EP1183387A2 - Reseau generique d'adnc ou de proteines destine a des bioanalyses personnalisees - Google Patents

Reseau generique d'adnc ou de proteines destine a des bioanalyses personnalisees

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Publication number
EP1183387A2
EP1183387A2 EP00919976A EP00919976A EP1183387A2 EP 1183387 A2 EP1183387 A2 EP 1183387A2 EP 00919976 A EP00919976 A EP 00919976A EP 00919976 A EP00919976 A EP 00919976A EP 1183387 A2 EP1183387 A2 EP 1183387A2
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EP
European Patent Office
Prior art keywords
molecule
capture
nucleic acid
group
solid support
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00919976A
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German (de)
English (en)
Inventor
Thomas M. Baer
Ann Bennett Lossing
Steven T. Kunitake
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Arcturus Engineering Inc
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Arcturus Engineering Inc
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Filing date
Publication date
Application filed by Arcturus Engineering Inc filed Critical Arcturus Engineering Inc
Publication of EP1183387A2 publication Critical patent/EP1183387A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • 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

Definitions

  • the invention relates generally to the field of detection of macromolecules. More particularly, the invention relates to a method and apparatus for generating a flexible array of spacial locations for the concurrent quantification of multiple macromolecules.
  • DNA and RNA sequence analysis and detection of protein molecules are useful in genetic and disease diagnosis, toxicology analysis, genetic research, and pharmaceutical and agricultural development. It is also desirable to concurrently analyze multiple molecules in a cost-effective and efficient manner. Often procedural variations in quantitative analysis can be reduced by concurrently analyzing multiple molecules from the same sample.
  • multiple target molecules can be detected to determine the presence of multiple pathogens that may be present in a test sample, or alternatively, multiple target nucleic acid sequences can be detected to quantify a target nucleic acid sequence present in a test sample.
  • methods for detecting multiple targets are somewhat limited by the methods for detecting the signal generating groups that are used to detect the molecules.
  • the molecules in order to detect multiple target molecules, the molecules must be distinguished from one another. While such distinctions can be made by detecting each target with different signal generating moieties, difficulties are presented in detecting the signals from these moieties. For example, when multiple fluorescent moieties are employed, each of the multiple moieties may have a distinct absorption and emission wavelength which can be employed to distinguish one sequence from another.
  • mRNA levels in a given cell provides insight into the function and differentiative state of that particular cell at any point in its life cycle.
  • a variety of assays have been developed to detect the presence of a particular nucleic acid sequence through selective hybridization of a hybridization probe to the target sequence.
  • mRNA levels do not always provide an accurate portrayal of a cell's functional state. It is the translated products of these mRNAs, such as the receptors, ion channels, enzymes, and structural proteins of the cell, that determine its function.
  • Techniques currently used to detect proteins are based on various types of immunoassays, such as ELISA, immunohistochemistry and radioimmunoassay, which utilize antibodies specific for the protein of interest. Standard labeling methods for detection of the antibody include fluorescence, radioisotopes, and enzymes such as peroxidase and phosphatase.
  • secondary antibodies are oftentimes biotinylated to increase their sensitivity.
  • a goal of the invention is to provide a flexible device that will allow the concurrent quantitation of multiple macromolecules in a sample.
  • One embodiment of the invention is based on an apparatus which includes a capture location on a solid support surface.
  • the capture location includes a capture molecule linked to the solid support surface by a first bond type.
  • a first complexing agent including (i) a probe molecule capable of specifically binding a target molecule and (ii) a linker molecule linked by a second bond type to the probe molecule is used, such that the linker molecule can be specifically bound to the capture molecule and the probe molecule is thus immobilized on the solid support surface.
  • the solid support surface is selected from the group consisting of glass, plastic, gel, celluloid, paper, magnetic resin, coated surfaces, plates, membranes, fibers, polyvinylidene-fluoride, nylon, nitrocellulose, and particulate matter.
  • the first bond type is a physical link selected from the group consisting of passive absorption, a Schiff base formed with silylated glass and a crosslink to a polymer.
