). Red curve = smoothened kernel density estimate. Local maxima are indicated. The expression of the individual genes measured separately is shown alongside.

Figure 6 presents exemplary scatter plots comparing combined tagged-bead collections (dual tag - Y axis) with individual tagged-bead collection (single tag - X axis). The linearity of the scatter plot regression analysis demonstrates that the detection is comparable.

Figure 7 presents one embodiment of an algorithm that determines peak calling using a Gaussian mixture model (GMM). The histogram indicates the raw distribution of intensities. Red curves = the two Gaussian distributions estimated via a GMM algorithm.

Figure 8 presents exemplary data showing the expression of optimal gene pairs selected by a gene-pairing algorithm.

Figure 9 presents exemplary data comparing optimal gene pairing (red curve) shown in Figure 8 and random gene pairings (blue curves). The optimally paired genes have larger absolute median deviations indicating larger separations between the pairs than the majority of randomly paired genes.

Figure 10 presents exemplary data collected from eight (8) genes.

Figure 10A: A calibration plot showing the relationship between HEK293 cell invariant gene set levels and measured intensity levels.

Figure 1 OB: A representative bead level distribution pattern.

Figure 11 presents exemplary data showing cell sample data from a 384 well plate.

Figure 11 A: An median expression analysis showing a trend of increasing intensity at each invariant transcript (Invset) level for each sample.

Figure 1 IB: A heat map analysis showing the relative even distribution of median invariant transcript (Invset) expression levels for each sample for each bead (i.e. G50, G100, G200, G400, G600, G1000, G2000, G3000, G5000, and G7000).

Detailed Description

The present invention is related to compositions and methods for the detection of analytes. Analytes capable of detection by this invention include, but are not limited to, nucleic acids, proteins, and/or small organic molecules. Any particular analyte may be detected and/or identified from a sample containing a plurality of other analytes. Further, the invention provides for a capability of simultaneously detecting and/or identifying all of the plurality of analytes contained within a sample (i.e., for example, a biological sample).

I. Solid Substrate Based Detection Systems

One method for differentiating between cell types within a heterogeneous cell mixture has been reported that generates a multimodal distribution pattern following simultaneous flow cytometric data collection. Specifically, multimodal/multispectral images of a population of cells were simultaneously collected, wherein photometric and/or morphometric features identifiable in the images were used to separate the population of cells into subpopulations. A multi-spectral flow cytometer was configured to detect light signals generated by a variety of labels such as, DAP I, FITC, dark field, PE, bright field, and Deep Red. These respective labels were conjugated to specific antibodies that had differential specific binding for normal cells versus diseased cells. Consequently, an abnormal ratio of detected cell patterns provides a basis for disease diagnosis. As such this method was limited to the ability to detect and label antigenic sites on biological cell surfaces that identified the cell's physiological state. Ortyn et al., "Blood And Cell Analysis Using An Imaging Flow Cytometer" United States Patent Application 2009/0190822 (herein incorporated by reference).

A qualitative and quantitative assessment of a plurality of analytes from a biological sample using microwell technology has been developed wherein the biological analytes are attached to a lithographic grid via known biological recognition elements. Identification of the analytes is accomplished by attaching luminescent labels having different emission wavelengths to either the analyte or the recognition element. Consequently, the assay may differentiate between analytes by using two or more labels having the same excitation wavelength, but differing in emission wavelength. Once the analytes are contacted with the lithographic grid, the analyte/recognition element complexes are detected using optically generated luminescent detection technology. Cross-reactivity between analytes could be differentiated by providing recognition elements having differing affinities for the respective analytes. Pawlak et al., "Kit and method for determining a plurality of analytes" United States Patent Number 7,396,675 (herein incorporated by reference).

A method specific for detecting circulating antibodies has been reported that uses microspheres conjugated to labeled antigens for the antibodies. The labeled antigens are usually other antibodies having specific affinity for species-specific Fc portions of a circulating antibody. The labels are described as generally fluorescent labels that are detected using a conventional flow cytometer. A multiplex calibration technique is described that uses several subsets of microspheres or beads, wherein the surface of each microsphere subset has a different concentration of the same antigen. This calibration procedure thereby generates "a standard curve" such that the concentration of a circulating antibody may be estimated.

Connelly et al., "Method and composition for homogeneous multiplexed microparticle-based assay" United States Patent 7,674,632 (herein incorporated by reference).

II. Solution -Based Detections Systems

Solution-based methods are generally based upon the use detectable target-specific bead sets which comprise a capture probe coupled to a detectable bead, where the capture probe binds to an individual labeled target nucleic acid. Each population of bead sets is a collection of individual bead sets, each of which has a unique detectable label which allows it to be distinguished from the other bead sets within the population of bead sets (i.e., for example, ranging from 5 -500 bead sets depending upon assay sensitivity parameters). Any labels or signals can be used to detect the bead sets as long as they provide unique detectable signals for each bead set within the population of bead sets to be processed in a single reaction. Detectable labels include but are not limited to fluorescent labels and enzymatic labels, as well as magnetic or paramagnetic particles (see, e.g., Dynabeads8 (Dynal, Oslo, Norway)). The detectable label may be on the surface of the bead or withi the interior of the bead.

The composition of the beads can vary. Suitable materials include, but are not limited to, any materials used as affinity matrices or supports for chemical and biological molecule syntheses and analyses, including but not limited to: polystyrene, polycarbonate,

polypropylene, nylon, glass, dextran, chitin, sand, pumice, agarose, polysaccharides, dendrimers, buckyballs, polyacrylamide, silicon, rubber, and other materials used as supports for solid phase syntheses, affinity separations and purifications, hybridization reactions, immunoassays and other such applications. Typically the beads have at least one dimension in the 5-10 mm range or smaller. The beads can have any shape and dimensions, but typically have at least one dimension that is 100 mm or less, for example, 50 mm or less, 10 mm or less, 1 mm or less, 100 pm or less, 50 pm or less, and typically have a size that is 10 pm or less such as, 1 pm or less, 100 nm or less, and 10 nm or less. In one embodiment, the beads have at least one dimension between 2-20 pm. Such beads are often, but not necessarily, spherical e.g. elliptical. Such reference, however, does not constrain the geometry of the matrix, which can be any shape, including random shapes, needles, fibers, and elongated. Roughly spherical, particularly microspheres that can be used in the liquid phase, also are contemplated. The beads can include additional components, as long as the additional components do not interfere with the methods and analyses herein.

