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WO2000060116A1 - Techniques ultrasensibles de detection a haut rendement - Google Patents

Techniques ultrasensibles de detection a haut rendement Download PDF

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Publication number
WO2000060116A1
WO2000060116A1 PCT/US2000/008465 US0008465W WO0060116A1 WO 2000060116 A1 WO2000060116 A1 WO 2000060116A1 US 0008465 W US0008465 W US 0008465W WO 0060116 A1 WO0060116 A1 WO 0060116A1
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WIPO (PCT)
Prior art keywords
target analyte
target
complex
cells
antibody
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.)
Ceased
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PCT/US2000/008465
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English (en)
Inventor
Alexander Aristarkhov
Christopher Martin
Michelle A. J. Palmer
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Tropix Inc
Original Assignee
Tropix Inc
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Publication date
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Priority to AU40513/00A priority Critical patent/AU4051300A/en
Publication of WO2000060116A1 publication Critical patent/WO2000060116A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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
    • C12Q1/6804—Nucleic acid analysis using immunogens
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    • 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
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    • 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
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    • B01J2219/00279—Features relating to reactor vessels
    • B01J2219/00306—Reactor vessels in a multiple arrangement
    • B01J2219/00313—Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks
    • B01J2219/00315—Microtiter plates
    • B01J2219/00317—Microwell devices, i.e. having large numbers of wells
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    • B01J2219/00603—Making arrays on substantially continuous surfaces
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    • B01J2219/0061—The surface being organic
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    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00614—Delimitation of the attachment areas
    • B01J2219/00621—Delimitation of the attachment areas by physical means, e.g. trenches, raised areas
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    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00623—Immobilisation or binding
    • B01J2219/00626—Covalent
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00623—Immobilisation or binding
    • B01J2219/0063—Other, e.g. van der Waals forces, hydrogen bonding
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00632—Introduction of reactive groups to the surface
    • B01J2219/00637—Introduction of reactive groups to the surface by coating it with another layer
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00659—Two-dimensional arrays
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    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00702—Processes involving means for analysing and characterising the products
    • B01J2219/00707—Processes involving means for analysing and characterising the products separated from the reactor apparatus
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

Definitions

  • the present invention relates to methods for performing high-throughput
  • biological assays including immunoassays, nucleic acid detection assays, and related
  • the target analyte to be detected is a specific mRNA
  • the assay monitors the impact of a particular substance or effector on transcription.
  • the target analyte may be a protein, hapten, non-biological chemical,
  • a target sample is inspected for the presence and/or amount of a particular analyte.
  • the signal must be strong, and easily detected.
  • Assays for detection of target analytes have high throughput requirements when a wide variety of possible targets must be screened.
  • nucleic acid detection In the case of nucleic acid detection,
  • HTS throughput screening
  • RNA When the target analyte is RNA, several methods, including cDNA macro
  • RNA labeling is required to detect the RNA.
  • a particular signal such as chemilumine scent, colorimetric, fluorescent, or some other visibly detectable signal.
  • HTS technology is addressed through two processes. One involves the
  • microplates These plates, typically plastic although they may be made from glass as well, have a large number of wells in the plate, typically 96, 384 or greater.
  • the well is coated with a reactant, for example, a DNA probe or an antibody, then the sample is added, with capture occurring if the target analyte is present in the sample.
  • a reactant for example, a DNA probe or an antibody
  • invention to provide a method and associated assay system for detecting a target analyte.
  • assay system for screening substances such as drugs, agents and the like, which have an effect on the level of expression of various genes.
  • analytes are detected by: (a) spotting multiple probes for the target analytes at a plurality
  • RNA wherein the cell lysates or non-purified RNA may comprise one or more of the
  • Yet another specific object of the invention is to provide a method of testing the ability of a drug or other entity to modulate the expression of a target analyte
  • Still another specific object of the invention is to provide a method of testing the ability of a drug or other entity to modulate the expression of target analytes
  • Figure 1 shows features of the mRNA detection assay.
  • a long 400-2000bp
  • FIG. 2 shows the covalent attachment of long single stranded DNA (ssDNA)
  • probes to solid surfaces including a microplate, particle and pin.
