WO2011127042A1 - Détection de substance à analyser de grande capacité - Google Patents

Détection de substance à analyser de grande capacité Download PDF

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Publication number
WO2011127042A1
WO2011127042A1 PCT/US2011/031232 US2011031232W WO2011127042A1 WO 2011127042 A1 WO2011127042 A1 WO 2011127042A1 US 2011031232 W US2011031232 W US 2011031232W WO 2011127042 A1 WO2011127042 A1 WO 2011127042A1
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Prior art keywords
analytes
subsets
analyte
distribution pattern
subset
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PCT/US2011/031232
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English (en)
Inventor
Aravind Subramanian
David D. Peck
Todd R. Golub
Rajiv Narayan
Willis Read-Button
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The Broad Institute
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Publication of WO2011127042A1 publication Critical patent/WO2011127042A1/fr
Priority to US13/646,294 priority Critical patent/US10619195B2/en
Priority to US16/794,036 priority patent/US20200347444A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures

Definitions

  • 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).
  • analytes i.e., for example, transcriptome-wide mRNA species
  • 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.
  • nucleic acid analytes both microarray and tag-sequencing techniques are associated with a number of significant problems.
  • 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.
  • 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.
  • 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).
  • 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.
  • the sample may be selected from the group comprising a biological sample, a soil sample, or a water sample.
  • 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.
  • the solid substrate population comprises a bead-set population.
  • 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.
  • 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.
  • 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
  • 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.
  • the solid substrate population comprises a label.
  • the label comprises a mixture of at least two different fluorophores.
  • the first proportion is different from the second proportion.
  • the first analyte is attached to the first subset with a first capture probe.
  • the second analyte is attached to the second subset with a second capture probe.
  • 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.
  • 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.
  • the related analytes comprise linked genes.
  • 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.
  • the rare event analyte portion is present in approximately less than 0.01% of said sample.
  • the rare event analyte comprises a small molecule or drug.
  • the rare event analyte comprises a nucleic acid mutation.
  • the rare event analyte comprises a diseased cell.
  • the rare event analyte comprises an autoimmune antibody.
  • the rare event analyte comprises a microbe.
  • 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.
  • the first labeled analyte comprises a normal cell.
  • the second labeled analyte comprises a tumor cell.
  • the multimodal intensity distribution pattern comprises a first peak corresponding to the first labeled analyte.
  • the multimodal intensity distribution pattern comprises a second peak corresponding to the second labeled analyte.
  • the unequal proportion is equivalent to a ratio of the first and second peaks.
  • analyte 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.
  • genes proteins, ligands (i.e., for example, peptides and/or small organic molecules, mRNA and/or microRNA.
  • ligands i.e., for example, peptides and/or small organic molecules, mRNA and/or microRNA.
  • the data provided herein utilizes genes as an exemplary analyte though the disclosed embodiments are equally applicable to any other type of analyte.
  • an analyte is capable of binding to a capture probe.
  • related analyte 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.
  • a particular disease i.e., for example, cancer
  • rare event analyte 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)
  • solid substrate population refers to any collection of particles that are capable of differential labeling such that subsets of solid substrates within the population may be identified.
  • the differential labeling may comprise various mixtures of fluorophores within the particles.
  • 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.
  • 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.
  • 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).
  • 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).
  • bead-set 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).
  • 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.
  • a capture probe may be an oligonucleotide, wherein the oligonucleotide is at least partially complementary to an analyte oligonucleotide.
  • 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.
  • unequal proportion refers to any mixture of multiple components, wherein the multiple components are not present in a 1 :1 ratio.
  • two components may be present in an approximate ratio of 1.25:0.75.
  • three components may be present in an approximate ratio of 1.25: 1.00:0.75.
  • four components maybe present in an approximate ratio of 1.25:1.00:0.75:0.5.
  • five components maybe present in an approximate ratio of 1.50:1.25:1.00:0.75:0.50.
  • six components may be present in an approximate ratio of
  • nine components may be present in an approximate ratio of
  • counts refers to the number of measured coupled beads that are attached or bound to at least one analyte.
  • 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.
  • luminescence refers to any spontaneous emission of photons in the range from ultraviolet to infrared, after optical or other than optical excitation, such
  • chemiluminescence bioluminescence, electroluminescence, and especially fluorescence and phosphorescence are included commonly under the term "luminescence”.
  • high throughput 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.
  • a means for detecting 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.
  • the means may be a flow cytometer that detects the solid particles using laser scanning.
  • disease 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.
  • affinity refers to any attractive force between substances or particles that causes them to enter into and remain in chemical combination.
  • 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.
  • derived from refers to the source of a compound or sequence.
  • a compound or sequence may be derived from an organism or particular species.
  • a compound or sequence may be derived from a larger complex or sequence.
  • protein 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.
  • peptide 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.
  • a peptide comprises amino acids having an order of magnitude with the tens.
  • purified 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.
  • substantially purified 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).
  • purified to homogeneity is used to include compositions that have been purified to
  • a purified composition is not intended to mean that some trace impurities may remain.
  • 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.
  • an isolated nucleic acid 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).
  • amino acid sequence and “polypeptide sequence” as used herein, are interchangeable and to refer to a sequence of amino acids.
  • 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.
  • 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.
  • 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.
  • 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.
  • telomere 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.
  • a particular structure i.e., for example, an antigenic determinant or epitope
  • an antibody is recognizing and binding to a specific protein structure rather than to proteins in general.