Commercially available beads which can be used in the methods of the present invention include, but are not limited to, bead-based technologies available from Luminex. Illumina, and/or Lynx. In one embodiment, microbeads can be labeled with different spectral properties and/or fluorescent (or colorimetric) intensities. For example, polystyrene microspheres are provided by Luminex Corp, Austin, Tex. that are internally dyed with two spectrally distinct fluorochromes. Using precise ratios of these fluorochromes, a large number of different fluorescent bead sets can be produced (i.e., for example, 5 - 100 bead sets). Each set of the beads can be distinguished by its spectral address, a combination of which allows for measurement of a large number of analytes in a single reaction vessel. Alternatively, a detectable target molecule may be labeled with a third fluorochrome. Because each of the different bead sets is uniquely labeled with a distinguishable spectral address, the resulting hybridized bead-target complexes will be distinguishable for each different target nucleic acid, which can be detected by passing the hybridized bead-target complexes through a rapidly flowing fluid stream. In the stream, the beads are interrogated individually as they pass two separate lasers. High speed digital signal processing classifies each of the beads based on its spectral address and quantifies the reaction on the surface. Thousands of beads can interrogated per second, resulting a high speed, high throughput and accurate detection of multiple different target nucleic acids in a single reaction. In addition to a detectable label, the bead sets may also contain a capture probe which can bind to an individual target analyte. For example, a capture probe may comprise a nucleic acid, a protein, a peptide, a biological receptor, an enzyme, a hormone, an antibody, a polyclonal antibody, a monoclonal antibody, and/or an Fab fragment. If the capture probe is a short unique DNA sequence, it may comprise uniform hybridization characteristics with a target nucleic acid analyte. The capture probe can be coupled to the beads using any suitable method which generates a stable linkage between probe and the bead, and permits handling of the bead without compromising the linkage using further methods of the invention. Nucleic acid coupling reactions include, but are not limited to, the use capture probes modified with a 5' amine for coupling to carboxylated microsphere or bead.

A. Luminex-Based Detection Systems

Most bead-based analyte detection systems are based upon Luminex colored beads, and/or the Luminex flow cytometric measurement system. The flow cytometric measurement system provides a summary report of median fluorescent intensity (MFI) values for each measured analyte as well as bead-level output data for each sample. The bead-level output data is usually stored in a standard flow cytometry data format, includes a set membership and fluorescent intensity of each individual bead that is detected. Although it is not necessary to understand the mechanism of an invention, it is believed that data collection and storage capability suggests that the capacity of the commercially available Luminex system can be expanded beyond its commonly accepted limitations of 500 bead-sets per well.

The Luminex xMAP^® technology is a commercially available bead-based system that has a limitation for simultaneous measurements of up to 500 analytes per sample.

Measurement instruments used to support Luminex technology are basically a flow cytometers capable of detecting and/or identifying 500 color bead set variations. Usually, each specific color bead variation provides a unique identification for an individual analyte. In particular, the system assigns each bead detected in a sample to a set based on its color. The system then summarizes the measurement value for each set by reporting the median fluorescent intensity (MFI) of all beads belonging to that set.

Recent advances in biotechnology, and in particular genomics, have exceeded the usefulness of data sets restricted to a 500 analyte assay. For example, in gene expression profiling, one might be interested in measuring the expression of more than 500 genes. One approach to overcome this limitation is to use two or more collections of the 500 bead sets, wherein each collection interrogates a different set of 500 genes. This approach requires measurement of the same sample in two separate wells to provide a complete assay. The problem with this approach is that it requires twice the amount of sample and takes twice the amount of time for detection. Duplicate sampling techniques is also prone to failures since failure of a single well also renders the data obtained from the duplicate sample well unusable. In addition, batch artifacts arise during the process of combining the wells that constitute a single sample.

The Luminex detector is analogous to a flow cytometer in that the instrument measures the fluorescent intensity of beads upon passage through a flow chamber.

Alternatively, the detector may be a charged coupled device. Generally, at least two fluorescence measurements are recorded from a maximum of 500 differentially colored bead sets. As a single analyte is usually attached to each differentially colored bead, the fluorescent counts can be used to uniquely identify individual analytes. In particular, the system assigns each bead detected in a sample to a set based on its color. A complete Luminex bead-set comprising these 500 differentially colored beads may be depicted using a three dimensional coordinate plot. See, Figure 1. It is generally believed that the number of differentially colored beads that can be accurately classified to a bead-color-region is limited by the overlapping spectral regimes of the different colors used. For example, a bead-color- region may include, but not limited to 500 beads each identified by a unique 3d coordinate using three classification laser measurements (CL1 , CL2 and CL3) See Figure 1;. In addition to classifying the beads, the instrument records another fluorescence measurement known as a "reporter" for each bead. The "reporter" measurement is used to quantify the chemical reaction of interest and/or determine the presence or absence of an analyte (i.e., for example, mRNA).

Microfluidic devices have also been suggested to be used with methods where labeled microspheres (Luminex beads) would simultaneously detect multiple analytes in one of several sample chambers. These devices are constructed by a process known as multilayer soft lithography (MSL) that create multilayer microfluidic systems, by binding multiple patterned layers of elastomers. For example, the presence of the multi-layered microchannels allow delivery a different labeled microparticle to a specific sample chamber where a different analyte is detected. Each microparticle is specifically functionalized to bind a particular analyte. Therefore, each microparticle in a given sample chamber is capable of analyzing an analyte different from the analyte each other microparticle in the same sample chamber. As the delivery of each microsphere is independently controlled, labeled microspheres may be added to their respective samples chambers in different proportions, presumably to optimize the detection of each specific analyte (i.e., for example, to prevent and/or overcome sample signal saturation). Diercks et al., "Multiplexed, microfluidic molecular assay device and assay method" United States Patent Application 2007/0183934 (herein incorporated by reference).