  • the capture molecule includes a first nucleic acid.
  • the linker molecule includes a second nucleic acid, the second nucleic acid having a sequence sufficiently complimentary to specifically hybridize to the first nucleic acid.
  • the capture molecule is selected from the group consisting of antigens, antibodies, biotin, biotin analogues, avidin, and avidin analogues.
  • the linker molecule can specifically bind the capture molecule and is selected from the group consisting of antigens, antibodies, biotin, biotin analogues, avidin, and avidin analogues.
  • the second bond type is a physical link selected from the group consisting of phosphodiester bond, bifunctional crosslinker, activated bead and macromolecule.
  • the target molecule is selected from the group consisting of nucleic acids, proteins and cell lysates.
  • the probe molecule is a nucleic acid having a nucleotide sequence sufficiently complimentary to specifically hybridize to a nucleic acid target molecule.
  • the probe molecule is an antibody specific for an antigen target molecule.
  • FIG. 1 illustrates a schematic view of a device, representing an embodiment of the invention.
  • FIG. 2 illustrates a schematic view of a device including an array of PNA or cDNA molecules specific for multiple, distinct targets, representing an embodiment of the invention. DESCRIPTION OF PREFERRED EMBODIMENTS
  • the purpose of this invention is to provide a flexible device that will allow the quantitation of macromolecules in a micro dissected sample. It is anticipated that many types of diagnostic assays will require the simultaneous measurement of several proteins, deoxyribonucleic acid (DNA), or ribonucleic acid (RNA) molecules in a given sample. It is desirable to have a flexible platform that would allow the measurement of multiple analytes using the same basic platform.
  • Some embodiments of the invention can include a plurality of spatially segregated sites, each of theses sites can be adapted to measure a specific analyte, thereby defining an n-dimensional array.
  • a device that includes such an array can be termed a multichannel device. Referring now to FIG. 1, a schematic view of the molecular components of a preferred embodiment of the invention is depicted.
  • a solid support 110 is comprised of material that serves as the substrate for all subsequent interactions. Any solid support to which a capture molecule may be attached may be used in the invention.
  • suitable solid support materials include, but are not limited to, silicates such as glass and silica gel, cellulose and nitrocellulose papers, nylon, polystyrene, polymethacrylate, latex, rubber, and fluorocarbon resins such as TEFLON.
  • the solid support material may be used in a wide variety of shapes including, but not limited to slides and beads.
  • Slides provide several functional advantages and thus are a preferred form of solid support. Due to their flat surface, probe and hybridization reagents can be minimized using glass slides. Slides also enable the targeted application of reagents, are easy to keep at a constant temperature, are easy to wash and facilitate the direct visualization of RNA and/or DNA immobilized on the solid support. Removal of RNA and/or DNA immobilized on the solid support is also facilitated using slides. It is estimated that a standard microscope glass slide can contain 50,000 to 100,000 cells worth of DNA. Beads, such as BioMag Strepavidin magnetic beads are another preferred form of solid support containing a second complexing agent.
  • Avidin may be chemically attached to glass using the N-hydroxysuccinamide active ester of avidin as taught by Manning, et al. Biochemistry 16:1364-1370 (1977) and may be attached to nylon by a carbodiimide coupling as taught by Jasiewicz, et al. Exp. Cell Res. 100:213-217 (1976). Magnetic microbeads labeled with avidin and streptavidin labeled beads may be obtained from
  • Capture molecules are fixed to solid substrates using any of a variety of methods such as passive absorption, formation of a Schiff base (aldehyde-amine) with silylated glass and crosslinking to polymer.
  • Solid substrates are commonly functionalized by exposing all or a portion of the substrate to a chemical reagent that fixes a chemical group to the surface which is reactive with a portion of the capture molecule.
  • groups that are suitable for attachment to a longer chain portion include, but are not limited to, amine, hydroxyl, thiol and carboxyl groups.
  • Aminoalkylsilanes and hydroxyalkylsilanes can be used to functionalize a variety of surfaces, such as a glass surface.
