WO2002038812A2 - Nouveau procede en matiere d'analyse quantitative de l'expression de genes et de proteines - Google Patents

Nouveau procede en matiere d'analyse quantitative de l'expression de genes et de proteines Download PDF

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Publication number
WO2002038812A2
WO2002038812A2 PCT/US2001/047277 US0147277W WO0238812A2 WO 2002038812 A2 WO2002038812 A2 WO 2002038812A2 US 0147277 W US0147277 W US 0147277W WO 0238812 A2 WO0238812 A2 WO 0238812A2
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molecules
immobilized
target
labeled
molecule
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PCT/US2001/047277
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WO2002038812A3 (fr
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Richard V. Denton
James O. Bowlby, Jr.
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Denton Richard V
Bowlby James O Jr
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Priority to AU2002235170A priority Critical patent/AU2002235170A1/en
Publication of WO2002038812A2 publication Critical patent/WO2002038812A2/fr
Publication of WO2002038812A3 publication Critical patent/WO2002038812A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

  • the present invention relates to improved methods, compositions and arrays for detection and quantitation of biological or chemical molecules, whether natural or synthetic, including but not limited to areas such as diagnostics of diseases, infection, or other medical conditions, detection of environment hazards, SNP genotyping, search for specific binding partners, such as small molecule drug discovery, determination of gene and protein expression, etc., all relevant to the field of plants, human and animal health, bacteria, viruses, archebacteria, fungi, rickettsia, prions, mycoplasmas, and parasitic organisms.
  • Arrays such as DNA and protein arrays have gained much popularity in the recent years especially for the conduct of genomics, proteomics and diagnostics research. Arrays are used, for example, in comparing the gene expression between normal cells and tumor cells.
  • Nucleic acids from a normal cell sample may carry one detectable label, for example, a fluorescent label, and nucleic acids from a tumor cell sample may carry a different detectable fluorescent label.
  • the two samples are mixed and hybridized to a set of oligonucleotide probes on an array.
  • One hybridization signal from a given spot on the array may indicate hybridization mostly with nucleic acid from normal cells, another hybridization signal may indicate hybridization mostly with nucleic acids from tumor cells, and a mixed hybridization signal may indicate hybridization with both normal and tumor cells.
  • Assays of this type are qualitative at best and assay results represent only an approximate indicator of gene expression.
  • variation in spot sizes and, thus, the number of probes at each spot renders any comparisons between relative expression levels even more difficult to interpret.
  • U.S. patents 6,040,138 and 6,004,755 also attempt to improve the measurement quantitation in differential expression.
  • a relatively small number of normalization controls are introduced.
  • Normalization controls are oligonucleotide probes that are perfectly complementary to labeled reference oligonucleotides that are added to the nucleic acid sample.
  • the signals obtained from the normalization controls after hybridization provide a control for variations in hybridization conditions, label intensity, "reading" efficiency and other factors that may cause the hybridization signal to vary between arrays.
  • the signals (e.g., fluorescence intensity) read from all other probes in the array are calibrated by the signal (e.g., fluorescence intensity) from the control probes thereby normalizing the measurements.
  • our method does not rely on extrapolation from a relatively small set of normalization control probes, with potential attendant extrapolation errors, but rather provides quantitative measurements (i.e. absolute calibration) for each and every site or bead as desired.
  • U.S. patents that address the measurement of analyte concentrations include U.S. patent 5,516,635 (EP 0608370B1) and U.S. patent 5,837,551. These involve both a capture binding agent and a developing binding agent.
  • Related patents in this series include U.S. 5,599,720 (EP 0134215), US 5,171,695 (EP 0271974), U.S. 5,834,319 (EP
  • a method of determining target molecule concentration in an array-based assay where the assay includes the steps of:
  • the substrate is in the form of a planar surface such as a sheet, or in the form of a bead or a microsphere.
  • the method as described herein where the array contains immobilized molecules that are situated at different spots of the array, and the immobilized molecules at different spots are the same or different.
  • the target molecule is labeled with a second tracer moiety that is indirectly measurable.
  • the labeled immobilized molecules and the unlabeled immobilized molecules herein may be the same or different molecular species.
  • a method of determining target molecule concentration in an array-based assay further including the step of providing or determining a dissociation constant for the bound target molecules
  • the second tracer moiety contains an indirectly measurable moiety.
  • the method of the present invention optionally includes the step of determining fractional occupancy of the immobilized molecules by the bound target molecules.
  • a method as described herein where the steps of labeling probe or target molecules and/or measuring probe and target signals are repeated sequentially for each target molecule concentration to be determined, and the target molecule concentrations obtained thereby are compared.
  • a method as described herein where target molecules of different dilutions are employed in a series of concentration determinations to arrive at a dissociation constant for the target molecules.
  • a. method of determining expression of a target molecule in a sample including the steps of: (a) providing a first composition having an array of molecules immobilized on a substrate, where the array contains a plurality of spots, at least one spot having more than one immobilized molecule, where the immobilized molecules are attached to the substrate, and at least one of the immobilized molecules is labeled with a first tracer moiety to form a labeled immobilized molecule that yields a probe signal; (b) providing a second composition having a plurality of target molecules, where at least one target molecule is directly labeled with a second tracer moiety that yields a target signal;
  • target molecules are genes, gene fragments or molecules resulting from expression or reverse transcription of genes or gene fragments; and where concentration of target molecules is related to expression thereof.
  • a method of comparing expression of a first target molecule with expression of a second target molecule including the steps of: (a) providing a first composition having an array of molecules immobilized on a substrate, where the array contains a plurality of spots, at least one spot having more than one immobilized molecule, where the immobilized molecules are attached to the
  • first and second target molecules are genes, gene fragments or molecules resulting from expression or reverse transcription of genes or gene fragments; and where concentration of target molecules is related to expression thereof.
  • the immobilized molecules and target molecules are each oligonucleotides or nucleic acids. Further, optionally, such methods are applicable to the determination of gene or protein expressions. Still optionally, the immobilized molecules situated at different spots of the array may be the same or different, such as in their specificities. Additionally, still optionally, the immobilized molecules that are different from spot to spot are all labeled with the same tracer moiety.
  • the plurality of target molecules in the second composition may be the same or different.
  • the tracer moieties for labeling the immobilized molecules and target molecules are also the same or different.
  • a method as described herein for an array-based assay where the array is supported by a substrate and the substrate has a planar surface, or is a bead or a microsphere.
  • the second composition contains target molecules from more than one sample population, where the tracer moiety for labeling the target molecules from one sample population are different from the tracer moiety for labeling target molecules from another sample population, and concenfration of the target molecules from the one sample population is compared with the concentration of target molecules from the other sample population.
  • a method of comparing gene or protein expression of two or more target molecules from two or more target sample populations where the target molecules from the different target population contains the same molecular species, where at least the target molecules from one of the sample populations are directly or indirectly labeled with a second tracer moiety.
  • At least one type of target molecules are labeled with a tracer moiety that carries a directly measurable or indirectly measurable moiety.
  • the second tracer moiety carries an indirectly measurable moiety
  • tlie target molecules are modified to link, by covalent bonding, to the indirectly measurable moiety.
  • kits that contains: (a) a composition having an array of molecules immobilized on a substrate, where the array contains a plurality of spots, at least one spot having more than one immobilized molecules, where the immobilized molecules are attached to the substrate; and (b) information on at least one dissociation constant for target molecules of one specificity bound to the immobilized molecules.
  • kits as described herein where the kit optionally includes a first tracer moiety for labeling or spiking the immobilized molecules, and/or instructions for at least a second tracer moiety for labeling the target molecules. Moreover, in another embodiment, the kit includes a plurality of different tracer moieties for labeling target molecules of different specificities. In a further embodiment, the kit as described herein includes instructions for determining dissociation constants for target molecules of different specificities bound to immobilized molecules, and/or instructions for determining gene or polypeptide expression.
  • kits containing an array as described herein where the array has for a substrate a planar surface, such as, for example, a sheet, a slide, a silicon wafer, or the substrate may be in the form of beads or microspheres, with one bead or microsphere representing one spot, for example.
  • kits having: (a) a composition that contains an array of molecules immobilized on a substrate, where the array includes a plurality of spots, at least one spot having more than one immobilized molecule, where the immobilized molecules are attached to the substrate; (b) information on at least one dissociation constant for one kind of target molecules bound to the immobilized molecules; (c) a first tracer moiety for labeling or spiking the immobilized molecules; (d) instructions for at least a second tracer moiety for labeling target molecules; (e) instructions for determination of dissociation constant for any target molecules; and (f) instructions for determination of target concentration or gene or protein expression.