  • 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.
  • small organic molecule refers to any molecule of a size comparable to those organic molecules generally used in pharmaceuticals.
  • 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.
  • 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.
  • siRNA refers to either small interfering RNA, short interfering RNA, or silencing RNA.
  • 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.
  • RNAi RNA interference
  • 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.
  • 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.
  • RISC RNA-induced silencing complex
  • miRNA 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.
  • mRNA messenger RNA
  • 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).
  • fluid e.g., blood, plasma and serum
  • solid e.g., stool
  • tissue e.g., tissue
  • liquid foods e.g., milk
  • solid foods e.g., vegetables
  • a pulmonary sample may be collected by bronchoalveolar lavage (BAL) which comprises fluid and cells derived from lung tissues.
  • BAL bronchoalveolar lavage
  • 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.
  • 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.
  • 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.
  • 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.
  • a partial degree of complementarity e.g., less than about 30% identity
  • homologous refers 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.
  • 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 ⁇ g/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.
  • 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.
  • high stringency e.g., increasing the temperature of the hybridization and/or wash steps, the use of formamide in the hybridization solution, etc.
  • 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 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.
  • 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)).
  • 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.
  • Tm 81.5 + 0.41 (% G+C)
  • 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.
  • conditions of "weak” or “low” stringency 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).
  • 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.”
  • sample template refers to nucleic acid originating from a sample which is analyzed for the presence of a target sequence of interest.
  • target sequence of interest refers to nucleic acid originating from a sample which is analyzed for the presence of a target sequence of interest.
  • 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.
  • PCR polymerase chain reaction
  • PCR polymerase chain reaction
  • 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
  • any oligonucleotide sequence can be amplified with the appropriate set of primer molecules.
  • the amplified segments created by the PCR process itself are, themselves, efficient templates for subsequent PCR amplifications.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • a nucleic acid sequence even if internal to a larger oligonucleotide, also may be said to have 5' and 3' ends.
  • 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.
  • 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.
  • 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.
  • 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.
  • regulatory element refers to a genetic element which controls some aspect of the expression of nucleic acid sequences.
  • 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.
  • operable combination 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.
  • 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.
  • Splicing signals mediate the removal of introns from the primary RNA transcript and consist of a splice donor and acceptor site.
  • 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.
  • 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.
  • 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.
  • nucleic acid molecule encoding refers 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.
  • 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:
  • RNA species complementary to the probe used are a standard tool of molecular biologists. J. Sambrook, J. et al. (1989) supra, pp 7.39-7.52.
  • reverse Northern blot 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.
  • 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.
  • 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).
  • structural gene refers to a DNA sequence coding for RNA or a protein.
  • regulatory genes are structural genes which encode products which control the expression of other genes (e.g., transcription factors).
  • 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.
  • mRNA messenger RNA
  • 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.
  • label or “detectable label” are used herein, to refer to any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • 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.
  • fluorescent dyes e.g., fluorescein, texas red, rhodamine, green fluorescent protein, and the like
  • 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.
  • small organic molecule refers to any molecule of a size comparable to those organic molecules generally used in pharmaceuticals.
  • 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.
  • 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
  • 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.
  • LMA ligand mediated amplification
  • 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).
  • GMM Gaussian mixture model
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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,
  • 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.
  • microbeads can be labeled with different spectral properties and/or fluorescent (or colorimetric) intensities.
  • 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.
  • 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.
  • the bead sets may also contain a capture probe which can bind to an individual target analyte.
  • 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.
  • 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.
  • MFI median fluorescent intensity
  • 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.
  • 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.
  • MFI median fluorescent intensity
  • 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.
  • the detector may be a charged coupled device.
  • at least two fluorescence measurements are recorded from a maximum of 500 differentially colored bead sets.
  • the fluorescent counts can be used to uniquely identify individual analytes.
  • 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.
  • 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;.
  • 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.
  • MSL multilayer soft lithography
  • 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
  • the microspheres can be spectrally encoded through incorporation of semiconductor nanocrystals (or SCNCs).
  • SCNCs semiconductor nanocrystals
  • 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.
  • 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
  • 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).
  • color coded beads comprising nucleic acid capture moieties capable of 'tandem hybridization' with target nucleotides.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • the present invention contemplates a solution-based method for highly multiplexed determination of populations of analyte levels present in a biological sample.
  • 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.
  • 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.
  • 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.
  • the method further comprises detecting the plurality of 500 bead-set collections using the single well.
  • the method further comprises generating a multi-modal fluorescent intensity distribution for each of the 500 bead color variations.
  • 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.
  • the multi-modal peak bead number matches the bead mixing proportion such that the specific analyte is identified.