  • the construction of such solid phase biopolymer arrays is described in the literature (see, e.g., Merrifield, J. Am. Chem. Soc, 85:2149-2154 (1963) (solid phase synthesis of peptides); Geysen, et al., J. Immun.
  • Non-chemical approaches for fixing capture molecules to solid supports may include heat or UV cross-linking.
  • the particular material selected as the solid support is not essential to the invention, as long as it provides the described function. Normally, those who make or use the invention will select the best commercially available material based upon the economics of cost and availability, the expected application requirements of the final product, and the demands of the overall manufacturing process.
  • Capture molecules 130, 132 and 134 are molecules bonded to the solid support that specifically bind to their counterpart linker molecules in solution.
  • the capture molecules may include nucleic acids with repeating sequences of oligonucleotides.
  • the repeating sequences of oligonucleotides may include (i) a repeating trimer, such as ACTACTACTACT..., (ii) a repeating tetramer, such as ACTGACTGACTG...., or (iii) a repeating pentamer, such as ACTGAACTGAACTGA
  • capture molecules 130, 132 and 134 may include proteins with specific binding properties, such as streptavidin (which specifically binds to biotin).
  • capture molecules 130, 132 and 134 may include small molecules with specific binding properties, such as biotin (which specifically binds to streptavidin).
  • peptide nucleic acid is used in place of oligonucleotides.
  • PNA is an analog of DNA in which the backbone is a pseudopeptide rather than a sugar. PNA mimics the behavior of DNA and binds complementary nucleic acid strands. The neutral backbone of PNA results in stronger binding and greater specificity than normally achieved.
  • the unique chemical, physical and biological properties of PNA have been exploited to produce powerful biomolecular tools, antisense and anti-gene agents, molecular probes and biosensors. Important new applications have emerged that could not be performed using oligonucleotides.
  • the capture and linker molecules used in the embodiment of the invention may be any pair of complexing agents which form a strong binding pair. Since elevated temperatures are generally required for hybridization, the binding pair should preferably be stable at temperatures at least up to about 37°C under hybridization conditions.
  • linker and capture molecule combinations can be used which are based upon numerous molecular interactions well described in the literature.
  • an immobilizable tag has a natural binder (e.g., biotin, protein A or protein G)
  • an appropriate capture molecule e.g., avidin, streptavidin, neutravidin the Fc region of an immunoglobulin, etc.
  • antibodies to molecules having natural capture properties, such as biotin are also widely available as appropriate capture molecules (see, SIGMA Immunochemicals 1999 catalogue, SIGMA Chemical Co. (St. Louis Mo.)).
  • any haptenic or antigenic compound can be used in combination with an appropriate antibody to form an immobilizable linker/capture molecule pair.
  • an appropriate antibody to form an immobilizable linker/capture molecule pair.
  • the antibody can serve as either the immobilizable tag binder or, in an indirect immobilization assay format, as the capture molecule.
  • the capture molecule is a first antibody that recognizes the immobilizable linker and the solid support has bound thereto a second antibody that recognizes the first antibody.
  • receptor-ligand interactions are also appropriate as immobilizable linker and capture molecule pairs.
  • agonists and antagonists of cell membrane receptors can be used in forming immobilizable linker and capture molecule pairs.
  • cell receptor-ligand interactions such as transferrin, c-kit, viral receptor ligands, cytokine receptors, chemokine receptors, interleukin receptors, immunoglobulin receptors and antibodies, the cadherein family, the integrin family, the selectin family, can all be employed in the methods of the invention (see, e.g., Pigott and Power, The Adhesion Molecule Facts Book (Academic Press New York, 1993)); Receptor Ligand Interactions: A Practical Approach, (Rickwood and Hames (series editors) Hulme (ed.) IRL Press at Oxford Press NY).
  • toxins, venoms, viral epitopes, hormones e.g., opiates, steroids, etc.
  • intracellular receptors e.g., receptors for various small ligands, including steroids, thyroid hormone, retinoids and vitamin D, peptides
  • drugs, lectins, sugars, nucleic acids both linear and cyclic polymer
  • oligosaccharides, proteins, phospholipids and antibodies can all interact with various cell receptors.