  • kits as described herein where the immobilized molecules and target molecules are nucleic acids or oligonucleotides.
  • the kit herein contains immobilized molecules that are polypeptides or proteins.
  • kits that contains: (a) a composition having an array of molecules immobilized on a substrate, where the array includes a plurality of spots, at least one spot having more than one immobilized molecule, where the immobilized molecules are attached to the substrate, and at least one immobilized molecule is labeled with a first tracer moiety to form a labeled immobilized molecule; (b) information on at least one dissociation constant for one kind of target molecules bound to the immobilized molecules; (c) instructions for at least a second tracer moiety for labeling target molecules; (d) instructions for determination of dissociation constant for any target molecules; and (e) instructions for determination of target concentration or gene expression.
  • kits containing: (a) a composition having an array of molecules immobilized on a substrate, where the array includes a plurality of spots, at least one spot containing more than one immobilized molecules, where the immobilized molecules are attached to the substrate, and at least one immobilized molecule is labeled with a first tracer moiety; and (b) information on at least one dissociation constant for target molecules of one specificity bound to the immobilized molecules.
  • the kit as described herein, further including instructions for determination of expression of a target molecule, where the target molecule is a gene or gene fragment, or result of expression or reverse transcription of a gene or gene fragment.
  • kits that contains: (a) a composition having an array of molecules immobilized on a substrate, where the array contains a plurality of spots, at least one spot having more than one immobilized molecules, where the immobilized molecules are attached to the substrate, and at least one immobilized molecule is labeled with a first tracer moiety; and (b) instructions on determining dissociation constant for target molecules bound to the immobilized molecules.
  • the immobilized molecules are any suitable probe for capturing a target molecule that is conventional in the art.
  • probes include: nucleotides, polynucleotides, DNA, cDNA, RNA, mRNA, cRNA, peptide nucleic acids, oligonucleotides, polypeptides, antibodies, enzymes, hormones, cytokines, antigens, other proteins, peptides displayed on phages and other peptides, carbohydrates, polymers containing alpha-, beta-, and omega-amino acids, polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, polyacetates, mixed polymers, small molecule drugs, fragments, analogs and optical isomers thereof, or combinations thereof that form chimeric molecules.
  • composition having an array of molecules immobilized on a substrate, where the array contains a plurality of spots, at least one spot having:
  • the composition described herein where the proportion is any proportion greater than about 0.16%.
  • proportions suitable herein may be one selected from the group of ranges of: about 0.16% to about 1%, about 1% to about 2%, about 2% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 50%, about 50% to about 70%, or about 70% to about 100%.
  • composition as described herein where the proportion of the first amount to the second amount can be the same or different for different spots of the array.
  • the array is supported by a substrate that conventional in the art.
  • the substrate may be planar in surface, such as on a glass slide or a flat sheet of nitrocellulose, or the substrate may be in the form of a bead or a particle such as a microsphere.
  • the substrate may be glass or silicon or a synthetic material. Examples of substrates include solid-phase synthesis supports, fibers, capillary tubes, silicon wafers, slides, membranes, filters or other sheets.
  • composition as described herein where the immobilized molecules may be attached to the substrate by covalent or non-covalent bonding, either directly or indirectly through a linker.
  • the first tracer moiety may be attached to the immobilized molecules also by covalent or non-covalent bonding, either directly or indirectly.
  • a composition as described herein where the first tracer moiety is removable from the immobilized molecules after attachment. This facilitates reading of the target signal without interference from the probe signal.
  • the first tracer moiety remains attached to the immobilized molecule, and the probe signal can be read in the presence or absence of the target signal.
  • the target signal can be determined by subtraction.
  • the first or second tracer moiety may be the same or different and are each directly measurable.
  • the first or second tracer moiety may be indirectly measurable.
  • the tracer moiety is optionally directly or indirectly measurable and can be any label that is conventional in the art.
  • the first and second tracer moiety may be a radioactive isotope, an enzyme including one catalyzing light emission such as luciferase , a luminescent label, or a bead or microsphere containing one or more of such.
  • luminescent labels include quantum dots, fluorescent labels, energy transfer dyes, chemiluminescent labels such as phosphorescent dyes, bioluminescent labels such as phycobilisomes, colorimetric labels, and combinations thereof.
  • the tracer moiety may be indirectly measurable using any indirectly measurable molecules conventional in the art, such as conventional binding pairs, one of which can carry a directly measurable moiety.
  • an indirectly measurable molecule may be one of a pair of binding partners, including but not limited to: an antibody/antigen pair, a biotin/avidin or strepavidin pair, a digoxigenin/anti-digoxigenin pair, a carbohydrate/lectin pair, a pair of complementary oligonucleotides or nucleic acids that hybridize to each other, a receptor/ligand pair, or a synthetic pair that is chemically synthesized to bind to each other with specificity.
  • Nucleic acid derivatives that are capable of hybridizing to a complementary molecule, such as peptide nucleic acids may also be used as indirectly measurable moieties.
  • binding pairs are used where the molecule to be labeled is chemically modified to comprise one member of the binding pair, the other member of the binding pair being linked or otherwise associated with a directly measurable moiety.
  • composition as described herein where the first tracer moiety is removable from the immobilized molecules and the removal is effected enzymatically, chemically, by light activation or other energy activation, or by a change in temperature.
  • the immobilized molecule is any molecule that is suitable for capturing a target molecule.
  • suitable immobilized molecules include, but is not limited to nucleotides, polynucleotides, DNA, cDNA, RNA, mRNA, cRNA, peptide nucleic acids, oligonucleotides, polypeptides, antibodies, enzymes, hormones, cytokines, other antigens or proteins, peptides displayed on phages or other peptides, carbohydrates, polymers containing alpha-, beta-, and omega-amino acids, polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, polyacetates, mixed polymers, fragments, analogs or optical isomers thereof, or combinations thereof that form chimeric probes.
  • compositions as described herein where the immobilized molecules situated at different spots of the array are the same or different in the specificities in recognizing or capturing different target molecules.
  • the target molecules situated at one spot may be the same or different in their specificities in binding to the immobilized molecules.
  • a method of determining target concentration in a sample in an array-based assay having the steps of:
  • a method of determining target concentration having the steps of:
  • target concentration determining target concentration.
  • target molecules are oligonucleotides or nucleic acids.
  • the target molecules are any target or analytes conventional in the art.
  • the target molecules may be nucleotides, polynucleotides, DNA, cDNA, RNA, mRNA, cRNA, peptide nucleic acids, oligonucleotides, polypeptides, antibodies, enzymes, hormones, cytokines, other antigens and proteins ' , peptides displayed on phages and other peptides, carbohydrates, polymers containing alpha-, beta-, and omega-amino acids, polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, polyacetates, mixed polymers, fragments, analogs and optical isomers thereof, and combinations thereof that form chimeric probes.
  • the target molecules may have originated from human or other animal host sources and may be derived from humans or other animals or from infectious or parasitic organisms, such as bacteria, viruses, fungi, prions, etc.
  • a method as described herein where the method is applied to the determination of concentration of a number of target molecules at one spot, where the target molecules may have the same or different binding specificities to different immobilized molecules.
  • a composition as described herein where the first tracer moiety is directly or indirectly attached to the immobilized molecule. Where the first tracer moiety is indirectly attached to the immobilized molecules, such attachment is through an intermediate molecule.
  • the method herein provides an intermediate molecule that is commonly shared among all the immobilized molecules for all the spots of the array.
  • the first tracer moiety is attached to an intermediate molecule, before or after a target molecule has contacted the immobilized molecules, and the probe signal is measured in the presence or absence of the target molecule.
  • FIG. 1 is a diagrammatic representation of an array of the present invention showing an expanded view of one spot on an array, as an example, showing target molecules (two represented here) that have been labeled with a tracer moiety (squares) binding to immobilized probes that have been labeled (4 immobilized probes out of 6 are shown labeled) with another tracer moiety (circles).
  • FIG. 2 is a diagrammatic representation of a process for measuring target signal and probe signal, as an example.
  • FIG. 2A is a- diagrammatic representation of an expanded view of one spot on an array, as an example, showing binding of target molecules that have been each labeled with a tracer moiety (squares) to immobilized molecules that have not been labeled.
  • FIG. 2B is a diagrammatic representation of an expanded view of one spot on an a ⁇ -ay, as an example, showing the addition of a plurality of tracer moieties (circles) to the array for the labeling of the immobilized molecules.