  • 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.
  • the present invention contemplates a method comprising interrogating multiple analytes, wherein said analytes are conjugated to individual, but identical, differentially colored beads.
  • a first analyte is conjugated to the individual, but identical, differentially colored bead that is selected from a first 500 bead-set.
  • a second analyte is conjugated to the individual, but identical, differentially colored variant that is selected from a second 500 bead-set.
  • 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.
  • 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.
  • MFI median fluorescent intensity
  • 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
  • Luminex data analysis methods ignore data wherein the bead count is less than thirty (30).
  • Luminex detectors 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.
  • 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.
  • the kernel algorithm may also ignore spurious outliers and/or identify analytes with low bead counts for further consideration.
  • the data presented herein show the differences between intensity distributions for two analytes between MFI values and kernel density based measurements. See, Figure 4.
  • the present invention contemplates a method comprising detecting peaks from a multi-modal fluorescent intensity distribution using an algorithm.
  • the algorithm recovers an expression value of each gene interrogated with each bead color variation.
  • the present invention contemplates a method for improving the accuracy of the peak detection algorithm.
  • the accuracy is improved by selecting paired genes.
  • the paired genes are frequently distant.
  • 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.
  • 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.
  • KDM Kernel Density Method
  • a kernel density method comprises a non-parametric method that does not make assumptions of the underlying distribution of the data.
  • 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.
  • GMM Gaussian Mixture Model
  • the Gaussian mixture models assumes that the signal is a mixture of two Gaussian populations.
  • 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 ⁇ , the variance ⁇ , and the mixing proportion ⁇ .
  • 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.
  • 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 present invention contemplates a peak detection algorithm further comprising a strategy to select paired genes for conjugation to individual, but identical differentially colored beads.
  • 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:
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • second differentially colored bead that is identical to the first differentially colored bead
  • an unambiguous assignment for each gene is made. See, Figure 5.
  • mRNA expression may be measured by any suitable method, including but not limited to, those disclosed below.
  • RNA is detection by Northern blot analysis.
  • Northern blot analysis involves the separation of RNA and hybridization of a complementary labeled probe.
  • 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.
  • 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
  • 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
  • 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.
  • 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.
  • 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.
  • various sequencing technologies have evolved which rely on a range of different detection strategies, such as mass spectrometry and array technologies.
  • 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).
  • ELIDA luciferase-based light detection reaction
  • 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
  • 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.
  • gene expression may be detected by measuring the expression of a protein or polypeptide.
  • Protein expression may be detected by any suitable method.
  • proteins are detected by immunohistochemistry.
  • 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.
  • radioimmunoassay e.g., ELISA (enzyme-linked immunosorbant assay), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immuno
  • antibody binding is detected by detecting a label on the primary antibody.
  • the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
  • the secondary antibody is labeled.
  • 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.
  • the analysis and presentation of results is also automated.
  • software that generates a prognosis based on the presence or absence of a series of proteins corresponding to cancer markers is utilized.
  • 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.
  • 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.
  • a sample e.g., a biopsy or a serum or urine sample
  • a profiling service e.g., clinical lab at a medical facility, genomic profiling business, etc.
  • 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
  • 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.
  • 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).
  • the profiling service 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the data is used for research use.
  • 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.
  • kits for the detection and characterization of proteins and/or nucleic acids contain antibodies specific for a protein expressed from a gene of interest, in addition to detection reagents and buffers.
  • the kits contain reagents specific for the detection of mRNA or cDNA (e.g., oligonucleotide probes or primers).
  • 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 can be optionally concentrated using a commercially available concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.
  • the concentrate can be applied to a suitable purification matrix as previously described.
  • a suitable affinity matrix can comprise a ligand or antibody molecule bound to a suitable support.
  • 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.
  • a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred.
  • RP- HPLC reversed-phase high performance liquid chromatography
  • hydrophobic RP-HPLC media e.g., silica gel having pendant methyl or other aliphatic groups
  • 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.
  • H1C hydrophobic interaction chromatography
  • HPLC high performance liquid chromatography
  • 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.
  • 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.
  • complete or incomplete Freund's adjuvant may be administered.
  • 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.
  • an individual animal whose antibody titer has been confirmed e.g., a mouse
  • 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]).
  • a fusion promoter for example, polyethylene glycol (PEG) or Sendai virus (HVJ), preferably PEG is used.
  • myeloma cells examples 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
  • 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.
  • 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.
  • a solid phase e.g., microplate
  • an anti-immunoglobulin antibody if mouse cells are used in cell fusion, anti-mouse immunoglobulin antibody is used
  • 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.
  • 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
  • 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.
  • 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% C0 2 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.
  • 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.
  • an immunogen an antigen against the protein
  • 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.
  • 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.
  • 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.
  • various condensing agents can be used for coupling of a hapten and a carrier.
  • 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.
  • 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.
  • a protein expressed resulting from a virus infection can be used as the immunogen.
  • 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.