  • Synthetic polymers, such as heteropolymers, in which a known drug is covalently bound to any of the above can also form appropriate immobilizable capture molecules.
  • Such polymers include, but are not limited to, polyurethane, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, and polyacetates. Numerous other immobilizable tag/capture moiety pairs that are useful in assay systems described herein will be readily apparent to one of skill in the art upon review of this disclosure.
  • Specific linker-capture molecule interactions will occur when the linker and capture molecules bind with a dissociation constant (K D ) of at least about 0.01 ⁇ M, preferably, at least about 0.001 ⁇ M or better and, most typically and preferably, 0.0001 ⁇ M or better under standard assay conditions.
  • K D dissociation constant
  • Linker molecules 140 and 142 are chosen as the binding counterpart to the capture molecules 132 and 134 respectively.
  • the capture molecule 132 may include a nucleic acid with a repeating sequence of oligonucleotides.
  • the repeating sequences of oligonucleotides in the capture molecule 132 includes a repeating trimer (e.g., ACT)
  • the corresponding linker molecule 140 will include a repeating trimer of the complementary sequence (e.g., TGA).
  • the capture molecule 132 is a repeating tetramer (e.g., ACTG)
  • the corresponding linker molecule 140 will include a repeating trimer of the complementary sequence (e.g., TGAC).
  • the linker molecule 142 is biotin, which specifically binds to streptavidin.
  • the linker molecule 142 is streptavidin.
  • the capture molecules 132, 134 and linker molecules 140, 142 create a binding pair for immobilizing subsequent molecules to the solid support 110.
  • a second type of bonds 150 and 152 represents the physical links connecting linker molecules 140 and 142 and a probe molecules 160 and 162.
  • Probe molecules 160 and 162 are selected for their specific binding ability to a target sequence.
  • Second bond types 150 and 152 may include a phosphodiester bond, a bifunctional crosslinker, an activated bead or a macromolecule.
  • bonds 150 and 152 include, but are not limited to, proteins, carbohydrates, lipids, peptides, polyester, nucleic acids and synthetic polymers.
  • Common second bond types include polypeptide sequences, such as poly-glycine sequences of between about 5 and about 200 amino acids.
  • proline residues are incorporated into the second bond type to prevent the formation of significant secondary structures by the second bond type itself.
  • Flexible second bond types are also suitable. For example, polyethylene glycol linkers are available from Shearwater Polymers, Inc. (Huntsville, Ala.).
  • a linker molecule 140 is preferably attached to a probe molecule 160 by a second bond type 150 which does not interfere with the ability of the probe molecule 160 to bind a target molecule 170.
  • Probe molecules 160 and 162 are selected from molecules that are specific for target molecules 170 and 172 respectively.
  • probe molecule 160 includes a nucleic acid or a peptide nucleic acid (PNA) having a sequence complimentary to a specific mRNA target 170.
  • probe molecule 162 includes an antibody with specific affinity for a biomolecular target 172.
  • a preferred embodiment includes a solid support 110 with multiple probe molecules immobilized on its surface.
  • the solid support 110 may include probe molecules 160 and 162 directed towards different target molecules 170 and 172.
  • the different target molecules 170 and 172 both include nucleic acids with different nucleotide sequences.
  • target 170 includes a nucleic acid and target 172 includes a protein.
  • a solid support 110 may include a PNA (or cDNA) as a probe molecule 160 having a nucleotide sequence complementary to a target HER-2/neu mRNA 170 and an anti-HER- 2/neu antibody as probe molecule 162 with specific binding affinity for a target HER-2/neu protein 172.
  • PNA or cDNA
  • the target molecule 170 or 172 is the specific molecule to be quantified and is selected from a specific nucleic acid, a specific cellular protein and any other cellular product.
  • the target molecule is identified with the aid of a detector molecule.
  • Detector molecules 180 and 182 are labeled molecules capable of binding to some common feature of target molecules 170 and 172 respectively.
  • the detector molecule 180 includes fluorescein-labeled poly dT nucleic acid molecule capable of binding to the polyA tail of mRNAs and used to detect target mRNA molecule 170.