Microspheres, such as Luminex beads, has been described as a platform to support the amplification of nucleic ands and production of proteins, in addition to the phototransfer from one substrate to another substrate. In particular, the microspheres can be spectrally encoded through incorporation of semiconductor nanocrystals (or SCNCs). A desired fluorescence characteristic maybe obtained by mixing SCNCs of different sizes and/or compositions in a fixed amount and ratio to create a solution having a specific fluorescence spectra. Therefore, a number of SCNC solutions can be prepared, each having a distinct distribution of semiconductor nanocrystal labeled microsphere size and composition, wherein each solution has a different fluorescence characteristic. Further, these solutions may be mixed in fixed proportions to arrive at a spectrum having predetermined ratios and intensities of emission from the distinct SCNCs suspended in that solution. Lim et al., "Methods for capturing nascent proteins" United States Patent Application 2010/0075374 (herein incorporated by reference).

Luminex bead systems have been described to improve the detection precision of a single analyte. A set of differently numbered microparticles (i.e., for example, belonging to different bead-sets or differential colors) are all coated with the same reagent so as to make them identical in sensitivity to the analyte being assayed. For example, an intra-assay titration curve may be constructed by coating the same fluorophore with different concentrations of labeled antibody, such that the same concentration of analyte is measured by detecting different signal magnitudes. Hanley B., " traplexing method for improving precision of suspended microarray assays" United States Patent 7,501,290 (herein incorporated by reference). The use of color coded beads has been described comprising nucleic acid capture moieties capable of 'tandem hybridization' with target nucleotides. Generally, a short capture probe is present on a color coded bead that binds a unique sequence of the target nucleic acid, while a longer labeled stacking probe has been preannealed to the target nucleic acid to facilitate subsequent detection. Each color coded bead therefore uniquely

distinguishes between specific target nucleotides based upon the capture moiety nucleic acid sequence. Beattie et al., "Nucleic acid analysis using sequence-targeted tandem

hybridization" United States Patent 6,268,147 (herein incorporated by reference).

A solution-based method for determining the expression level of a population of labeled target nucleic acids has been developed that is based upon capturing the labeled target nucleic acids with color coded beads. Each bead is conjugated to a specific capture probe that binds to an individual labeled target nucleic acid. Usually, the capture probes are nucleic acids capable of hybridization to the labeled target nucleic acids such that their respective expression level may be determined within a biological sample. The method describes specific populations of target-specific bead sets, wherein each target- specific bead set is individually detectable hybridizes to only one target nucleic acid. Specifically, the target- specific bead sets are described as having at least 5 individual bead sets that can bind with a corresponding set of target nucleic acids. As such, the bead population of a target-specific bead set can contain at least 100 individual beads that bind with a corresponding set of target nucleic acid. Golub et al., "Solution-based methods for RNA expression profiling" United States Patent Application 2007/0065844 (herein incorporated by reference).

B. Multimodal Signal Peak Analyte Identification

In one embodiment, the present invention contemplates a solution-based method for highly multiplexed determination of populations of analyte levels present in a biological sample. For example, the population of target analytes can be a collection of individual target nucleic acids of interest, such as a member of a gene expression signature or just a particular gene of interest. Alternatively, the population of target analytes can be a collection of individual target proteins and/or peptides. Each individual target analyte of interest is conjugated to a detectable solid substrate (i.e., for example, a differentially colored bead) in a quantitative or semi-quantitative manner, such that the level of each target analyte can be measured using a detectable signal generated by the detectable solid substrate. The detectable signal of the detectable solid substrate is sometimes referred to as the target molecule signal or simply as the target signal. The method also involves a population of target-specific bead sets, where each target-specific bead set is individually detectable and has a capture probe which corresponds to an individual analyte. The population of analytes is attached in solution with the population of detectable solid substrates to form a solid substrate-analyte complex. To determine the level of the population of target analytes present, one detects the solid substrate signal for each solid-substrate-analyte complex, such that the level of the solid substrate signal indicates the level of the target analyte, and the location of the solid substrate signal within a multi-modal signal distribution pattern indicates the identity of the analyte being detected.

Limitations of existing bead-based systems is that, due to relatively large microliter- scale volume of sample used per well, each analyte must be assayed with multiple beads of the same type to prevent signal saturation. Similar beads will compete with each other to bind to the same analyte. This situation decreases the sensitivity of the assay because the target analyte present in the sample is distributed over all of the beads specific for that analyte; and each bead will be reporting only a fraction of the analyte concentration. The mean value of the analyte concentration will, therefore, have a large standard error due to variable concentration values reported by each bead. The improvements of bead-based analyte detection described below make possible a highly accurate, and sensitive, high capacity analyte detection system wherein an analyte may be detected using a single bead.

In one embodiment, the present invention contemplates a method comprising combining a plurality of 500 bead-set collections in a single well, wherein each collection interrogates a different set of 500 genes. In one embodiment, the method further comprises detecting the plurality of 500 bead-set collections using the single well. In one embodiment, the method further comprises generating a multi-modal fluorescent intensity distribution for each of the 500 bead color variations. Although it is not necessary to understand the mechanism of an invention, it is believed that the number of beads that support each multi- modal peak may be determined by determining the local height and width. In one

embodiment, the method further comprises comparing the number of beads within a specific multi-modal peak to the mixing proportion of a bead for a specific gene. In one embodiment, the multi-modal peak bead number matches the bead mixing proportion such that the specific analyte is identified.

1. Improved Simultaneous Analyte Detection

As detailed above, the standard commercially available high capacity analyte detection systems are limited to simultaneously processing 500 analytes. While the ability of measuring up to 500 analytes may be sufficient for many applications, this limitation is restrictive for most practical genomics applications. For example, in assessing transcriptome- wide gene expression profiling a practical assay requires a simultaneous processing of much more than 500 genes.

One obvious approach to solve this problem would be to detect more than 500 analytes (i.e., for example, 1,000 genes) by using two wells per sample (i.e., for example, 500 genes per well x 2 wells). This technique would then assay a complete collection of 500 differentially dyed bead sets in both wells, where the bead sets in the first well are coupled to genes 1-500 and the bead sets in the second well are couples to genes 501-1000.

Consequently, equal aliquots of a biological sample are added to each well and detected separately. In order to determine the final result, the data from the two separate detections would have to be combined. See, Figure 2.

Several disadvantages are inherent in this approach including but not limited to: i) logistically cumbersome; ii) requires twice as much sample; iii) takes twice as much detection time; iv) loss of one well compromises both wells of data; or v) susceptible to batch artifacts which makes it difficult to re-constitute the whole sample.