  • FIG. 2C is a diagrammatic representation of an expanded view of one spot on an array, as an example, showing the bound target molecules each labeled with a tracer moiety (squares) binding to immobilized molecules, some of which have been labeled with another tracer moiety (circles).
  • FIG. 3 is a diagrammatic representation of a process for measuring target signal and probe signal, as an example.
  • FIG. 3 A represents an expanded view of one spot on an array, where some of the immobilized molecules are each labeled with a tracer moiety (circles).
  • FIG. 3B represents the removal of the tracer moieties (circles) from the immobilized molecules and addition of target molecules that have been labeled with a tracer moiety (squares) onto the spot of FIG. 3 A.
  • FIG. 3C represents the same spot after addition of labeled target molecules, showing binding to immobilized molecules.
  • FIG. 4 A represents an expanded view of one spot on an array, as an example, where the immobilized molecules on one spot are.
  • chimeras each containing a "universal key" at the distal end that is common to all the immobilized molecules on that spot as well as for all the spots on the array, although all the immobilized molecules at a given spot may be unique to that spot.
  • FIG. 4B represents the same spot as FIG. 4A, showing the binding of target molecules to the chimeras, where the target molecules are shown as being labeled with a tracer moiety (squares).
  • FIG. 4C represents the same spot as in FIG. 4A and FIG. 4C, showing the labeling of the chimeras with another tracer moiety (circles) after removal of the target molecules .
  • FIG. 5 is a graphical representation of one example, showing the relationship between fractional occupancy and log concentration for a target molecule that is a 20-mer oligonucleotide.
  • FIG. 6 is a graphical representation of one example, showing the relationship between fractional occupancy of one target plotted against the fractional occupancy of a second target.
  • FIG. 7 is a graphical representation of one example showing the relationship between the concentration of one target plotted against the concentration of the second target, where the error bars for the measurements are also shown.
  • the present invention involves the labeling of the target molecules, reading the target signal in the presence or absence of the probe label or vice versa, determination of fractional occupancy of the immobilized molecules by the target molecules, and the use of K D> the dissociation constant for the dissociation of bound target molecules under equilibrium conditions, in the determination of target molecule concentration.
  • An “array” shall mean a collection of spots that can be one-dimensional, two- dimensional or three-dimensional, and that are supported by a solid substrate onto which immobilized molecules and target molecules are placed and reactions, such as binding, are allowed to occur.
  • a three-dimensional array includes an array of beads or particles.
  • immobilized molecule shall mean any molecule that can be immobilized on a substrate by any means conventional in the art.
  • a "molecule” shall mean a natural or synthetic, a biological or chemical molecule, particularly one that has significance in plants, human or animal health, bacteria, viruses, archebacteria, fungi, rickettsia, prions, mycoplasmas, and parasitic organisms.
  • Such molecules include, but are not limited to single molecules and synthetic or natural polymers, such as, nucleotides, polynucleotides, DNA, cDNA, RNA, mRNA, cRNA, peptide nucleic acids ("PNA"), oligonucleotides, polypeptides, antibodies, kinases, phosphatases and other enzymes, hormones, cytokines, antigens, cell surface receptors and other proteins, peptides displayed on a phage and other peptides, carbohydrates, polymers including those containing ⁇ -, ⁇ -, or ⁇ -amino acids, polymers such as polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, polyacetates, mixed polymers, small molecules (for example, drug candidates), analogs, fragments and optical isomers thereof, and combinations thereof that form chimeric molecules.
  • PNA peptid
  • a "molecular species" in reference to the labeled and unlabeled immobilized molecules shall refer to the nature of a molecule.”
  • the molecular species are the same when the underlying molecules are the same.
  • an unlabeled immobilized molecule that is an antibody to Her2 a breast cancer antigen, has the same molecular species as a labeled immobilized molecule that is a labeled antibody to Her2.
  • the molecular species are different when the underlying molecules are different.
  • an unlabeled immobilized molecule that is an oligonucleotide having a sequence complementary to a fragment of the Her2 gene has a different molecular species from a labeled immobilized molecule that is an oligonucleotide having a sequence complementary to a fragment of a prostate cancer gene.
  • a “probe” or “capture probe” shall mean any immobilized molecule or molecules that are capable of capturing one or more target molecules by specifically binding thereto or by specific hybridization thereto to create bound target molecules.
  • a probe that is labeled with a tracer moiety gives rise to a probe signal that is detectable.
  • Specific binding shall mean the inherent or artificially created property of a molecule to recognize and selectively bind another molecule ("its binding partner").
  • specific binding include, but are not limited to, antigen/antibody binding, biotin/avidin or strepavidin binding, receptor/ligand binding, hybridization of complementary oligonucleotides, polynucleotides, or nucleic acids, or synthetic molecules chemically synthesized to bind to other molecules, for example, peptoids.
  • Substrate shall mean any surface conventional in the art that supports an array and on which molecules are allowed to interact, and their interaction detected without degradation of or reaction with the surface.
  • the substrate can be planar, such as a glass slide or a nitrocellulose filter or in the form of beads or particles, such as microspheres or nanobeads.
  • Substrate can be made of glass, silicon or a synthetic, such as plastic. It can be permeable or impermeable. Additionally, the substrate can be, for example, a solid- phase synthesis support, a fiber, such as glass fiber, a capillary tube, or a silicon wafer.
  • target molecule shall mean any molecule that can be captured by an immobilized molecule, labeled directly or indirectly, detected and/or quantified.
  • target molecules may be of any origin including, but not limited to: plants, humans, other animals, other vertebrates or invertebrates, microbial, including bacterial, archebacterial, viral, or fungal, parasitic, or from mycoplasmas or prions.
  • a "tracer moiety” is a molecule that contains any detectable label that is conventional in the art.
  • the tracer moiety may be removable without damaging the molecule to which it binds.
  • the tracer moiety may further be directly or indirectly measurable.
  • directly measurable tracer moieties include, but are not limited to: radioactive isotopes, energy transfer dyes, enzymes, quantum dots (sometimes referred to as semiconductor nanocrystals) and luminescent labels.
  • Luminescent labels may be fluorescent labels, chemiluminescent labels, such as a phosphorescent label, bioluminescent labels such as phycobilisomes, colorimetric labels or combinations of such.
  • the label is a fluorescent label such as a fluorescein, a rhodamine, a polymethine dye derivative, a phosphor, an energy transfer dye, and the like.
  • the tracer moiety may be in the form of a bead or microsphere containing a directly measurable label, such as a fluorescent label.
  • the tracer moiety may be indirectly measurable, such that it is modified, chemically or otherwise, to associate with one member of a binding pair, with the opposite member of the binding pair being linked to a directly measurable moiety.
  • binding pairs include, but are not limited to: biotin/avidin or strepavidin; oligonucleotides, polynucleotides or nucleic acids for which a complementary molecule carrying a detectable label can be constructed to hybridize therewith; antibody/antigen; receptor/ligand; and molecules chemically synthesized to bind fo each other, such as by combinatorial chemistry.
  • sample shall mean a biological or other sample that is being assayed or tested for presence or for quantitation of "target molecules.”
  • the methods, kits and compositions of the present invention arise from the recognition that the concept of fractional occupancy, as determined by the Langmuir isotherm (see, for example, Kittel C, "Thermal Physics,” Wiley & Sons, 1969, pp. 341- 345), can be utilized to accurately determine the concentration of target molecules in solution, in an array-based assay in which a fraction of the target molecules are captured by immobilized molecules attached to a substrate supporting the array, under conditions in which the concentration of the immobilized molecules constitutes a small fraction of the concentration of the target molecules, and when provided with K D , the dissociation constant for the bound target molecules under equilibrium conditions.
  • any inconsistency or variation in the number of immobilized molecules deposited at each spot of an array can easily be taken into consideration, and comparisons between samples can be more accurately determined.
  • a special application of the present invention is in the area of diagnostics for diseases, infection or other medical conditions, detection of environmental hazards or conditions, SNP genotyping, small molecule drug discovery, discovery research, and determination and comparison of gene and protein expression in the areas of genomics and proteomics.
  • SNP genotyping small molecule drug discovery
  • discovery research determination and comparison of gene and protein expression in the areas of genomics and proteomics.
  • present invention may be applicable to other areas as well.
  • the present invention includes methods of determining target molecule concentration in an array-based assay, methods of determining expression of target molecules, methods of comparing expression of one or more target molecules, kits containing labeled or unlabeled arrays of molecules and information on dissociation constants for determination of concentration of target molecules, as well as compositions containing labeled arrays and labeled target molecules for determination of fractional occupancy of capture probes and, ultimately, concentration of target molecules through use of a dissociation constant, K D , which is unique to each bound target molecule.