  • Figure 3 shows a method of detection with a reagent with enhanced signal
  • a dendromer may be coated with (1) a secreted alkaline phosphatase
  • SEAP selectively mutagenized alkaline phosphatase
  • AP selectively mutagenized alkaline phosphatase
  • FIG. 4 shows steps involved in an enzyme-linked immunosorbant assay
  • ELISA ELISA-type assay using a branched antibody/ AP complex as a detection reagent.
  • Figure 5 shows cell-based HTS monitoring of the level of a specific mRNA in reference to a "housekeeping gene” and several other genes.
  • Figure 6 shows HTS using magnetic pin technology.
  • Figure 7 shows the membrane/particle approach in a capture-type assay.
  • FIG. 8 shows HTS using the membrane/particle approach.
  • Figure 9 shows a mini-gene array in a 96- well microplate.
  • Figure 10 shows model oligo detection formats.
  • Figure 11 shows RNA quantitation in an array format with anti-hybrid detection.
  • Figure 12 shows model systems for RNA quantitation with anti -hybrid detection -
  • Figure 13 shows a Zip-Code array with anti-hybrid detection.
  • Figure 14 shows a method for performing a high throughput mRNA screening
  • Figure 15 shows a method of signal amplification using an RNA/DNA hybrid
  • Figure 16 is a graphical comparison of the features of the invention compared
  • a "DNA molecule” refers to the polymeric form of deoxyribonucleotides
  • double-stranded DNA found, wter alia, in linear DNA molecules (e.g., restriction
  • oligonucleotide as used herein in referring to the probe of the present
  • ribonucleotides preferably more than three. Its exact size will depend upon many factors which, in turn, depend
  • oligonucleotide upon the ultimate function and use of the oligonucleotide.
  • primer refers to an oligonucleotide, whether occurring
  • nucleotides is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA
  • the primer may be either single- stranded or double-stranded and must be sufficiently long to prime the synthesis of the
  • the primer will depend upon many factors, including temperature, source of primer and use
  • the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.
  • the primers herein are selected to be “substantially” complementary to different
  • the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primers must be sufficiently complementary
  • primer sequence need not reflect the exact sequence of the template.
  • a non- complementary nucleotide fragment may be attached to the 5' end of the primer, with the
  • non-complementary bases or longer sequences can be interspersed into the primer
  • the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template for the synthesis of the
  • Two DNA sequences are "substantially homologous" when at least about 75%
  • substantially homologous can be identified by comparing the sequences using standard
  • Antisense nucleic acids are DNA or RNA molecules that are complementary to at
  • Two amino acid sequences are "substantially homologous" when at least about
  • amino acid residues preferably at least about 80%, and most preferably at
  • an “antibody” is any immunoglobulin, including antibodies and fragments
  • the term encompasses polyclonal, monoclonal, and chimeric antibodies, the last mentioned described in further detail in U.S. Patent
  • an "antibody combining site” is that structural portion of an antibody molecule
  • Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin
  • Fab and F(ab') 2 portions of antibody molecules are prepared by the proteolytic reaction of papain and pepsin, respectively, on substantially intact antibody molecules by
  • a monoclonal antibody thus typically displays a single binding
  • a monoclonal antibody may be any monoclonal antibody having affinity for any antigen with which it immunoreacts.
  • a monoclonal antibody may be any monoclonal antibody.
  • each immunospecific for a different antigen e.g., a bispecific (chimeric) monoclonal antibody.
  • standard hybridization conditions refers to salt and temperature
  • Standard hybridization conditions is whether the two sequences hybridizing are RNA- RNA, DNA-DNA or RNA-DNA. Such standard hybridization conditions are easily
  • the degree of similarity between the nucleic acid sequences of two polynucleotides may be measured by determining whether the two polynucleotide
  • one aspect of the invention is directed to polynucleotide molecules capable of selectively
  • hybridization conditions may be varied so that the hybridization interaction
  • Tm melting temperature
  • Tm is defined as the temperature at which half the duplex molecules have
  • stranded polynucleotide may be determined empirically or by reference to well known formulas that take into account hybridization conditions that influence Tm.
  • the '"stringency" of a hybridization may be defined as degrees centigrade below the Tm of the probe nucieotide sequence.