In one embodiment, the present invention contemplates a method comprising interrogating multiple analytes, wherein said analytes are conjugated to individual, but identical, differentially colored beads. In one embodiment, a first analyte is conjugated to the individual, but identical, differentially colored bead that is selected from a first 500 bead-set. In one embodiment, a second analyte is conjugated to the individual, but identical, differentially colored variant that is selected from a second 500 bead-set.

The Luminex bead-level intensity data distributions suggested that expansion of the system's capacity might be possible by combining two collections of 500 bead-sets in a single well, wherein each 500 bead-set collection interrogates a different set of 500 genes. This approach would allow detection of a single sample in a single well. In some

embodiments, various analytical methods are applied to the resulting bead level intensity data to obtain the correct identity for all 1,000 analytes. See, Figure 3.

2. Improved Intensity Summarization

Usually, colored bead intensities belonging to a particular bead set are summarized as a single value, wherein a median fluorescent intensity (MFI) is reported as the data point. For example, when the measured analytes are genes, the MFI of a particular bead set color represents the expression value of a particular gene. A significant disadvantage to the median-based algorithm is the presence of inaccuracy if the number of outliers is

significantly large (e.g. if a number of beads have an intensity value close to zero), or where low bead counts could lead to misleading MFI values. For example, suggested Luminex data analysis methods ignore data wherein the bead count is less than thirty (30).

In addition to the MFI value, however, Luminex detectors also make available data for each individual bead (e.g., bead-level data). These data are stored in a standard flow cytometry data format (i.e., for example, an LXB file) and include information such as, set membership and/or a fluorescent intensity of each individual bead that is detected. Certain embodiments of the present invention have taken advantage of this alternative data by developing a kernel density based intensity summarization method as an alternative to the default MFI summarization method. In a kernel density method, a smoothed Gaussian density estimate is first fit to the data. A peak detection algorithm then detects local maxima. The most prominent peak (defined as the peak comprising the highest bead count) is reported as the summary intensity value. Unlike the standard MFI algorithm, the kernel algorithm may also ignore spurious outliers and/or identify analytes with low bead counts for further consideration. For example, the data presented herein show the differences between intensity distributions for two analytes between MFI values and kernel density based measurements. See, Figure 4.

3. Peak Detection Improvements

Detection and analysis of multimodal peaks have been discussed in relation to mass spectrometry analysis. Old et al., "Methods and systems for peak detection and quantitation" United States Patent Number 7,279,679 (herein incorporated by reference). However, some embodiments of the present invention provide significant improvements that provide superior detection of analytes.

In one embodiment, the present invention contemplates a method comprising detecting peaks from a multi-modal fluorescent intensity distribution using an algorithm. In one embodiment, the algorithm recovers an expression value of each gene interrogated with each bead color variation.

In one embodiment, the present invention contemplates a method for improving the accuracy of the peak detection algorithm. In one embodiment, the accuracy is improved by selecting paired genes. In one embodiment, the paired genes are frequently distant. Although it is not necessary to understand the mechanism of an invention, it is believed that a linear programming approach may be employed to maximize the pairwise distances across all genes. Peak detection usually involves the identification of sufficient statistics comparing different populations from a multimodally distributed signal pattern. For example, the statistical analysis may identify two different populations from a bi-modal distribution signal pattern. Generally, a first step in peak identification involves assigning each data point (i.e., for example, a bead-level data point) to its most salient population. Once these data points have been mapped to their respective population, suitable statistics may be computed (i.e., for example, a median or mean) to summarize the values localized to a population of interest.

a. Kernel Density Method (KDM)

A kernel density method comprises a non-parametric method that does not make assumptions of the underlying distribution of the data. In general, the steps of the KDM algorithm may be performed in the following manner: i) log transform the data; ii) obtain a smoothed Gaussian kernel density estimate. An optimal bandwidth for the kernel is chosen automatically; iii) detect local maxima by comparing each element of the smoothed estimate to its neighboring values. If an element is larger than both of its neighbors, it is a local peak; iv) assign every data point to the nearest peak. The support for a peak is the number of points that are assigned to it; and 5) rank order the peaks according to the support.

The data presented herein shows an example output of a KDM analysis. See, Figure 5. The central panel shows the dual tag signal for a single analyte. The histogram shows the distribution of intensities, wherein two component populations are clearly seen. The red curve indicates the smoothened kernel density estimate. Local maxima are indicated. The expression of the individual genes measured separately is shown alongside. The algorithm correctly detects the two component peaks. These data were further compared between dual tag and control single tag intensities of 1000 analytes for two samples. The high correlations of the scatter plot data indicate that the component signals can be recovered accurately. See, Figure 6.

b. Gaussian Mixture Model (GMM)

Another method, the Gaussian mixture models, assumes that the signal is a mixture of two Gaussian populations. In general, the steps of the GMM algorithm may be performed in the following manner: i) log transform the data; and ii) assuming a mixture of two Gaussians, estimate the mean μ, the variance σ, and the mixing proportion π. q>6(y) is the normal density evaluated at y given Θ = {μ, σ} as follows;

(a) Take initial guess at Qi = {μ_1; σι}, θ₂ = {μ₂, σ₂}, and π. (b) Compute the membership probability, 5j, of each data point y.

via

Update the parameter vectors θι and θ₂ with the following update equations

Return to step (b) until convergence.

It should be noted that a GMM parameter estimation can be sensitive to non-Gaussian components of the signal. Consequently, exploratory data analysis has resulted in a definition of a set of heuristics coupled with GMM estimation, which produce accurate peak calls. For example, the data presented herein shows an example output of the GMM for a single analyte measured using the dual tag approach. See, Figure 7. The histogram indicates the raw distribution of intensities. The red curves indicate the two Gaussian distributions estimated via

the GMM algorithm.

c. Gene Paring

In one embodiment, the present invention contemplates a peak detection algorithm further comprising a strategy to select paired genes for conjugation to individual, but identical differentially colored beads. In one embodiment, the paired genes are frequently distant. For example, a linear programming approach is used to maximize the pairwise distances across all genes. The optimization prolem can be stated as:

Maximize:

M M

^■I ί

Where d(I,j) is the pairwise distance between the ith and jth gene, x is a

symmetric binary matrix whose x(i,j) =1 is the ith and jth gene are paired. Subject to:

M

¥

f

The data presented herein shows gene pairs selected by an algorithm for one example dataset, wherein the gene pairing obtained by the algorithm is compared to random pairs. See, Figure 8 and Figure 9, respectively.