  • the present invention enables calibration by measuring target signal, and separately or simultaneously, measuring probe signal to determine the ratio of the bound sample to the overall number of immobilized molecules at any spot, this ratio being known as the fractional occupancy.
  • This invention contains a variety of embodiments for determining the target concentration by measuring the fractional occupancy f, and also presents several means to obtain the dissociation constant K D that appears in the Langmuir equation.
  • a composition in one aspect of the present invention, contains an array supported by a substrate, where the array contains a number of spots. Usually, the spots are addressable spots. Immobilized molecules for the capture of target molecules are placed or made in situ in one or more spots of the array. One or more or all of the spots of the array may contain immobilized molecules that are either the same or different and that are directly attached to the substrate or, optionally, are attached to the substrate through a linker.
  • the present invention takes into consideration the amount of immobilized molecules attached at a given spot ("probes" for short) that are available for capture of target molecules. This may be achieved in a number of ways known to persons skilled in the art. For example, one way of determining the amount of probes at a spot is by direct or indirect attachment of a quantifiable marker to that spot that is in a known proportion to the immobilized molecules at that spot. In practice, a known or set portion of immobilized molecules labeled with tracer moieties may be mixed with a known or set portion of the same immobilized molecules but unlabeled prior to attaching the mixture of labeled and unlabeled immobilized molecules at a spot on the substrate.
  • the composition of molecules to be immobilized (i.e., probes) on a spot may be spiked with a different molecular species that has a detectable label and that is capable of immobilizing to the substrate with the same efficiency as the probes.
  • the spot will contain an amount of label that is proportional to the total number of immobilized molecules.
  • the amount of tracer moiety or detectable label at such a spot may be preset or predetermined to be a desired proportion relative to the amount of immobilized molecules at that spot.
  • the array therefore, may be constructed with different spots containing the same or different proportion of tracer moiety or detectable label as compared to the immobilized molecules.
  • Patent No. 6,245,518 issued June 12, 201 to Hyseq, Inc., entitled " Polynucleotide Arrays and Methods of Making and Using the Same.”
  • such proportions may be between about 0.16% to about 100%.
  • persons skilled in the art may utilize other ranges including: 0.16% to
  • the tracer moiety is attached to, incorporated within, or otherwise associated with the same type of molecules as that to be immobilized.
  • the molecules to be immobilized may be spiked with an amount of labeled molecules of the same type, forming labeled immobilized molecules.
  • microarrays may be constructed using a Biodot spotting apparatus and aldehyde-coated glass slides. Techniques for immobilizing the probes on the array are also well known to those skilled in the art as can be seen, for example, in Lindroos, K.
  • Protein arrays that are known and conventional in the art may also be used herein, as described in WO00/056926, entitled “Methods for detection of nucleic acid polymorphisms using peptide-labeled oligonucleotides and antibody arrays”; WO99/39210, entitled “High density arrays for proteome analysis and methods and compositions therefore”; and WO00/004389, entitled “Arrays of protein-capture agents and methods of use thereof.”
  • the arrays herein may contain "addressable" spots in that each spot can be located and/or identified by its coordinates or address on the array for a one-dimensional or two- dimensional array, such that by virtue of their positions in the array, the identity of the probes or targets will become known.
  • the array is a three-dimensional array containing beads or particles, such beads or particles may be coded for identification purposes or otherwise identified by its properties, such as fluorescence or size or magnetic properties or combinations of such.
  • the immobilized molecules herein are attached to a substrate directly or indirectly.
  • Direct attachment can be, for example, by covalent bonding to the substrate.
  • Indirect attachment can be, for example, covalent bonding to a linker affixed to the substrate.
  • the nature of the immobilized molecules is such that they are any molecules capable of acting as capture molecules for any target molecules. In a particular application, they are designed to bind specifically to the target molecules. They include, but are not limited to, molecules or sequences capable of specific binding to or hybridization with target molecules.
  • the immobilized molecules may be antibodies, including polyclonal antibodies, monoclonal antibodies, single chain antibodies, fragments or chimeras thereof, or they may be synthetic molecules, including peptides, peptoids or peptide nucleic acids, generated combinatorially or otherwise, to recognize and bind specific target molecules.
  • the immobilized molecules may be, for example, the complementary oligonucleotides, polynucleotides or nucleic acids, or hybridizing analogs or mimetics thereof, including nucleic acids in which the phosphodiester linkage has been replaced with a substitute linkage, such as phosphorothioate, methylimino, methylphosphonate, phosphoramidate, guanidine and the like; nucleic acids in which the ribose subunit has been substituted, such as hexose phosphodiester; and peptide nucleic acids.
  • immobilized molecules may include, but are not limited to: nucleotides, polynucleotides, DNA, cDNA, RNA, mRNA, cRNA, peptide nucleic acids, oligonucleotides, polypeptides, antibodies, enzymes, hormones, cytokines, antigens, other proteins, peptides displayed on phages and other peptides, carbohydrates, polymers containing alpha-, beta-, and omega-amino acids, polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, polyacetates, small molecule drugs, mixed polymers, fragments, analogs and optical isomers thereof, and combinations thereof that form chimeric molecules.
  • the length of the immobilized molecules in instances where they are nucleotides, polynucleotides, nucleic acids or similar polymers, will usually range between 5 to 1000 nucleotides, optionally 5 to 500 nucleotides, further optionally 5 to 250 nucleotides, still optionally, 5 to 100 nucleotides; still further optionally, 5 to 50 oligonucleotides.
  • the polynucleotide, oligonucleotide or nucleic acid probes may be double or single stranded, or PCR fragments amplified from cDNA.
  • the immobilized molecules may be tailored to specifically bind to or hybridize with specified target molecules. For example, if the array is used to determine expression of a particular gene from a cDNA library that has been reverse transcribed from mRNA molecules, the immobilized molecules will be constructed with a sequence complementary or otherwise capable of recognizing the gene, gene fragment or expression products of such gene or gene fragments.
  • the nucleic acids may be derived from any biological sources including, but is not limited to, human, animal, plants, bacterial, fungal, viral, environmental or other sources.
  • the substrate supporting the array may be any solid substrate of any suitable configuration that is conventional in the art for the construction of arrays.
  • the substrate may be substantially planar, such as * a glass slide, a silicon wafer, or a nitrocellulose membrane, or the substrate may be in the form of a bead or particle, such as a microsphere or a nanoparticle.
  • the array may be a one-dimensional, two- dimensional or three-dimensional array.
  • the substrate may be constructed of any suitable materials that provide the array with a solid support and yet is unreactive towards any of the components of the assay including the immobilized molecules, the tracer moieties, and the target molecules.
  • suitable materials for the substrate herein includes, but are not limited to: plastics, ceramics, metals, gels, membranes, glass, silicon and other synthetic materials.
  • Substrates of interest herein include, but are not limited to: solid-phase synthesis support, fibers, capillary tubes, silicon wafers, slides, membranes, and filters.
  • the immobilized molecules herein are usually spotted onto a substrate to form an array that have at 1 spot, optionally, at least 5 spots, optionally, at least 10 spots, further optionally, at least 100 spots.
  • the array of the present invention may contain at least 1000 spots, optionally, at least 10,000 spots and further optionally, at least 100,000 spots.
  • the substrate is in the form of beads or particles
  • immobilized molecules are attached to the beads and microparticles, each bead or particle being treated as a spot of the array.
  • the beads or microparticles herein may be coded for identification purposes or they may be identified through their chemical or physical properties.
  • the immobilized molecules herein may be situated at "addressable" spots in that the type of immobilized molecules at each spot is known or can be determined. Accordingly, expression of a certain gene or protein may be traced to an "address" on the array for identification of the gene or protein sequence being expressed.
  • immobilized molecules situated at different spots of the array may be the same.
  • the immobilized molecules situated at different spots of the array are different.
  • the immobilized molecules herein may be labeled directly, or optionally through a linker, with one or more tracer moieties, by covalent or non-covalent bonding. Such tracer moieties are conventional in the art as described, for example, U.S. Patent No. 4,366,241.
  • the tracer moiety that is suitable for use herein may be a directly measurable moiety or an indirectly measurable moiety.
  • the directly measurable moiety is a detectable or measurable label, which is conventional in the art.