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Abstract

La présente invention améliore les compositions et les procédés connus pour une détection à haut rendement de substances à analyser. Les substances à analyser peuvent être issus d'échantillons, comprenant, mais sans s'y limiter, des échantillons biologiques, de telle sorte que la détection d'acides nucléiques, de protéines, de peptides et/ou de petites molécules organiques, est facilitée. Différentes substances à analyser peuvent être fixées à des particules solides ayant des caractéristiques spectrales identiques, les substances à analyser étant identifiés par un nombre différentiel de particules (c'est-à-dire, par exemple, des sous-ensembles de particules identiques sont testés dans des proportions inégales). La détection et/ou l'identification simultanées des substances à analyser contenues dans un échantillon est facilitée par une analyse algorithmique de motifs de distribution multimodaux.
PCT/US2011/031232 2010-04-06 2011-04-05 Détection de substance à analyser de grande capacité WO2011127042A1 (fr)

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US9486770B2 (en) 2012-07-20 2016-11-08 National University Of Singapore Combinatoric encoding methods for microarrays
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Publication number Priority date Publication date Assignee Title
US9486770B2 (en) 2012-07-20 2016-11-08 National University Of Singapore Combinatoric encoding methods for microarrays
US10203324B2 (en) 2012-07-20 2019-02-12 National University Of Singapore Combinatoric encoding methods for microarrays
US11567926B2 (en) * 2020-03-17 2023-01-31 Noodle Analytics, Inc. Spurious outlier detection system and method

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