  • the degree of stringency of hybridization is
  • nucleic acid hybridization probes for the detection of a target mRNA should be used.
  • Hybridization probes may be labeled by a variety of reporter groups, including, but not limited to, radionuclides such as 32 P or 35 S, or enzymatic labels such as horseradish peroxidase or alkaline phosphatase coupled to the probe via
  • Probes for hybridization may be synthesized by both enzymatic and in vitro
  • Short hybridization probes are preferably synthesized by in vitro methodology on commercially available DNA synthesizers such as the machines sold by
  • vitro synthesis may be readily spliced together using generally known ligation
  • the oligonucleotide probes will generally comprise between about 10
  • an optimal probe is about 100-10,000 nucleotides, preferably 200-5000 nucleotides, and
  • target analyte-specific hybridization probes include the cloning of nucleic acid sequences of the target analyte into vectors for the production of
  • RNA probes Such vectors are known in the art, commercially available, and may be used to synthesize RNA probes in vitro.
  • the vector of interest is labeled by addition of
  • RNA polymerases such as T7 or SP6 RNA polymerase
  • labeled nucleotides including, but not limited to, fluorescent or radioactive
  • the present invention concerns the identification of a target
  • Advantages of the present invention include high sensitivity and the ability to
  • the assay may utilize one of two general formats: (1) wherein cells are cultured at a plurality of
  • probes for the target analyte are spotted at a plurality of positions to form a
  • micro-array and lysed cells or non-purified fractions or components of cells (e.g., non- purified RNA) are contacted therewith. Both formats preferably utilize probes which are
  • antibody(ies) to the target analyte in particular to an RNA/DNA hybrid, can be produced and isolated by standard methods including the well
  • DNA/RNA hybrid will be referred to herein as Ab, and antibody(ies) raised in another
  • a target analyte including a specific mRNA
  • Three such procedures which are especially useful utilize either the target analyte labeled with a detectable label, a DNA which binds
  • the target analyte forms complexes with one or more
  • the binding partner is a
  • DNA probe and one member of the complex is labeled with a detectable label.
  • Ab 2 may be raised
  • Ab 2 therefore would be anti-rabbit antibody raised in goats.
  • Ab will be referred to as a
  • Ab 2 will be referred to as a secondary or anti-Ab, antibody.
  • the labels most commonly employed for these studies are radioactive elements, enzymes, chemicals which fluoresce when exposed to ultraviolet light, and others.
  • Lucifer Yellow A particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate.
  • the target analyte or its binding partner(s) can also be labeled with a radioactive
  • the radioactive label can be detected by any of the currently selected radioactive material
  • the preferred isotope may be selected from 3 H, 14 C, 32 P,
  • Enzyme labels are likewise useful, and can be detected by any of the presently
  • the enzyme is conjugated to the selected particle by reaction
  • bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like.
  • bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like.
  • Many enzymes which can be used in these procedures are known and can be
  • the preferred are peroxidase, ⁇ -glucuronidase, ⁇ -D-glucosidase,
  • ⁇ -D-galactosidase urease, glucose oxidase plus peroxidase and alkaline phosphatase.
  • test kits suitable for use by
  • a medical specialist may be prepared to determine the presence or absence of a predetermined target analyte in suspected target cells. In accordance with the testing
  • binding partner to the target analyte for instance a probe or an antibody specific thereto
  • kits may also contain peripheral reagents such as
  • test kit may be prepared for the demonstration of the presence or
  • the diagnostic test kit may comprise:
  • test kit may be prepared and used for the purposes
  • ligand or an immobilized ligand, which ligand is selected from the group consisting of:
  • the prospective drug may be introduced into a cell culture, and the culture thereafter
  • the invention utilizes a probe (e.g., a denatured PCR product, a single-
  • the probe may be covalently bound directly to a solid support, which provides
  • the solid support is a 96 well microtiter plate or nylon membrane.
  • the probe can be attached to a "pin" or columnar device
  • the probe may be biotinylated.
  • the biotin can then be
  • an unlabeled avidin may be
  • cells may be cultured, or non-purified RNA may be spotted onto a solid support, and a DNA probe for the target
  • the DNA probe may be attached to the solid surface,
  • RNA/DNA complexes may be added.