  • Suitable tracer moieties are disclosed, for example, in U.S. Patent No. 5,800,992 issued September 1, 1998 to Fordor et al., entitled “Method of Detecting Nucleic Acids,” and include, but are not limited to: a radioactive isotope, an energy transfer dye, an enzyme, a luminescent label, a quantum dot, or a bead or particle containing such..
  • a luminescent label includes, for example, a fluorescent label, a chemiluminescent label such as a phosphorescent dye, a bioluminescent label such as a phycobilisome, or a colorimetric label.
  • Fluorescent molecules that are of interest herein include: fluorescein, rhodamine, Texas Red, cyanine dyes and the like. Radioisotopes that are of interest herein include: 35 S, 32 P, 3 H, 125 1, 14 C and the like.
  • the indirectly measurable moiety is a molecule that is not directly measurable on its own but that will interact with a directly measurable moiety to yield a detectable signal.
  • the immobilized molecule is linked to one member of a binding pair, such as, for example, a biotin/avidin or strepavidin pair, an antibody/antigen pair, a receptor/ligand pair, an enzyme/substrate pair, a hybridizing oligonucleotide, polynucleotide or nucleic acid pair, and the like.
  • a directly measurable moiety is then linked to the other member of the binding pair.
  • the immobilized molecule becomes measurable.
  • the tracer moiety herein may be attached to immobilized molecules through a detachable or removable linkage. This is advantageous in that either the probe signal from the labeled immobilized molecules or the target signal from the labeled target molecules may be read without interference from the other signal, whether the two tracer moieties are the same or different.
  • a removal tracer moiety may be attached in such a way that it is removable enzymatically, chemically, by light or other energy activation, or by a change in temperature.
  • linkages are conventional in the art. For example, a simple labeling procedure is offered by Pierce rwww.piercenet.com).
  • the target molecules of the present invention are any analytes that can be captured, labeled and measured.
  • these target molecules are biological or chemical molecules from natural sources, such as human, animals, plants, bacteria, viruses, fungi, prions, mycoplasmas, and rickettsia.
  • the target molecules may be synthetic molecules, for example, combinatorially synthesized chemical compounds that are small molecule drug candidates.
  • Target molecules from natural sources include, but are not limited to: oligonucleotides, polynucleotides, or nucleic acids, such as DNA, cDNA, RNA, mRNA, cRNA; proteins or polypeptides such as antibodies, antigens, enzymes, hormones, cytokines; carbohydrates; factors, cofactors, analogs, fragments or combinations thereof.
  • oligonucleotides, polynucleotides, or nucleic acids such as DNA, cDNA, RNA, mRNA, cRNA
  • proteins or polypeptides such as antibodies, antigens, enzymes, hormones, cytokines
  • carbohydrates factors, cofactors, analogs, fragments or combinations thereof.
  • Examples of-some target molecules suitable herein can be found in U.S. Patent No. 4,366,241 issued December 28, 1982 to Syva Company, entitled “Concentrating Zone Method in Heterogeneous Immunoassays.”
  • the target molecules herein may also be labeled with
  • the tracer moiety for labeling target molecules may be a directly or indirectly measurable moiety as described above.
  • the target molecule when the target molecule is labeled with an indirectly measurable moiety, the target molecule may be chemically modified to contain one member of a binding pair that can be recognized by the other member of the binding pair carrying a directly measurable moiety. Any such binding pair that is conventional in the art may be used herein. An example of such a binding pair is the biotin/avidin or strepavidin pair.
  • the present invention includes a method of determining target molecule concentration in an array-based assay.
  • the invention makes use of the measured fractional occupancy at each spot to obtain an absolute calibration of a target concentration, for example, to determine a gene expression profile, typically using cDNA molecules, reverse transcribed from mRNA, as target molecules.
  • two or more samples can be compared to one another by comparing the absolute calibration thereof, thereby obtaining, for example, differential comparison of gene expression among the samples.
  • a single sample will exhibit a range of fractional occupancies corresponding to the range of concentrations of each type of molecules in the sample.
  • a probe signal emitting from the labeled immobilized molecules can be first detected and quantified.
  • a composition containing the target molecules, labeled with a tracer moiety is allowed to contact the immobilized molecules of the array. After incubation or sufficient passage of time to allow specific binding between the target molecules and the immobilized molecules to occur, excess unbound target molecules are removed, the array washed, and the target signal is detected and measured. Alternatively, the probe and target signal can both be measured after hybridization of the target molecules and washing of the array.
  • the steps, reagents, and conditions for the conduct of an assay using an array such as the ones described herein are conventional in the art, depending on the immobilized molecules utilized and the target molecules to be captured. Such steps, reagents and conditions may be found in the patents cited herein.
  • the experimental conditions are such as to allow specific hybridization to occur, such as under high stringency conditions.
  • the immobilized molecules are antibodies and the target molecules are cancer antigens, for example, then experimental conditions are such as to allow antibody and antigen to form a complex.
  • the target molecules situated on any or all spots of an array may be the same or different and the target signals can be measured with or without removal of the tracer moiety from the labeled immobilized molecules, or in the presence or absence of the tracer moiety from the labeled or unlabeled immobilizes molecules.
  • the method herein may be useful in the determination of the expression and concentrations of each type of target molecule in the sample population.
  • the method herein is useful for the comparison of expression of the target molecules in the two or more sample populations.
  • a sample containing target molecules to be analyzed may contain a mixture of 2 samples, one taken from sample 1 containing target molecules 1 that are labeled with target tracer 1, and the other taken from sample 2 containing target molecules 2 that are labeled with target tracer 2.
  • target molecule 1 and target molecule 2 both contain the same molecular species and differ only in the tracer moiety used to label the targets.
  • the method of the present invention may be utilized to obtain a quantitation of probe signal, target molecule 1 signal and a target molecule 2 signal. The fractional occupancy and, thus, concentration of the target molecule 1 and target molecule 2 may be determined, provided that K D , the dissociation constant, for each of the bound target molecule 1 and 2 are known, provided or determined.
  • This method may be applied to the comparison of two samples, for example, by determination of target molecule concentration in each sample sequentially, one sample after another and, thus, obtaining two separate target signals in multiple steps or by combining aliquots of the two samples together to form one sample, in equal or known proportions, after labeling the target molecules in each sample in a distinguishing manner so as to obtain two different target signals upon binding between the respective target molecules to the immobilized molecules.
  • An example of this application is the comparison in gene or protein expression using genes and proteins, fragments thereof or molecules resulting from expression or reverse transcription of such genes, proteins and fragments.
  • Suitable sources of genes, proteins, fragments thereof or results of expression or reverse transcription thereof for application of the present methods include, but are not limited to: normal cells, tumor cells, diseased cells, infected cells, bacteria, viruses, prions, fungi, cells at different stages of division, cells at different stages of growth, cells at different stages of growth arrest, cells at different stages of cell death, and tissues at different stages of development from all biological sources including humans and other animals, plants and microorganisms.
  • Results of expression or reverse transcription of genes, proteins and fragments include, but is not limited to: mRNA, cDNA, cRNA, proteins and polypeptides and fragments thereof, including those resulting from natural post-transcriptional and/or post-translational processing.
  • the target composition may be a mixture containing target molecules taken from more than two sample populations, such as, up to N sample populations, where N is any positive integer greater than 1, and each sample population of target molecules are labeled with a distinguishing label .yielding distinguishing target signals.
  • target molecules from each of N sample populations may be detected and measured sequentially and the concentration of each is compared after separately determining the concentration thereof. Variations in the sequence of measuring probe signal and target signal are within the contemplation of the present invention. For example, if the tracer moiety for the immobilized molecules and the target molecules are the same, the probe signal may be first measured, then the combined probe and target signals are measured following binding of target molecules to immobilized molecules and removal of excess target molecules. The target signal is then obtained from subtracting the probe signal from the combined signal.
  • the probe and target signals are measured separately and/or independently.
  • the probe signal from the labeled immobilized molecules may be measured first and then removed. Then labeled target molecules are allowed to contact the immobilized molecules. Target signal from bound target molecules can then be measured without interference from the probe signal.
  • the tracer moiety labeling the immobilized molecules and target molecules may be the same or different.
  • a proportion of the immobilized molecules may be labeled with tracer moieties a second time, with or without removal of the target molecule, and the probe signal measured or determined again, either as a check for consistency or to obtain an average value for the probe signal.
  • the fracer moiety from the immobilized molecule is not removed and the target signal is measured in the presence of the probe signal.
  • the immobilized molecules are labeled with a tracer moiety that differs from the tracer moiety labeling the target molecule in its emission spectra or other measurable characteristics.