Further data was collected from genes derived from HEK293 cells. The data show that measuring a sample in which eight (8) gense are attached to each of 10 different beads (i.e., eight (8) genes per bead and ten (10) such beads, a total of eight genes were assayed simultaneously. Specifically, the data shows median and variance intensities from a sample of HEK293 cells. Figure 10A. Further data shows the bead level distribution of one of the eight levels. The data show that most beads from an analyte cluster towards a central value. Figure 10B, arrow. As a result of this technique is may be concluded that: i) as many as eighty (80) genes may be assessed in each sample by using ten (10) analytes; and b) increased robustness may be gained for any given level because the measurements are averaged over eight (8) genes. While the above data was collected using control genes, the use of signature genes would be expected to result in equivalent compact representation and robustness, wherein each level would represent one signature.

Data presented herein was collected from an experiment where the cells were cultured on a 384-well plate. Most samples generated data showing an ascending shape of the invariant genes. The flat profiles shown are from negative controls. Figure 1 IB. These data show that at least two tags per analyte (e.g., per bead) may be analyzed thereby improving the efficiency and robustness of the assay due to the redundancy.

In one embodiment, the present invention contemplates a peak detection algorithm further comprising a strategy under circumstances where it is difficult to achieve exact mixing proportions of beads, the actual bead counts are measured and then employed as priors within the peak assignment algorithm.

In one embodiment, the detected peak signal may be improved by conjugating every member of an analyte set to the same differentially colored bead. Although it is not necessary to understand the mechanism of an invention, it is believed that multiple analytes on the same bead color will increase the signal-to-noise ratio.

4. Peak Assignment Improvements

Once peaks within a multimodal distribution pattern have been detected, the peaks need unambiguous assignment to specific genes. In one embodiment, the present invention contemplates a method for unambiguous gene assignment comprising combining a plurality of bead-set collections, wherein each differentially colored bead is present in an unequal proportion between each bead-set collection. In one embodiment, a first differentially colored bead may be present in a proportion that is 1.25 times the standard volume selected from a first bead-set collection, while second differentially colored bead, that is identical to the first differentially colored bead, may be present in a proportion that is 0.75 times the standard volume selected from a second bead-set collection. Then, by examining the support for each peak (e.g. peak height, neighboring bead count or mixing proportion) and using the prior knowledge of the mixing proportion of a bead for a specific gene, an unambiguous assignment for each gene is made. See, Figure 5.

III. Detection Methodologies

A. Detection of Nucleic Acids

mRNA expression may be measured by any suitable method, including but not limited to, those disclosed below.

In some embodiments, RNA is detection by Northern blot analysis. Northern blot analysis involves the separation of RNA and hybridization of a complementary labeled probe.

In other embodiments, RNA expression is detected by enzymatic cleavage of specific structures (INVADER assay, Third Wave Technologies; See e.g., U.S. Pat. Nos. 5,846,717, 6,090,543; 6,001,567; 5,985,557; and 5,994,069; each of which is herein incorporated by reference). The INVADER assay detects specific nucleic acid (e.g., RNA) sequences by using structure-specific enzymes to cleave a complex formed by the hybridization of overlapping oligonucleotide probes.

In still further embodiments, RNA (or corresponding cDNA) is detected by hybridization to a oligonucleotide probe. A variety of hybridization assays using a variety of technologies for hybridization and detection are available. For example, in some

embodiments, TaqMan assay (PE Biosystems, Foster City, Calif; See e.g., U.S. Pat. Nos. 5,962,233 and 5,538,848, each of which is herein incorporated by reference) is utilized. The assay is performed during a PCR reaction. The TaqMan assay exploits the 5'-3' exonuclease activity of the AMPLITAQ GOLD DNA polymerase. A probe consisting of an oligonucleotide with a 5'-reporter dye (e.g., a fluorescent dye) and a 3'-quencher dye is included in the PCR reaction. During PCR, if the probe is bound to its target, the 5'-3' nucleolytic activity of the AMPLITAQ GOLD polymerase cleaves the probe between the reporter and the quencher dye. The separation of the reporter dye from the quencher dye results in an increase of fluorescence. The signal accumulates with each cycle of PCR and can be monitored with a fluorimeter.

In yet other embodiments, reverse-transcriptase PCR (RT-PCR) is used to detect the expression of RNA. In RT-PCR, RNA is enzymatically converted to complementary DNA or "cDNA" using a reverse transcriptase enzyme. The cDNA is then used as a template for a PCR reaction. PCR products can be detected by any suitable method, including but not limited to, gel electrophoresis and staining with a DNA specific stain or hybridization to a labeled probe, hi some embodiments, the quantitative reverse transcriptase PCR with standardized mixtures of competitive templates method described in U.S. Pat. Nos.

5,639,606, 5,643,765, and 5,876,978 (each of which is herein incorporated by reference) is utilized.

B. Sequencing Of Nucleic Acids

The method most commonly used as the basis for nucleic acid sequencing, or for identifying a target base, is the enzymatic chain-termination method of Sanger. Traditionally, such methods relied on gel electrophoresis to resolve, according to their size, wherein nucleic acid fragments are produced from a larger nucleic acid segment. However, in recent years various sequencing technologies have evolved which rely on a range of different detection strategies, such as mass spectrometry and array technologies.

One class of sequencing methods assuming importance in the art are those which rely upon the detection of PPi release as the detection strategy. It has been found that such methods lend themselves admirably to large scale genomic projects or clinical sequencing or screening, where relatively cost-effective units with high throughput are needed.

Methods of sequencing based on the concept of detecting inorganic pyrophosphate (PPi) which is released during a polymerase reaction have been described in the literature for example (WO 93/23564, WO 89/09283, W098/13523 and WO 98/28440). As each nucleotide is added to a growing nucleic acid strand during a polymerase reaction, a pyrophosphate molecule is released. It has been found that pyrophosphate released under these conditions can readily be detected, for example enzymically e.g. by the generation of light in the luciferase-luciferin reaction. Such methods enable a base to be identified in a target position and DNA to be sequenced simply and rapidly whilst avoiding the need for electrophoresis and the use of labels.