  • the detector molecule 182 includes fluorescein-labeled anti -mouse IgG antibodies for the detection of a mouse antibody target molecule 172.
  • Detector molecules 180 and 182 are molecules such as nucleic acids, antibodies, small molecules, etc. which are able to specifically bind target molecules 170 and 172 respectively, in solution. Detector molecules 180 and 182 also include an analytically detectable marker that can be attached to or incorporated into the detector molecules 180 and 182.
  • any analytically detectable marker that can be attached to or incorporated into a molecule may be used in the invention.
  • An analytically detectable marker refers to any molecule, moiety or atom which can be analytically detected and quantified.
  • Methods for detecting analytically detectable markers include, but are not limited to, radioactivity, fluorescence, absorbance, mass spectroscopy, EPR, NMR, XRF, luminescence and phosphorescence.
  • any radioactive label which provides an adequate signal and a sufficient half-life may be used as a detectable marker.
  • Commonly used radioisotopes include 3 H, 14 C, 32 P and I25 I.
  • l4 C is used as the detectable marker and is detected by accelerator mass spectroscopy (AMS). I4 C is preferred because of its exceptionally long half-life and because of the very high sensitivity of AMS for detecting 14 C isotopes.
  • Other isotopes that may be detected using AMS include, but are not limited to, 3 H,
  • Fluorescent molecules such as fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbeliferone and acridimium, and chemiluminescent molecules such as luciferin and 2,3-dihydrophthalazinediones may also be used as detectable markers.
  • the detectable marker may also be a molecule which, when subjected to chemical or enzymatic modification, becomes analytically detectable such as those disclosed in Leafy, et al., Proc.
  • detectable markers include protein binding sequences which can be detected by binding proteins, such as those disclosed in U.S. Pat. No. 4,556,643 the entire contents of whichwhich is incorporated herein by reference.
  • the probe sequence 160 includes a detectable marker. After the probe molecule 160 binds and immobilizes the target molecule
  • Molecules which bind to an analytically detectable marker may also be covalently attached to or incorporated into the probe molecule, for example, as taught by Ward,
  • the probe molecule is detected by adding an analytically detectable marker which specifically binds to the probe molecule, thereby enabling detection of the probe molecule.
  • a typical assay starts with a microdissection (e.g., laser capture microdissection) of the biopsy sample to choose cells of interest.
  • the cell surfaces are digested using standard buffers containing proteinase-K or guanidinium iso-thiocyanate (GITC), releasing the cell contents into a buffer solution.
  • GITC guanidinium iso-thiocyanate
  • the probe sequences can contain intercalator dyes that can be fluorescent when hybridized with the targets and are non-fluorescent when the probe is not hybridized.
  • an intercalator dye specific for double stranded targets could be added after hybridization to quantitate the amount of hybridized probe in the sample.
  • the detectable marker may be detected by a variety of methods known in the art, depending on the particular detectable marker employed.
  • AMS may be used when the detectable marker is a radioisotope such as ,4 C
  • liquid scintillation may be used when the detectable marker is 3 H-thymidine
  • standard fluorescence or spectroscopic methods may be used when the detectable marker is a fluorescent molecule or the DNA itself.
  • the quantity of the target molecule that is present may be determined based on the signal generated from the detectable marker using a calibration curve.
  • the calibration curve may be formed by analyzing a serial dilution of a sample of nucleic acids having a known concentration of the target sequence.
  • a calibration curve may be generated by analyzing a series of known amounts of cells from a cell line in which the concentration of the target sequence is known.
  • samples of cells may be analyzed according to the method of the invention and according to a method known in the art for quantifying the target nucleic acid sequence.
  • Alternative methods for generating a calibration curve are within the level of skill in the art and may be used in conjunction with this method.
  • an array of PNA or cDNA capture locations is created on the solid support 110.
  • a capture molecule 132 has a sequence optimized to anneal to a complementary linker sequence 140.