  • the "label” or “reporter molecule” may be an enzyme for chemiluminescent detection of a fluorescent label for direct detection.
  • the label may be a
  • luminescent molecule such as a dioxetane, which may be activated by enzymes.
  • the enzyme is alkaline phosphatase, particularly secreted alkaline phosphatase (SEAP), and the reporter substrate on which the enzyme
  • AMPPD® a dioxetane
  • CSPD® a dioxetane
  • the image signal from the label or reporter molecule can be read with a sensitive
  • multiple enzyme molecules may be associated with a single binding probe.
  • dendromers available from Polyprobe, Inc., are coated with multiple enzyme molecules and at least one antibody. The ideal ratio of enzyme to antibody is determined based on the nature of the assay.
  • a DNA sequence available from Polyprobe, Inc.
  • probe is attached to a column with a restriction site built-in such that the DNA can be
  • the DNA linker is biotinylated.
  • the column is
  • HTS HTS
  • each well may be exposed to a
  • the target sample for example cells, are placed in the well and lysed to release the suspected target analyte.
  • the target mRNA if
  • the pins are removed from the first well, and a second well is provided which contains the antibody/enzyme detection reagent, the two being incubated together. The pins may be removed again, and washed
  • reporter substrate such as a dioxetane
  • the pins may either be plastic or coated glass, for each of attachment, or the
  • particles coated by the ss DNA probe may be magnetic, and magnetic pins may be used.
  • pins may be cleaned and reused.
  • particles coated with the probe such as the ss DNA probe discussed above, are provided.
  • the particles are provided with nucleic acid that will hybridize to specific mRNA. These particle bound probes are allocated into each well of a multiple well microplate provided
  • microplates are available from Nalge Nunc
  • Target cells are grown on the membrane bottom of the microplate wells, in the
  • Hybridization conditions are
  • the mixture is contacted with the enzyme/antibody conjugate, and again incubated, to
  • size is selected to be slightly less than the particle size, washing under vacuum filtration
  • the substrate (the dioxetane or other luminescent
  • the cell lysis preparation may be done
  • RNA or cell lysates can be used.
  • the target mRNA is detected with high sensitivity. Further, the proposed device has the advantage of measuring gene expression from multiple genes for a large
  • multiple probes for specific mRNAs are spotted in a mini-array in the bottom of a microplate.
  • the plate bottom surface can
  • nylon PVDF
  • other material with either a flow-through or a solid bottom design.
  • the array has applications for gene expression analysis, including, for example,
  • low density macroarrays high density cDNA arrays, high density oligo arrays and novel HTS formats.
  • the array can also be applied to toxicology or pathway determination by
  • the array can also be used for DNA sequencing (sequencing by hybridization or SBH) for use in genomics research or diagnostic sequencing. It can
  • the array is also be used for genetic mapping, including assessment of single nucieotide polymorphisms (SNPs) and disease linkage.
  • SNPs single nucieotide polymorphisms
  • the array is also useful for pharmacogenetics (SNP and other).
  • the arrays may also be comprised of peptide/protein
  • the array may be used in combination with a "stripped down" Northstar system.
  • 12 x 8 cm membrane arrays can be made with 96-5,000 features. Detection may be performed using chemiluminescence.
  • arrays are useful for increased information content for high-throughput screening.
  • a spot volume on the membrane may be 1 - 5 nL (Foster City, S&T).
  • Membranes which may be used include, but are not limited to PVDF .45 um (MSI),
  • 6,6 membranes including Biodyne A .45 um, Biodyne Plus .45 um, XuXu neutral unsupported nylon .45 um*, NY0011 .2 um unsupported Biodyne B/.8 um supported Biodyne B*, Biodyne A composite .04 um*, Biodyne A .1 um*, Biodyne B, H support
  • the oligonucleotide attachment may be performed by means of UV X-link or
  • plasma gas derivatization (ammonia, oxygen or hydrogen and ammonia) covalent
  • the goals of using an array for HTS include measuring an expression level of 10- 100 genes in a high throughput pharmaceutical screening format, the utilization of non- purified RNA or cell lysates, and achieving adequate sensitivity for the target mRNA of
  • Array panels may be developed which represent a pharmaceutically relevant
  • the assay is designed to be sensitive enough to detect a single copy of a specific
  • mRN A/cell Maximum signal amplification is based on an antibody against a specific DNA/RNA hybrid. Detection monitors changes in the level of a specific message
  • the assay is referred to as a "housekeeping gene” as well as several other genes.