At its most basic, a PPi-based sequencing reaction involves simply carrying out a primer-directed polymerase extension reaction, and detecting whether or not that nucleotide has been incorporated by detecting whether or not PPi has been released. Conveniently, this detection of PPi-release may be achieved enzymatically, and most conveniently by means of a luciferase-based light detection reaction termed ELIDA (see further below).

It has been found that dATP added as a nucleotide for incorporation, interferes with the luciferase reaction used for PPi detection. Accordingly, a major improvement to the basic PPi-based sequencing method has been to use, in place of dATP, a dATP analogue

(specifically dATP_ai_phaS) which is incapable of acting as a substrate for luciferase, but which is nonetheless capable of being incorporated into a nucleotide chain by a polymerase enzyme (W098/13523).

Further improvements to the basic PPi-based sequencing technique include the use of a nucleotide degrading enzyme such as apyrase during the polymerase step, so that unincorporated nucleotides are degraded, as described in WO 98/28440, and the use of a single-stranded nucleic acid binding protein in the reaction mixture after annealing of the primers to the template, which has been found to have a beneficial effect in reducing the number of false signals, as described in WO00/43540.

C. Detection of Protein

In other embodiments, gene expression may be detected by measuring the expression of a protein or polypeptide. Protein expression may be detected by any suitable method. In some embodiments, proteins are detected by immunohistochemistry. In other embodiments, proteins are detected by their binding to an antibody raised against the protein. The generation of antibodies is described below.

Antibody binding may be detected by many different techniques including, but not limited to, (e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (e.g., using colloidal gold, enzyme or radioisotope labels, for example), Western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays, etc.), complement fixation assays, immunofluorescence assays, protein A assays, and Immunoelectrophoresis assays, etc.

In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled.

In some embodiments, an automated detection assay is utilized. Methods for the automation of immunoassays include those described in U.S. Pat. Nos. 5,885,530, 4,981,785, 6,159,750, and 5,358,691, each of which is herein incorporated by reference. In some embodiments, the analysis and presentation of results is also automated. For example, in some embodiments, software that generates a prognosis based on the presence or absence of a series of proteins corresponding to cancer markers is utilized.

In other embodiments, the immunoassay described in U.S. Pat. Nos. 5,599,677 and 5,672,480; each of which is herein incorporated by reference.

D. Remote Detection Systems

In some embodiments, a computer-based analysis program is used to translate the raw data generated by the detection assay (e.g., the presence, absence, or amount of a given marker or markers) into data of predictive value for a clinician. The clinician can access the predictive data using any suitable means. Thus, in some preferred embodiments, the present invention provides the further benefit that the clinician, who is not likely to be trained in genetics or molecular biology, need not understand the raw data. The data is presented directly to the clinician in its most useful form. The clinician is then able to immediately utilize the information in order to optimize the care of the subject.

The present invention contemplates any method capable of receiving, processing, and transmitting the information to and from laboratories conducting the assays, wherein the information is provided to medical personal and/or subjects. For example, in some embodiments of the present invention, a sample (e.g., a biopsy or a serum or urine sample) is obtained from a subject and submitted to a profiling service (e.g., clinical lab at a medical facility, genomic profiling business, etc.), located in any part of the world (e.g., in a country different than the country where the subject resides or where the information is ultimately used) to generate raw data. Where the sample comprises a tissue or other biological sample, the subject may visit a medical center to have the sample obtained and sent to the profiling center, or subjects may collect the sample themselves (e.g., a urine sample) and directly send it to a profiling center. Where the sample comprises previously determined biological information, the information may be directly sent to the profiling service by the subject (e.g., an information card containing the information may be scanned by a computer and the data transmitted to a computer of the profiling center using an electronic communication systems). Once received by the profiling service, the sample is processed and a profile is produced (i.e., expression data), specific for the diagnostic or prognostic information desired for the subject.

The profile data is then prepared in a format suitable for interpretation by a treating clinician. For example, rather than providing raw expression data, the prepared format may represent a diagnosis or risk assessment for the subject, along with recommendations for particular treatment options. The data maybe displayed to the clinician by any suitable method. For example, in some embodiments, the profiling service generates a report that can be printed for the clinician (e.g., at the point of care) or displayed to the clinician on a computer monitor.

In some embodiments, the information is first analyzed at the point of care or at a regional facility. The raw data is then sent to a central processing facility for further analysis and/or to convert the raw data to information useful for a clinician or patient. The central processing facility provides the advantage of privacy (all data is stored in a central facility with uniform security protocols), speed, and uniformity of data analysis. The central processing facility can then control the fate of the data following treatment of the subject. For example, using an electronic communication system, the central facility can provide data to the clinician, the subject, or researchers.

In some embodiments, the subject is able to directly access the data using the electronic communication system. The subject may chose further intervention or counseling based on the results. In some embodiments, the data is used for research use. For example, the data may be used to further optimize the inclusion or elimination of markers as useful indicators of a particular condition or stage of disease.

E. Detection Kits

In other embodiments, the present invention provides kits for the detection and characterization of proteins and/or nucleic acids. In some embodiments, the kits contain antibodies specific for a protein expressed from a gene of interest, in addition to detection reagents and buffers. In other embodiments, the kits contain reagents specific for the detection of mRNA or cDNA (e.g., oligonucleotide probes or primers). In preferred embodiments, the kits contain all of the components necessary to perform a detection assay, including all controls, directions for performing assays, and any necessary software for analysis and presentation of results. IV. Purification Processes

Samples (i.e., for example, biological samples) can be optionally concentrated using a commercially available concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a suitable purification matrix as previously described. For example, a suitable affinity matrix can comprise a ligand or antibody molecule bound to a suitable support. Alternatively, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification. Alternatively, a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred.

Finally, one or more reversed-phase high performance liquid chromatography (RP- HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify an IL-IR composition. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially pure recombinant protein.

Protein may be isolated by initial extraction from cell pellets, followed by one or more concentration, salting-out, hydrophobic interaction chromatography (H1C), aqueous ion exchange or size exclusion chromatography steps. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps. Most biological cells can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.

V. Antibodies

The present invention provides isolated antibodies (i.e., for example, polyclonal or monoclonal). In one embodiment, the present invention provides antibodies that specifically bind to a subset of a solid particle population. These antibodies find use in the detection methods described above.