  • Each capture location has a capture molecule with a unique and distinct nucleotide sequence optimized to anneal to a specific complementary sequence. The sequences are chosen so that the various complementary sequences are optimized for non-cross reactivity (i.e. linker sequences anneal preferentially to a specific location and do not anneal to the other locations).
  • a typical assay reagent would consist of a synthesized probe nucleic acid 160 including sequences with linker tail section 140 that has a sequence complementary to a specific capture molecule 132 on the slide 110. These probe sequences would be ligated to a section that was the complement to the chosen target molecules in the sample.
  • RNAse ribonuclease enzyme
  • a bridge sequence that has a specific pattern that could be cleaved by a specific enzyme could be included in the sequence. This sequence could be chosen so that it was cleaved as a single strand but not cleaved when the target was hybridized to the probe.
  • an array of protein capture molecules could be similarly constructed by using distinct antibodies optimized for low cross reactivity located in a spatial array, similar to the PNA or cDNA array indicated above. These antibodies would be conjugated with probe antibodies that specifically bind the antigen target molecule of interest.
  • General protein indicator dyes could be used as detector molecules to detect the specific proteins or a sandwich assay with fluorescent indicators as detector molecules could be used.
  • Another method for constructing an array of protein capture molecules would be to conjugate the probe antibodies to PNA linkers complementary to the PNA capture molecules at the various capture locations on the assay slide.
  • the PNA probes would provide the method for localizing the antibodies at specific locations.
  • the protein array would then be exposed to the digested sample and the target proteins would conjugate selectively with their associated antibodies.
  • the total amount of target could be measured by performing a sandwich assay using a generic antihuman antibody conjugated to a fluorescent dye, for example.
  • the invention also relates to a kit for providing some, or all, of the components of the invention, and optionally for performing the invention. Unless otherwise specified, the components of the kit can be the same as those used in the invention.
  • the kit includes a solid support surface and a linker molecule capable of specifically binding to a capture molecule that is linked to a solid support surface by a first bond type.
  • the kit may include the solid support and reagents for attaching one or more capture molecules to the solid support.
  • the kit further includes reagents capable of linking the linker molecule by a second bond type to a probe molecule.
  • a probe molecule capable of specifically binding a target molecule is also provided.
  • a second complexing agent including detector molecules that specifically bind the target molecule and include a detectable marker may also be provided. It is preferred that the detectable marker included on the second complexing agent mixture be of a known concentration relative to the second complexing agent.
  • the kit may also include apparatus and written instructions for practicing the assay(s) and one or more target molecules for use in the preparation of a calibration curve(s).
  • Example 1 Fig. 2 illustrates a PNA or cDNA array with each location having a separate capture sequence optimized to anneal to a complementary sequence.
  • Such an array of capture locations may be generated by starting with an avidin labelled solid support prepared by the methods described in Manning, et al. Biochemistry 16:1364-1370 (1977) and Jasiewicz, et al. Exp. Cell Res. 100:213-217 (1976).
  • Biotinylated nucleic acids prepared by chemically modifying the oligonucleotides comprising the capture location sequences to incorporate biotin according to the method of Pinkel, et al, Proc. Natl. Acad. Sci. (USA) 83:2934-2938 (1986).
  • sequences of the capture locations 210, 212, 214 and 216 are chosen so that the various complementary sequences are optimized for non-cross reactivity, i.e. sequences anneal preferentially to a specific location and do not anneal to the other locations.
  • Capture location 210, immobilized in column 1 has a sequence AC AC AC AC AC optimized to anneal to a complementary linker sequence TGTGTGTGTG present in the complexing agent 220.
  • capture location 212, immobilized in column 2 has a sequence GTGTGTGT optimized to anneal to a complementary linker sequence CACACACACA present in the complexing agent 222
  • capture location 214, immobilized in column 3 has a sequence ATATATATAT optimized to anneal to a complementary linker sequence TAT AT AT ATA present in the complexing agent 224
  • capture location 216, immobilized in column 4 has a sequence AGAGAGAG optimized to anneal to a complementary linker sequence TCTCTCTC present in the complexing agent 226.
  • Each capture location has a capture molecule with a unique and distinct nucleotide sequence optimized to anneal to a specific complementary sequence.