  • the assay is
  • DNA probe in antisense to specific mRNA is used.
  • the probe is attached directly to a solid support.
  • a detection reagent with high amplification power per single binding event is used.
  • An easy washing step is performed and one can obtain a multiple gene read out from the same cell population after signal application.
  • a long ssDNA probe (close to the full size of the mRNA target) is covalently
  • reagent can be obtained by using probe coated particles.
  • Methods of producing the probe include run-off PCR using a 5' activated primer
  • the detection reagent has enhanced signal amplification power and is based on a
  • DNA/RNA hybrid specific antibody so as to maximize signal amplification from a
  • the amplification enzyme (alkaline phosphatase) is
  • a DNA/RNA hybrid-specific ELISA-type assay Preferably, a DNA/RNA hybrid-specific ELISA-type assay.
  • a DNA/RNA hybrid-specific ELISA-type assay Preferably, a DNA/RNA hybrid-specific ELISA-type assay.
  • Formed matrix was used as the detection reagent in an mRNA detection assay.
  • the matrix synthesized with corresponding antibody can also be used in any type of
  • Example 3 Cell based HTS monitoring level of specific mRNA in reference to housekeeping gene
  • a microplate lid with pins oriented down was used to create an active surface on
  • the pins were coated with DNA corresponding to the housekeeping gene and mRNA
  • Detection reagent was added to each well and incubated.
  • Pins were placed in the 384 well microplate with a substrate for alkaline
  • Particles are prepared in bulk with a reagent which changes as a result of the
  • mRNA are attached to the particles and an aliquot is placed into each well of a membrane bottom microplate (96 well, 384 well, etc.)
  • Hybridization is conducted at 65°C for 2 hours.
  • AP/antibody conjugate is added and incubated. The wells are then washed 3 times with EW buffer at 53°C, and then washed 2 times with HCII buffer. AP substrate is then
  • Cells can be grown in a well of a 96 well culture microplate, induced, lysed, and
  • Detection reagent was added to the reaction mix and incubated.
  • Alkaline phosphatase substrate was added to microplate wells. After incubation, the microplate was read in a luminometer.
  • Particles with an enhanced surface were used. This can be considered an
  • membrane was limited to holding particles during washing. Therefore, membrane pore size was chosen to be slightly less than particle size.
  • alkaline phosphatase substrate with the membrane was lowered.
  • a detection reagent an antibody specific for DNA/RNA hybrids, conjugated to alkaline phosphatase, was used to detect
  • Particles were coated with a peptide containing a phosphorylation site specific for
  • a detection reagent based on an antibody directed to that phosphorylated site was used to detect the results of the reaction.
  • extension type assay In general the approach can be applied to any assay where the reaction product is fixed to the surface, which should be spatially separated from the
  • RNA sample is added and the assay is performed with standard 96 well
  • the plate bottom surface can be nylon, PVDF, or other
  • the signal can be imaged
  • a high throughput mRNA screening assay may be performed using Xpress- ScreenTM (Tropix Inc.) with applications for the RIANA module ( Figure 14).
  • the assay may be performed using Xpress- ScreenTM (Tropix Inc.) with applications for the RIANA module ( Figure 14).
  • RNA was synthesized in vitro from the T7 promoter using most of the 9000 bp of
  • RNA/phage DNA was hybridized to the synthetic RNA.
  • the RNA/phage DNA was hybridized to the synthetic RNA.
  • hybrid was mixed with cell lysate, two 100 bp single stranded biotinylated capture probes and incubated 3 hours for hybridization of the DNA probes and mRNA target.