An antibody against a protein of the present invention may be any monoclonal or polyclonal antibody, as long as it can recognize the protein. Antibodies can be produced by using a protein of the present invention as the antigen according to a conventional antibody or antiserum preparation process.

The present invention contemplates the use of both monoclonal and polyclonal antibodies. Any suitable method may be used to generate the antibodies used in the methods and compositions of the present invention, including but not limited to, those disclosed herein. For example, for preparation of a monoclonal antibody, protein, as such, or together with a suitable carrier or diluent is administered to an animal (e.g., a mammal) under conditions that permit the production of antibodies. For enhancing the antibody production capability, complete or incomplete Freund's adjuvant may be administered. Normally, the protein is administered once every 2 weeks to 6 weeks, in total, about 2 times to about 10 times. Animals suitable for use in such methods include, but are not limited to, primates, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, etc.

For preparing monoclonal antibody-producing cells, an individual animal whose antibody titer has been confirmed (e.g., a mouse) is selected, and 2 days to 5 days after the final immunization, its spleen or lymph node is harvested and antibody-producing cells contained therein are fused with myeloma cells to prepare the desired monoclonal antibody producer hybridoma. Measurement of the antibody titer in antiserum can be carried out, for example, by reacting the labeled protein, as described hereinafter and antiserum and then measuring the activity of the labeling agent bound to the antibody. The cell fusion can be carried out according to known methods, for example, the method described by Koehler and Milstein (Nature 256:495 [1975]). As a fusion promoter, for example, polyethylene glycol (PEG) or Sendai virus (HVJ), preferably PEG is used.

Examples of myeloma cells include NS-1, P3U1, SP2/0, AP-1 and the like. The proportion of the number of antibody producer cells (spleen cells) and the number of myeloma cells to be used is preferably about 1 : 1 to about 20: 1. PEG (preferably PEG 1000- PEG 6000) is preferably added in concentration of about 10% to about 80%. Cell fusion can be carried out efficiently by incubating a mixture of both cells at about 20°C to about 40°C, preferably about 30°C to about 37°C for about 1 minute to 10 minutes.

Various methods may be used for screening for a hybridoma producing the antibody

(e.g., against a tumor antigen or autoantibody of the present invention). For example, where a supernatant of the hybridoma is added to a solid phase (e.g., microplate) to which antibody is adsorbed directly or together with a carrier and then an anti-immunoglobulin antibody (if mouse cells are used in cell fusion, anti-mouse immunoglobulin antibody is used) or Protein A labeled with a radioactive substance or an enzyme is added to detect the monoclonal antibody against the protein bound to the solid phase. Alternately, a supernatant of the hybridoma is added to a solid phase to which an anti-immunoglobulin antibody or Protein A is adsorbed and then the protein labeled with a radioactive substance or an enzyme is added to detect the monoclonal antibody against the protein bound to the solid phase. Selection of the monoclonal antibody can be carried out according to any known method or its modification. Normally, a medium for animal cells to which HAT

(hypoxanthine, aminopterin, thymidine) are added is employed. Any selection and growth medium can be employed as long as the hybridoma can grow. For example, RPMI 1640 medium containing 1% to 20%, preferably 10% to 20% fetal bovine serum, GIT medium containing 1% to 10% fetal bovine serum, a serum free medium for cultivation of a hybridoma (SFM-101, Nissui Seiyaku) and the like can be used. Normally, the cultivation is carried out at 20°C to 40°C, preferably 37°C for about 5 days to 3 weeks, preferably 1 week to 2 weeks under about 5% C0₂ gas. The antibody titer of the supernatant of a hybridoma culture can be measured according to the same manner as described above with respect to the antibody titer of the anti-protein in the antiserum.

Separation and purification of a monoclonal antibody can be carried out according to the same manner as those of conventional polyclonal antibodies such as separation and purification of immunoglobulins, for example, salting-out, alcoholic precipitation, isoelectric point precipitation, electrophoresis, adsorption and desorption with ion exchangers (e.g., DEAE), ultracentrifugation, gel filtration, or a specific purification method wherein only an antibody is collected with an active adsorbent such as an antigen-binding solid phase, Protein A or Protein G and dissociating the binding to obtain the antibody.

Polyclonal antibodies may be prepared by any known method or modifications of these methods including obtaining antibodies from patients. For example, a complex of an immunogen (an antigen against the protein) and a carrier protein is prepared and an animal is immunized by the complex according to the same manner as that described with respect to the above monoclonal antibody preparation. A material containing the antibody against is recovered from the immunized animal and the antibody is separated and purified.

As to the complex of the immunogen and the carrier protein to be used for immunization of an animal, any carrier protein and any mixing proportion of the carrier and a hapten can be employed as long as an antibody against the hapten, which is crosslinked on the carrier and used for immunization, is produced efficiently. For example, bovine serum albumin, bovine cycloglobulin, keyhole limpet hemocyanin, etc. may be coupled to an hapten in a weight ratio of about 0.1 part to about 20 parts, preferably, about 1 part to about 5 parts per 1 part of the hapten.

In addition, various condensing agents can be used for coupling of a hapten and a carrier. For example, glutaraldehyde, carbodiimide, maleimide activated ester, activated ester reagents containing thiol group or dithiopyridyl group, and the like find use with the present invention. The condensation product as such or together with a suitable carrier or diluent is administered to a site of an animal that permits the antibody production. For enhancing the antibody production capability, complete or incomplete Freund's adjuvant may be administered. Normally, the protein is administered once every 2 weeks to 6 weeks, in total, about 3 times to about 10 times.

The polyclonal antibody is recovered from blood, ascites and the like, of an animal immunized by the above method. The antibody titer in the antiserum can be measured according to the same manner as that described above with respect to the supernatant of the hybridoma culture. Separation and purification of the antibody can be carried out according to the same separation and purification method of immunoglobulin as that described with respect to the above monoclonal antibody.

The protein used herein as the immunogen is not limited to any particular type of immunogen. For example, a protein expressed resulting from a virus infection (further including a gene having a nucleotide sequence partly altered) can be used as the immunogen. Further, fragments of the protein may be used. Fragments may be obtained by any methods including, but not limited to expressing a fragment of the gene, enzymatic processing of the protein, chemical synthesis, and the like.