  • the combined probe and target signal following binding of target molecules to immobilized molecules may be measured first and the tracer moiety from either the immobilized molecules or the target molecules are then removed. The signal intensity of the remaining molecules is then measured.
  • signal intensity of the target molecules may be measured first after the composition containing target molecules is allowed to contact the immobilized molecules of the array, prior to the immobilized molecules being labeled. Then the immobilized molecules are labeled, followed by measurement of combined probe and target signal. Thereafter, through subtraction, the probe signal can be determined.
  • the target molecules may be allowed to interact with immobilized molecules before the immobilized molecules are labeled.
  • Target signal from bound target molecules may be read and the target molecules or their fracer moieties may be removed.
  • the immobilized molecules may be labeled with the same or different tracer moiety, and the probe signal measured without interference from the target molecules.
  • immobilized molecules may be attached to a universal linker.
  • a universal linker may be attached to all immobilized molecules, of the same or different specificities, on one or more or all spots of an array.
  • fracer moieties may be attached to the universal linker on the immobilized molecules and the probe signal measured on all the spots on the array, regardless of binding specificities of the immobilized molecules.
  • the resultant binding between the immobilized molecules and the tracer molecule or between the immobilized molecules and labeled target molecules may be visualized in a number of ways conventional in the art.
  • K D may be calculated based on a generally accepted formula (see Example 6 below).
  • K D may be determined experimentally, such as by conducting the method of the present invention for each of several different dilutions of a target molecule and plotting the target concenfration against the function f / (1-f ), where f is the fractional occupancy.
  • K D is the slope of the line through all points that minimizes the squared distance between the line and the points.
  • the present invention includes a kit that contains, in one aspect, an array of molecules immobilized on a substrate as described above and certain information relevant to the determination of target molecule concenfration, where the array contains a number of spots, at least one spot having more than one immobilized molecules, and where the molecules to be immobilized or already immobilized on the substrate of the array are labeled or as yet unlabeled.
  • the kit herein may further contain one or more of the following: (a) instructions for determining dissociation constant, K D , for any target molecules bound to immobilized molecules; (b) instructions for determination of gene or protein expression; (c) instructions for comparing gene or protein expression; (d) instructions for determination of target molecule concentration; (e) instructions regarding an appropriate tracer moiety to use for labeling target molecules so as to optimize the spectral separation between the target signal and probe signal.
  • the kit will contain instructions for more than one type of tracer moieties for labeling more than one type of target molecules.
  • the kit may contain a fracer moiety for labeling the immobilized molecules or a detectable label that is suitable for spiking the immobilized molecules.
  • the kit will contain a substrate suitable for attaching probes to produce an array, a solution of probes to be immobilized, a solution of labeled molecules for spiking the probes, and information regarding K D .
  • Example 1 Derivation of formula for determination of target molecule concenfration as a function of fractional occupancy, f.
  • Fractional occupancy is a ratio representing the total number of target molecules bound to immobilized molecules at a given spot on an array divided by the total number of immobilized molecules available at that spot.
  • the actual number of bound target molecules at a spot on average, will depend on the number of capture molecules available under a given set of experimental conditions.
  • the average number of bound target molecules is the fractional occupancy times the number of molecules located at the spot. Since arrays generally consist of many spots, with the immobilized molecules generally being different from one spot to another, the fractional occupancy and number of bound targets will generally differ from one spot to another.
  • [bound target] - [free molecule] + [target] the fractional occupancy, f , for bound target at a given spot equals [bound target] / ([bound target] + [free molecule]).
  • [bound target] is the concenfration of target molecules bound to the immobilized molecules at a spot
  • [free molecule] is the concenfration of unbound immobilized molecules at that spot
  • [target] is the concentration of the free target molecules in the sample solution.
  • the second te ⁇ n in the denominator is the dissociation constant K D for the reaction, so the fractional occupancy has the simple form:
  • the free target molecule concenfration in this formula changes if there are a large amount of immobilized molecules at a spot that can bind the free target molecules, it is usually convenient to have sufficiently large target sample concentration so that the number of target molecules is large compared to the number of immobilized molecules at the spot (or spots, if several spots are identical). In this limit, the concenfration of target does not change substantially as a result of binding events.
  • the target concenfration is determined based on this formula.
  • FIG. 1 a composition of the present invention is diagrammatically represented by the expanded view of one spot 7 on an array 8. Other spots are not shown here.
  • Labeled target molecules 1 are shown labeled with tracer moiety 2 (squares) and the labeled immobilized molecules 3 are shown labeled witi another fracer moiety 4 (circles), where . the immobilized molecules are attached to a substrate 5.
  • Unlabeled target molecules are not shown here, but unlabeled immobilized molecules 6 are shown without any fracer moieties attached.
  • FIGS. 1-4 This may be viewed, for example, in terms of cDNA molecules or oligonucleotides as target molecules 1, being hybridized to its complementary nucleic acid molecules 3 and 6 on the array 8.
  • Use of DNA in FIGS. 1-4 is not meant to limit the broad applicability of the present invention, since " these figures also apply to any other target molecules and the complementary immobilized molecules at a spot.
  • the immobilized molecules at the spot are shown as being partially labeled in FIG. 1. Since the immobilized molecules at a spot can be large in comparison to the bound target molecules, this partial labeling of the immobilized molecules can be useful so that the signal from the immobilized molecules does not mask the signal from the target molecules.
  • the total number of immobilized molecules at the spot is readily determined once the number of labeled immobilized molecules is known.
  • the target molecules in a sample to be assayed are labeled with a different tracer moiety than that used for the immobilized molecules.
  • the target molecules are shown as being completely labeled, since their concentration and corresponding assay signal strength will usually be low, although the target molecules can also be partially labeled. So long as the proportion of partial labeling of target molecules is known, then the total amount of target can be dete ⁇ nined from the amount of partially labeled targets.
  • the ratio of the bound target molecules to the total immobilized molecules at the spot, the fractional occupancy, is described by the Langmuir isotherm introduced above. As discussed in Example 1, it is convenient to work in the limit that the number of free target molecules is large compared to the number of immobilized molecules at the spot (or spots, if several spots are identical). Under such conditions, the concentration of target does not change substantially as a result of binding events.
  • the signal associated with the bound target molecules at a spot is measured. The probe signal can be measured either before or after the hybridization of the target molecules.
  • the fractional occupancy f is by definition the ratio of the bound target molecules divided by the total number of immobilized molecules.
  • the reading instrument does not necessarily need to be calibrated to separately determine the actual number of target and immobilized molecules. Instead, the instrument can be calibrated on a relative basis to adjust for efficiency differences in reading the signals associated with the target molecules versus reading the signals associated with the probes.
  • Calibration of a label signal intensity to number of molecules, starting from a standard sample with known amount of labeled molecules is a straight forward exercise in physical chemistry: The emission photon flux can be accurately measured, the fluorescent efficiency is known, and the numerical aperture and detection loss is part of any good imaging design.
  • the number of labeled probe molecules N p can be inferred from the signal intensity I p of the label used for the probe molecules, and the amount of labeled bound target N t can be infe ⁇ red from the intensity I t of the label associated with the bound target.
  • the concentration of target is then given by inserting this ratio into the formula for target concentration:
  • [target] K D f/ (l - f).
  • This embodiment of the invention has the probe label attached to the immobilized molecules that in turn can bind to the target molecules.
  • the label can be attached directly to the spot or indirectly to the spot by means of other molecules that are attached to the spot, where the other molecules do not interact with the target. In doing so, there needs to be a proportionality between amount of label at the spot and the amount of immobilized molecules that can interact with the target molecules.
  • a single tracer moiety may be used for labeling both the immobilized molecules and the target molecules. In doing so, the probe signal for a spot is measured first.
  • the target is hybridized to the immobilized molecules, excess target molecules are removed, and a composite signal consisting of the probe signal and the target signal at the spot is measured.
  • the actual target signal is then the incremental signal above the probe signal, that is, a simple difference of the second (composite) signal and the probe signal gives the target signal.
  • corrections need to be made to account for partial labeling of the immobilized molecules or of the target.
  • the target can be measured first and the probe labels are indirectly attached, in that they can be added after the prior measurement of the target signal.
  • the tracer moieties described in this example are conventional luminescent labels, both for the probes attached at each spot and for the target molecules.
  • FIG. 2 Another embodiment of the present invention is diagrammatically represented in FIG. 2 portraying one variation in the sequence of labeling of the probes.
  • FIG. 2 portraying one variation in the sequence of labeling of the probes.