  • Alkaline phosphatase conjugated antibody against the DNA/RNA hybrid was added. After a 1 hour incubation and washing of unbound antibody and probes, alkaline
  • phosphatase substrate was added. After a 30 minute incubation, chemiluminescent

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Abstract

L'invention concerne des techniques qui permettent d'effectuer des analyses biologiques à haut rendement, notamment des dosages immunologiques, des tests de détection d'acide nucléique, etc. Dans l'un des modes de réalisation préférés, l'analyte cible à détecter est un ARNm spécifique. Dans un autre mode de réalisation, l'objectif de l'analyse est de surveiller l'impact d'une substance ou d'un effecteur particulier sur la transcription. L'analyte cible peut aussi être une protéine, un haptène, un produit chimique non biologique, une substance pharmaceutique ou un autre matériel cible. La technique assure une amplification élevée permettant de maximiser le signal et fait appel à des échantillons qui ne nécessitent pas de purification très poussée.
PCT/US2000/008465 1999-04-02 2000-03-31 Techniques ultrasensibles de detection a haut rendement Ceased WO2000060116A1 (fr)

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AU40513/00A AU4051300A (en) 1999-04-02 2000-03-31 High throughput and high sensitivity detection assays

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US60/127,480 1999-04-02
US16961899P 1999-12-08 1999-12-08
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WO2001036681A3 (fr) * 1999-11-15 2001-12-13 Digene Corp Detection immunologique d'arn: hybrides d'adn sur jeux ordonnes de microechantillons
GB2401942A (en) * 2003-05-22 2004-11-24 Bioct 5 Ltd Polypeptide and polynucleotide assay methods and apparatus
WO2005017193A1 (fr) * 2003-08-13 2005-02-24 Tsinghua University Procede de detection rapide de molecules d'acide nucleique
WO2005080602A3 (fr) * 2003-12-11 2005-11-03 Digene Corp Detection immunologique d'hybrides d'arn:adn sur microreseaux
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EP2360273A1 (fr) * 2001-06-30 2011-08-24 Enzo Life Sciences, Inc. Compositions et procédés pour la détection d'analyte, la quantification et l'amplification
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US9689047B2 (en) 2010-01-29 2017-06-27 Qiagen Gaithersburg Inc. Methods and compositions for sequence-specific purification and multiplex analysis of nucleic acids
US9777312B2 (en) 2001-06-30 2017-10-03 Enzo Life Sciences, Inc. Dual polarity analysis of nucleic acids
US9797000B2 (en) 2009-05-01 2017-10-24 Qiagen Gaithersburg Inc. Non-target amplification method for detection of RNA splice-forms in a sample
US9885092B2 (en) 2011-02-24 2018-02-06 Qiagen Gaithersburg Inc. Materials and methods for detection of HPV nucleic acids
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US6686151B1 (en) 1998-02-06 2004-02-03 Digene Corporation Immunological detection of RNA:DNA hybrids on microarrays
US7399589B2 (en) 1998-02-06 2008-07-15 Digene Corporation Immunological detection of RNA:DNA hybrids on microarrays
WO2001036681A3 (fr) * 1999-11-15 2001-12-13 Digene Corp Detection immunologique d'arn: hybrides d'adn sur jeux ordonnes de microechantillons
US7439016B1 (en) 2000-06-15 2008-10-21 Digene Corporation Detection of nucleic acids by type-specific hybrid capture method
US8389219B2 (en) 2000-06-15 2013-03-05 Qiagen Gaithersburg, Inc. Detection of nucleic acids by type-specific hybrid capture method
US7645571B2 (en) 2000-06-15 2010-01-12 Qiagen Gaithersburg, Inc. Detection of nucleic acids by type-specific hybrid capture method
US7601497B2 (en) 2000-06-15 2009-10-13 Qiagen Gaithersburg, Inc. Detection of nucleic acids by target-specific hybrid capture method