Claims

A method, comprising:

a) providing;

i) a sample comprising a plurality of analytes;

ii) a plurality of solid substrate populations, wherein each of said solid substrate populations comprise a plurality of subsets, and wherein each subset is present in an unequal proportion from every other subset in the same solid substrate population; iii) a plurality of capture probes capable of attaching to said

plurality of analytes, wherein each subset comprises a different capture probe; and

vi) a means for detecting said plurality of subsets that is capable of creating a multimodal intensity distribution pattern;

b) detecting said plurality of subsets with said means, wherein a

multimodal intensity distribution pattern is created;

c) identifying said plurality of analytes from said multimodal distribution pattern.

The method of Claim 1, wherein said sample may be selected from the group comprising a biological sample, a soil sample, or a water sample.

The method of Claim 1 , wherein said plurality of analytes may be selected from the group comprising nucleic acids, proteins, peptides, biological receptors, enzymes, antibodies, polyclonal antibodies, monoclonal antibodies, or Fab fragments.

The method of Claim 1, wherein said solid substrate population comprises a bead-set population.

The method of Claim 1, wherein said unequal proportions comprise two subsets in an approximate ratio of 1.25:0.75.

6. The method of Claim 1, wherein said unequal proportions comprise three subsets in an approximate ratio of 1.25:1.00:0.75.

7. The method of Claim 1, wherein said unequal proportions comprise four subsets in an approximate ratio of 1.25:1.00:0.75:0.50.

8. The method of Claim 1, wherein said unequal proportions comprise five subsets in an approximate ratio of 1.50:1.25: 1.00:0.75:0.50.

9. The method of Claim 1 , wherein said unequal proportions comprise six subsets in an approximate ratio of 1.75:1 :50:1.25:1.00:0.75:0.50.

10. The method of Claim 1, wherein said unequal proportions comprise seven subsets in an approximate ratio of 2.00: 1.75: 1 :50: 1.25: 1.00:0.75:0.50.

11. The method of Claim 1 , wherein said unequal proportions comprise eight subsets in an approximate ratio of 2.00: 1.75 : 1 :50: 1.25: 1.00:0.75 :0.50:0.25.

12. The method of Claim 1 , wherein said unequal proportions comprise nine subsets in an approximate ratio of 2.25:2.00:1.75:1 :50:1.25:1.00:0.75:0.50:0.25.

13. The method of Claim 1, wherein said unequal proportions comprise ten subsets in an approximate ratio of 2.50:2.25:2.00:1.75:1 :50:1.25:1.00:0.75:0.50:0.25.

14. A method, comprising:

a) providing;

i) a solid substrate population comprising a first subset and a second subset, wherein the first subset is present in a first proportion and the second subset is present in a second proportion;

ii) a first analyte attached to said first subset;

iii) a second analyte attached to said second subset; and vi) a means for detecting said first subset and second subset that is capable of creating a multimodal intensity distribution pattern; b) detecting said first subset and said second subset with said means, wherein a multimodal intensity distribution pattern is created; and c) identifying said first analyte and said second analyte from said

multimodal distribution pattern.

The method of Claim 14, wherein said solid substrate population comprises a label.

The method of Claim 15, wherein said label comprises a mixture of at least two different fluorophores.

The method of Claim 14, wherein said first proportion is different from said second proportion.

18. The method of Claim 14, wherein said first analyte is attached to said first subset with a first capture probe.

19. The method of Claim 14, wherein said second analyte is attached to said second subset with a second capture probe. 20. The method of Claim 14, wherein said multimodal intensity distribution pattern

comprises a first peak corresponding to said first subset.

21. The method of Claim 14, wherein said multimodal intensity distribution pattern

comprises a second peak corresponding to said second subset.

22. A method, comprising:

a) providing;

i) a solid substrate population comprising a plurality of subsets; ii) a sample comprising a plurality of analytes, wherein at least one portion of said plurality of analytes comprise related analytes; and

iii) a means for detecting said subsets that is capable of creating a multimodal intensity distribution pattern; b) attaching each of said related analyte portions to one of said plurality of subsets;

c) detecting said plurality of subsets with said means, wherein a

multimodal intensity distribution pattern is created; and

d) identifying said related analytes from said multimodal distribution pattern.

The method of Claim 22, wherein said related analytes comprise linked genes.

A method, comprising:

a) providing;

i) a solid substrate population comprising a plurality of subsets; ii) a sample comprising a plurality of analytes, wherein at least one portion of the plurality of analytes comprise rare event analytes; and

iii) a means for detecting said subsets that is capable of creating a multimodal intensity distribution pattern;

b) attaching a portion of said plurality of analytes comprising one or more of the rare event analytes to one of the plurality of subsets;

c) detecting said plurality of subsets with said means, wherein a

multimodal intensity distribution pattern is created; and

d) determining if said rare event analytes occur in said multimodal

distribution pattern. 25. The method of Claim 24, wherein said rare event analyte portion is present in

approximately less than 0.01% of said sample.

The method of Claim 24, wherein said rare event analyte comprises a small molecule or drug.

The method of Claim 24, wherein said rare event analyte comprises a nucleic acid mutation.

The method of Claim 24, wherein said rare event analyte comprises a diseased cell.

29. The method of Claim 24, wherein said rare event analyte comprises an autoimmune antibody.

30. The method of Claim 24, wherein said rare event analyte comprises a microbe.

31. A method, comprising:

a) providing;

i) a solid substrate population comprising a plurality of subsets; ii) a sample comprising a first labeled analyte and a second

labeled analyte; and

b) attaching said first and second labeled analytes in an unequal

proportion to one of said plurality of subsets;

c) detecting said plurality of subsets with said means, wherein a

multimodal intensity distribution pattern is created; and

d) identifying said first and second labeled analytes from said multimodal distribution pattern.

32. The method of Claim 31, wherein said first labeled analyte comprises a normal cell.

33. The method of Claim 31 , wherein said second labeled analyte comprises a tumor cell.

34. The method of Claim 31 , wherein said multimodal intensity distribution pattern

comprises a first peak corresponding to said first labeled analyte.

35. The method of Claim 31 , wherein said multimodal intensity distribution pattern

comprises a second peak corresponding to said second labeled analyte.

36. The method of Claim 31, wherein said unequal proportion is equivalent to a ratio of said first and second peaks.