  • the labeled target molecules 1 that are labeled with tracer moiety 2 are hybridized to unlabeled probes 6 immobilized on substrate 5 at a spot 7 of an array 8. After removal of excess target molecules, the target signal is measured. Following this, in FIG. 2B, tracer moieties 4 are added to label the probes, using a suitable linker mechanism, such as a biotin/avidin combination.
  • FIG. 2C the labeled target molecules 1 are shown hybridized to labeled immobilized molecules 3 and unlabeled immobilized molecules 6 on a substrate 5 at a spot 7 of the array 8. The probe signal from a spot 7 is then measured, the fractional occupancy is determined, and the target concenfration is determined as before through use of the Langmuir equation.
  • the fracer moieties for the target molecules and for the immobilized molecules can be the same or different.
  • the two signals may be read separately, preferably the target signal is measured first to avoid any noise background due to the probe signal.
  • a combined probe and target signal may be measured after labeling of the probe, and the probe signal can be arrived at by subfraction of target signal from the combined signal.
  • FIG. 3 shows use of the removable tracer moiety in connection with labeling of the immobilized molecules.
  • FIG. 3 A diagrammatically represents the labeling of the immobilized molecules 6 with a removable tracer moiety 4, to form labeled immobilized molecules 3 at a spot 7 (other spots not shown) of an array 8, for example, using a removable disulfide linker (Pierce EZ-Link Sulfo-NHS-SS-Biotin).
  • the probe signal from the labeled immobilized molecules 3 at the spot is measured first.
  • the removable tracer moieties 4 are then removed from the labeled immobilized molecules 3 to become unlabeled immobilized molecules 6.
  • the target molecules 1 with labels 2 are then brought into contact with the unlabeled immobilized molecules to allow binding. Excess target molecules (those that are not bound) are removed, and the target signal is then measured. Removing the fracer moieties from the probe following measurement of the probe signal permits use of the same tracer moieties for both probe and target molecules in this embodiment. Of course, two different tracer moieties may be used here as well. Apart from the detail of using a removable tracer moiety for labeling the immobilized molecules, this embodiment is very similar to the earlier two embodiments, including any corrections that are needed for partial probe labeling prior to computation of the target concenfration. This variation in labeling and measuring target and probe signals flips around the process described in Example 3 above, where the probe signal is measured first in this Example 4, then the tracer moiety is removed from the probe, then the labeled target molecules are added.
  • FIG. 4 is a diagrammatic representation of another embodiment of the present invention in which an intermediate molecule 10 is used for labeling of the immobilized molecules attached to the substrate 5 to form composite molecules 9.
  • FIG. 4A shows immobilized composite molecules in which a portion of each composite molecule 9 is complementary to the target molecule and is made to be unique for one or more spots of the array 8 (Other spots not shown herein.). Another portion of the composite molecule 9 is made to be identical or "universal" for all the spots of the array.
  • the composite molecules 9 may be referred to as chimeric probes.
  • FIG. 4B shows labeled target molecules 1 being brought into contact with composite molecules 9and binding thereto. In practice, excess target molecules are removed. The target signal at the spot is then measured. The number of immobilized molecules at a spot is then dete ⁇ nined by bringing the universal portion of the immobilized composite molecules in contact with a solution that contains the labeled complement molecule to the universal portion of the immobilized molecules. If the amount of labeled complement molecules are in significant excess, compared to the number of immobilized molecules, experimental conditions including the temperature can be adjusted so that virtually all of the immobilized molecules will be bound to their labeled complement. The signal from the labeled complement is then measured.
  • the concentration of target can be calculated as before.
  • One variation to this fourth embodiment is to reverse the order of measuring the target signal and the probe signal.
  • the same fracer moiety can be used for both the target and the immobilized composite molecules.
  • only a fraction of the immobilized molecules have a universal portion, so a correction needs to be made as discussed before.
  • only a portion of the targets may be labeled. In such a case a correction here also would be necessary.
  • the present invention has been described in which single target molecule populations are measured separately and compared to the signal from the probes for absolute calibration, in determining the target concentration. Often the interest is in a differential comparison of the two concentrations.
  • the method described herein can equally well be extended to include several target populations that are run simultaneously, each of which uses a different tracer moiety and for each of which the calibration is achieved using the absolute calibration provided by the immobilized molecules at each spot (which in turn have is labeled by another fracer moiety which differs from the fracer moieties used for labeling the target molecules). By running two or more populations simultaneously and calibrating them to the immobilized molecules, they can subsequently be compared to each other to arrive at the differential comparison.
  • the invention is applicable to analysis of many polymers, including polypeptides, carbohydrates, and synthetic polymers, including alpha-, beta-, and omega-amino acids, polyurethanes, polyesters, polycarbonates, polyureas, polyamides, proteins, antibodies, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, polyacetates, and mixed polymers.
  • polymers including polypeptides, carbohydrates, and synthetic polymers, including alpha-, beta-, and omega-amino acids, polyurethanes, polyesters, polycarbonates, polyureas, polyamides, proteins, antibodies, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, polyacetates, and mixed polymers.
  • Various optical isomers e.g., various D- and L- forms of the monomers, may be used.
  • antibody probes will be generated which specifically recognize particular subsequences found on a polymer.
  • antibodies may be generated which are specific for recognizing a three contiguous amino acid sequence, and monoclonal antibodies may be preferred. Optimally, these antibodies would not recognize any sequences other than the specific three amino acid stretch desired and the binding affinity should be insensitive to flanking or remote sequences found on a target molecule.
  • antibodies specific for particular carbohydrate linkages or sequences may be generated.
  • a similar approach may be used for preparing specific reagents that recognize other polymer subunit sequences. These reagents would typically be site specifically localized to a substrate matrix pattern where the regions are closely packed.
  • the specific reagents will often be polypeptides. These polypeptides may be protein binding domains from enzymes or other proteins that display specificity for binding. Usually an antibody molecule may be used, and monoclonal antibodies may be particularly desired.
  • K D dissociation constant
  • the K D in moles can be expressed as a function of the free energy G:
  • the free energy G can be related to the well-measured melting temperature T m by noting that T m is the temperature at which the free energy is zero.
  • the free energy G equals H - TS, where H and S are the enthalpy and entropy, respectively.
  • the melting temperature of oligonucleotides is closely approximated by an increase of 3 deg Celsius for every nucleotide (Strachan T, Human Molecular Genetics):
  • the enthalpy is calculated from the average number (2.5) of hydrogen bonds (approx. 3 Kcal/mole) for each Watson-Crick DNA pair:
  • K D More precise calculations of K D are readily available.
  • a variety of calculations express the melting temperature T m in terms of enthalpy, entropy, sodium content, and GC content including nearest neighbor interactions for a given molar concentration of the strands (see for example the melting temperature calculator in www.genosys.com, also for DNA see Breslauer et al, Proc. Natl. Acad. Sci USA 83: 3746-3750 (1986)). Since the melting temperature is by definition when the fractional occupancy equals one-half, this is when K D equals the strand concenfration. Therefore, replacing the strand concenfration with K D in the chosen melting temperature equation and inverting this equation gives K D to sufficiently high precision for many applications.
  • Example 7 Fractional occupancy versus concentration of target cDNAs in solution binding to 20-mer oligo probes.
  • Example 1 The K D obtained in Example 1 is used to generate the results shown herein in Table 1. These results are also graphically represented in FIG. 5. Note a target typically has a range of cDNA concentrations, to which correspond to a range of fractional occupancies.
  • the fractional occupancies are independently determined by, for example, labeling the target molecules and the probes at each spot and calculating the ratio of the respective signal intensities, the point to this invention, then the concentration of the target corresponding to the probes at that spot is readily determined.
  • samples from different populations are compared to a given sample as a reference sample. In such cases, only one reference sample needs to be run and different populations are run separately thereafter. The total number of runs is therefore quite close to the total number of runs needed for competitive hybridization as described in U.S. Patent 5,800,992, that is, there is no need to run the reference sample every time a new target sample is mn.
  • Example 8 Comparison of gene expression between two different targets and determination of the error bars associated with this comparison.
  • Example 7 was for only one type of labeled target molecule in solution while the other label is associated with the complementary probes at a spot.
  • “competitive hybridization” for example, U.S. Patent 5,800,992
  • differential gene expression measurements separate targets are measured using two different arrays, and comparison is made by using normalization control genes or other target molecules.
  • the present invention affords an absolute calibration of two or more types of target molecules, and comparison of each of the at least two types of target molecules against this absolute calibration, and provides a means to then compare the concenfration of each target molecule type against each other, thereby, obtaining differential gene expression.