  • Each complexing agent also includes a synthetic probe nucleic acid having a nucleotide sequence complementary to the chosen target molecules in the sample.
  • a probe 220 corresponding to target sequence #la is immobilized on column 1 by virtue of the attached linker TGTGTGTGTG hybridizing to the capture PNA or DNA molecule 210 having the nucleotide sequence ACACACACAC bound to column 1.
  • probe 222 corresponding to target sequence #2a is bound to column 2
  • probe 224 corresponding to target sequence #3a is bound to column 3
  • probe 226 corresponding to target sequence #4a is bound to column 4.
  • Fig. 2 The results of applying cell lysates from six different samples along rows 'a' through 'f are depicted in Fig. 2.
  • a lack of hybridization signal indicates the absence of the particular target molecule in the sample.
  • a decreased hybridization signal indicates a partial expression of the target molecule in the sample.
  • the sample in row 'e' lacks targets #2a and #4a.
  • the sample in row 'c' has partial expression of the target #3a and the sample in row 'f indicates partial expression of the target 4a.
  • RNAse ribonuclease
  • a further enhancement of this approach could entail use of a reagent, such as the enzyme ribonuclease (RNAse) that specifically degrades the unhybridized probes before the slide is exposed to the reagents. This would eliminate any probe not hybridized to target from binding to the slide.
  • a bridge sequence that has a specific pattern that could be cleaved by a specific enzyme could be included in the sequence. This sequence could be chosen so that it was cleaved as a single strand but not cleaved when the target was hybridized to the probe.
  • the complexing agents 230, 232, 234 and 236 indicate variations of the complexing agents 220, 222, 232, 234 respectively wherein a single stranded cleavage site is introduced between the linker moiety and the probe moiety such that susceptibility to cleavage when target is not hybridized to probe can be used to eliminate probe molecules that do not bind target. This arrangement is particularly useful where the detectable markers are attached to the probe molecules.
  • Example 2 This example includes the simultaneous detection of HER-2/neu mRNA and protein.
  • An avidin labelled solid support is prepared by the methods described in Manning, et ⁇ .Biochemistry 16:1364-1370 (1977) and Jasiewicz, et al. Exp. Cell Res. 100:213-217 (1976), each of which are incorporated herein by reference.
  • Biotinylated hybridization probes are prepared by chemically modifying a nucleic acid having a sequence complementary to the HER-2/neu mRNA to incorporate biotinylated uridine according to the method of Pinkel, et al, Proc. Natl. Acad. Sci. (USA) 83:2934-2938 (1986), which is incorporated herein by reference.
  • a portion of the avidin-labelled solid support is designated for treatment with biotinylated probe for HER-2/neu mRNA creating an array of probe locations for HER-2/neu mRNA.
  • Mouse antibody raised against HER-2/neu protein is conjugated with biotin using conventional methods.
  • the remaining portion of the avidin-labelled solid support is designated for treatment with biotinylated antibody with specific binding affinity for HER- 2/neu protein.
  • the assay starts with a microdissection of a biopsy sample from a breast cancer patient to choose cells of interest. Following lysis of the cell surfaces with proteinase k or GITC-containing buffer, the cell lysates are mixed with the biotinylated reagents to allow hybridization of the probe molecules to the targets. Each type of target molecule is thus separately immobilized at specific locations on the solid support.
  • the HER-2/neu mRNA may then be quantified using fluorescein-labeled oligo-dT and the HER-2/neu protein may be quantified using a sandwich assay with labeled antibodies.
  • HER2 human epidermal growth factor receptor-2
  • HER-2/neu and c-erbB-2 encodes a growth factor receptor that been found to play an important role in breast cancer.
  • the HER2 gene is amplified and its associated receptor protein is overexpressed on the tumor cell surface.
  • Overexpression of HER-2/neu has been shown to play important role in the malignant transformation and clinical aggressiveness of breast cancer. This has led to the hypothesis that amplification and overexpression of HER2 is an early event in the development of breast cancer.