  • Table 2 2 samples of 5 cDNA. Sample 2 loaded at 1.5 x sample 1. Third cDNA has 2x expression.
  • ⁇ f (f * (l ' -f) /probe#) ⁇ 0.5 for binomial distribution (One standard deviation.)
  • fractional occupancy depends strictly on the number of fixed molecules of each type, whether the molecules are at one array site or many, or on one bead or many.
  • the statistics for fractional occupancy are quite robust: The total number of hybridized cDNA of a given type is divided by the total number of available oligos for that type. In the case where the same oligo or cDNA is located at multiple microarray sites or beads and all of the measurement data is "good,” this model states that the best answer is to take the total signal from all the hybridized cDNA and divide it by the total signal from all the fixed oligos or cDNA of that type, rather than (for example) computing a ratio for each site or bead and averaging the ratios from any replicates.
  • the data goodness can be analyzed through an initial processing step in which, for each cDNA type, a regression fit is generated of the hybridized cDNA signal versus the calibration signal from the fixed oligos or cDNA. (Each of the relevant sites or beads contributes a data point to this regression fit.) This allows any data outliers from the regression fit to be rejected before summing up the total signal as indicated above.
  • Example 9 Cancellation of dissociation constant K D errors in differential comparisons.
  • the target concentration is linear in the dissociation constant
  • fr_occ2 and fr_occl can be used to form the respective f / (1- f ) value corresponding to each of the expression concenfrations shown in the rows.
  • the ratio of these values gives a differential expression value of 1.5.
  • the ratio of the f / (1- f ) values gives a value of 3.0. This is consistent with the concentration assumptions used in this example.
  • the ratio of the f / (1- f ) values results in the concentration ratios, or differential expression ratio, independent of K D .
  • the following example describes an antibody as the immobilized molecule being bound to a solid support, and a protein interacting with the immobilized antibody as the target molecule.
  • the antibody can be any molecule having the characteristics of an antibody, including a monoclonal or polyclonal antibody, and can be derived from human (e.g.: IgG), non-human (e.g.: IgY) or mixed (humanized Ab) sources.
  • Antibodies have been well characterized and are known to those skilled in the art. They consist of two chains: a light chain and a heavy chain, and resemble in structure a "Y". The variable domains inside the "V of the "Y" contain complementary determining regions (CDRs) which bind to antigens.
  • the binding capacity is substantially increased by covalent binding.
  • high binding levels are achieved at much lower concentrations than with passive binding. This is the result of the irreversible nature of the covalent binding process; monolayer coverage can be achieved at low concenfrations, limited only by the diffusion rate of the antibodies to the surface. Monolayer coverage is usually about 300 ng. per square centimeter, and can be achieved with concentrations containing twice that amount of protein in agitated systems.
  • the presence of surfactant in a protein solution does not inhibit covalent binding- with XENOBINDTM plates as it does with passive binding.
  • Antibody-antigen dissociation constants vary due to the specificity of the variable domain. Typical values range between 10 "6 - 10 "7 . Protein affinity determinations, or 1/ K D , can be found in www.biacore.com, or see also NHRC Publication 84-37 (Griswold,
  • the fractional occupancy can be detected by any of the methods described above.
  • the immobilized antibody can be labeled with one dye and the target antigen protein labeled with a spectrally dissimilar dye.
  • the same process can be used to label both the antibody and the protein since the antibody is a protein and contains numerous N-termini.
  • a simple labeling procedure is offered by Pierce (www.piercenet.com).
  • the EZ-LinkTM Suylfo-NHS-LC-Biotinylation Kit allows biotin labeling of proteins or antibodies in 30 minutes at room temperature.
  • the biotin labeled proteins or antibodies can be reacted with streptavidin-conjugated dyes (e.g.:
  • the probe signal from the dye attached to the antibody must be calibrated to the number of dye molecules.
  • the signal from the spectrally dissimilar dye attached to the target protein (antigen) must be calibrated to the number of protein molecules. This can easily be done by one skilled in the art by measuring the signal associated with known concentrations of antibodies and proteins, respectively.
  • the fractional occupancy f is calculated by dividing the number of bound protein molecules by the number of antibody molecules.
  • K D can easily be measured by those skilled in the art.
  • One method is to use varying concenfrations of protein, measure the associated fractional occupancy f and then plot the protein concenfration as a function of the fraction f/(l-f), analogous to Example 10
  • the slope of a line through the origin minimizing the squared difference between the line and the concentration is the best estimate of K D .
  • SNPs Single Nucleotide Polymo ⁇ hisms
  • the concentration calculated for one spot is plotted on the x- axis, and the other on the y-axis, and this is done for a population of individuals, there will tend to be a scatter plot between the positive x and y axes due to the noise sources.
  • the variation in concenfrations can be large, and at times it is hard to tell which are the homozygotes and which are the heterozygotes.
  • this invention improves precision by removing variability due to spot size. Therefore, the scatter plots are reduced in their spread, allowing easy identification of SNP type.

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Abstract

L'invention concerne des procédés, des trousses et des compositions permettant de déterminer toutes concentrations moléculaires cibles dans des essais à base de jeux d'échantillons ordonnés utilisant le principe d'occupation fractionnelle conformément à l'isotherme Langmuir.
PCT/US2001/047277 2000-11-07 2001-11-07 Nouveau procede en matiere d'analyse quantitative de l'expression de genes et de proteines WO2002038812A2 (fr)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988001058A1 (fr) * 1986-08-06 1988-02-11 Roger Philip Ekins Determination de la concentration d'analyte a l'aide de deux substances de marquage
WO1993008472A1 (fr) * 1991-10-15 1993-04-29 Multilyte Limited Methode de dosage par liaison a l'aide d'un reactif marque
EP0742286A2 (fr) * 1995-05-08 1996-11-13 Roche Diagnostics GmbH Détection quantitative d'acides nucléiques
WO1997043450A1 (fr) * 1996-05-16 1997-11-20 Affymetrix, Inc. Tests d'hybridation sur des alignements d'oligonucleotides
WO2000024940A1 (fr) * 1998-10-28 2000-05-04 Vysis, Inc. Reseaux cellulaires et methodes de detection et d'utilisation de marqueurs de troubles genetiques
WO2000034523A1 (fr) * 1998-12-11 2000-06-15 Hyseq, Inc. Groupes de polynucleotides, leurs techniques de production, et leur utilisation
WO2002012855A2 (fr) * 2000-08-10 2002-02-14 Nanobiodynamics, Inc. Procede et systeme de reconnaissance biomoleculaire rapide des sequences d'acides amines et de proteines

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988001058A1 (fr) * 1986-08-06 1988-02-11 Roger Philip Ekins Determination de la concentration d'analyte a l'aide de deux substances de marquage
WO1993008472A1 (fr) * 1991-10-15 1993-04-29 Multilyte Limited Methode de dosage par liaison a l'aide d'un reactif marque
EP0742286A2 (fr) * 1995-05-08 1996-11-13 Roche Diagnostics GmbH Détection quantitative d'acides nucléiques
WO1997043450A1 (fr) * 1996-05-16 1997-11-20 Affymetrix, Inc. Tests d'hybridation sur des alignements d'oligonucleotides
WO2000024940A1 (fr) * 1998-10-28 2000-05-04 Vysis, Inc. Reseaux cellulaires et methodes de detection et d'utilisation de marqueurs de troubles genetiques
WO2000034523A1 (fr) * 1998-12-11 2000-06-15 Hyseq, Inc. Groupes de polynucleotides, leurs techniques de production, et leur utilisation
WO2002012855A2 (fr) * 2000-08-10 2002-02-14 Nanobiodynamics, Inc. Procede et systeme de reconnaissance biomoleculaire rapide des sequences d'acides amines et de proteines

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Title
EKINS R P ET AL: "MULTIANALYTE MICROSPOT IMMUNOASSAY-MICRONALYTICAL COMPACT DISK OF THE FUTURE" CLINICAL CHEMISTRY, AMERICAN ASSOCIATION FOR CLINICAL CHEMISTRY. WINSTON, US, vol. 37, no. 11, 1991, pages 1955-1966, XP002939391 ISSN: 0009-9147 *
EKINS R P: "LIGAND ASSAYS: FROM ELECTROPHORESIS TO MINIATURIZED MICROARRAYS" CLINICAL CHEMISTRY, AMERICAN ASSOCIATION FOR CLINICAL CHEMISTRY. WINSTON, US, vol. 44, no. 9, 1998, pages 2015-2030, XP002939389 ISSN: 0009-9147 *

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