  • Overexpression of HER-2 has also been found to be associated with increased resistance to chemotherapy. Thus, patients with elevated levels of HER-2 respond poorly to many drugs.
  • HER-2 status is important to predict response to endocrine therapy and chemotherapy.
  • HER-2/neu as a Prognostic and Predictive Marker for Response to Treatment, H. B. Muss, Miami Breast Cancer Conference, 1999.
  • HER-2 gene amplification using immunohistochemical (IHC) methods Assays for HER2 in breast cancer have employed a variety of reagents, tissue preparation techniques, and scoring system. Questions about reliability, reproducibility and the best predictive method for determining HER2 status have limited the use of HER2 in clinical-decision making.
  • a practical application of the invention that has value in determining HER2 status is the ability to concurrently detect both HER-2/neu mRNA and protein from a single cell lysate in a single assay plate. Results obtained from use of this invention is likely to be more reliable and reproducible and useful in clinical-decision making for breast cancer treatment. There are innumerable other uses for the invention, all of which need not be detailed here.
  • a device representing an embodiment of the invention, can be cost effective and advantageous for at least the following reasons.
  • the invention provides a flexible, multichannel device that will allow the quantitation of macromolecules in a microdissected sample. Diagnostic assays often require the simultaneous measurement of several proteins, deoxyribonucleic acid (DNA), or ribonucleic acid (RNA) molecules in a given sample.
  • the invention provides a flexible platform that would allow the measurement of multiple analytes using the same basic platform. All the disclosed embodiments of the invention described herein can be realized and practiced without undue experimentation. Although the best mode of carrying out the invention contemplated by the inventors is disclosed above, practice of the invention is not limited thereto. Accordingly, it will be appreciated by those skilled in the art that the invention may be practiced otherwise than as specifically described herein.
  • the individual molecular components need not be provided in the disclosed forms, or hybridized in the disclosed configurations, but could be provided in virtually any form, and hybridized in virtually any configuration.
  • all the disclosed elements and features of each disclosed embodiment can be combined with, or substituted for, the disclosed elements and features of every other disclosed embodiment except where such elements or features are mutually exclusive. It will be manifest that various additions, modifications and rearrangements of the features of the invention may be made without deviating from the spirit and scope of the underlying inventive concept. It is intended that the scope of the invention as defined by the appended claims and their equivalents cover all such additions, modifications, and rearrangements.
  • the appended claims are not to be interpreted as including means-plus- function limitations, unless such a limitation is explicitly recited in a given claim using the phrase "means-for.” Expedient embodiments of the invention are differentiated by the appended subclaims.

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Abstract

L'invention concerne des systèmes et des procédés pour une évaluation quantitative de multiples macromolécules dans un dispositif flexible. Le dispositif comprend un emplacement de capture sur une surface support solide. L'emplacement de capture comprend une molécule de capture liée à la surface support solide au moyen d'un premier type de liaison. Un premier agent complexant comprend a) une molécule sonde capable de lier spécifiquement une molécule cible et b) une molécule de liaison liée à la molécule sonde au moyen d'un second type de liaison, qui est utilisée de façon que la molécule de liaison puisse être liée spécifiquement à la molécule de capture, immobilisant ainsi la molécule sonde sur la surface support solide. Les systèmes et procédés sont avantageux en ce qu'ils permettent à un utilisateur de créer un réseau d'emplacements de capture pour l'évaluation quantitative de macromolécules, comprenant des acides nucléiques et des protéines. L'invention concerne également un kit comprenant des réactifs capables de lier la molécule de liaison à une molécule sonde.
EP00919976A 1999-04-09 2000-03-31 Reseau generique d'adnc ou de proteines destine a des bioanalyses personnalisees Withdrawn EP1183387A2 (fr)

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US538862 1990-06-15
US12857899P 1999-04-09 1999-04-09
US128578P 1999-04-09
US53886200A 2000-03-29 2000-03-29
PCT/US2000/008611 WO2000061806A2 (fr) 1999-04-09 2000-03-31 Reseau generique d'adnc ou de proteines destine a des bioanalyses personnalisees

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