US9234234B2 (en) 2001-06-30 2016-01-12 Enzo Life Sciences, Inc. Detection and quantification process for more than one nucleic acid in library
US9790621B2 (en) 2001-06-30 2017-10-17 Enzo Life Sciences, Inc. Composition of matter comprising library of first nucleic acid analyte copies
US9765387B2 (en) 2001-06-30 2017-09-19 Enzo Biochem, Inc. Process for detecting or quantifying nucleic acids in a library
US9771667B2 (en) 2001-06-30 2017-09-26 Enzo Life Sciences, Inc. Arrays comprising chimeric compositions
EP2360273A1 (fr) * 2001-06-30 2011-08-24 Enzo Life Sciences, Inc. Compositions et procédés pour la détection d'analyte, la quantification et l'amplification
US9873956B2 (en) 2001-06-30 2018-01-23 Enzo Biochem, Inc. Compositions and processes for analyte detection, quantification and amplification
US9777312B2 (en) 2001-06-30 2017-10-03 Enzo Life Sciences, Inc. Dual polarity analysis of nucleic acids
US9745619B2 (en) 2001-06-30 2017-08-29 Enzo Biochem, Inc. Process for detecting or quantifying nucleic acids in a library
US9777406B2 (en) 2001-06-30 2017-10-03 Enzo Biochem, Inc. Process for detecting or quantifying nucleic acids in a library
GB2401942A (en) * 2003-05-22 2004-11-24 Bioct 5 Ltd Polypeptide and polynucleotide assay methods and apparatus
GB2401942B (en) * 2003-05-22 2007-12-19 Bioct 5 Ltd Assay method and apparatus
US7700373B2 (en) 2003-05-22 2010-04-20 Nalia Systems Ltd. Assay method and apparatus
WO2005017193A1 (fr) * 2003-08-13 2005-02-24 Tsinghua University Procede de detection rapide de molecules d'acide nucleique
WO2005080602A3 (fr) * 2003-12-11 2005-11-03 Digene Corp Detection immunologique d'hybrides d'arn:adn sur microreseaux
US8901287B2 (en) 2004-10-20 2014-12-02 Qiagen Gaithersburg, Inc. Detection of nucleic acids by target-specific hybrid capture method
US9115410B2 (en) 2004-10-20 2015-08-25 Qiagen Gaithersburg, Inc. Detection of nucleic acids by target-specific hybrid capture method
US8877436B2 (en) 2008-10-27 2014-11-04 Qiagen Gaithersburg, Inc. Fast results hybrid capture assay on an automated platform
US8735564B2 (en) 2008-10-27 2014-05-27 Qiagen Gaithersburg, Inc. Fast results hybrid capture assay and system
US8288520B2 (en) 2008-10-27 2012-10-16 Qiagen Gaithersburg, Inc. Fast results hybrid capture assay and system
US9797000B2 (en) 2009-05-01 2017-10-24 Qiagen Gaithersburg Inc. Non-target amplification method for detection of RNA splice-forms in a sample
US9410146B2 (en) 2009-09-14 2016-08-09 Qiagen Gaithersburg Inc. Compositions and methods for recovery of nucleic acids or proteins from tissue samples fixed in cytology media
US9689047B2 (en) 2010-01-29 2017-06-27 Qiagen Gaithersburg Inc. Methods and compositions for sequence-specific purification and multiplex analysis of nucleic acids
US9605303B2 (en) 2010-01-29 2017-03-28 Qiagen Gaithersburg, Inc. Method of determining and confirming the presence of an HPV in a sample
US9422593B2 (en) 2010-05-19 2016-08-23 Qiagen Gaithresburg, Inc Methods and compositions for sequence-specific purification and multiplex analysis of nucleic acids
US9376727B2 (en) 2010-05-25 2016-06-28 Qiagen Gaithersburg, Inc. Fast results hybrid capture assay and associated strategically truncated probes
US9885092B2 (en) 2011-02-24 2018-02-06 Qiagen Gaithersburg Inc. Materials and methods for detection of HPV nucleic acids
WO2018133008A1 (fr) * 2017-01-19 2018-07-26 Yantai Ausbio Laboratories Co., Ltd. Système, procédé et porte-échantillon pour dosage
CN110268271A (zh) * 2017-01-19 2019-09-20 烟台澳斯邦生物工程有限公司 用于测定的系统、方法和样品载体
JP2020505597A (ja) * 2017-01-19 2020-02-20 ヤンタイ・アウスビオ・ラボラトリーズ・カンパニー・リミテッド アッセイのためのシステム、方法およびサンプルキャリア
US11426733B2 (en) 2017-01-19 2022-08-30 Yantai Ausbio Laboratories Co., Ltd. System, method and sample carrier for assaying
WO2019169395A1 (fr) * 2018-03-02 2019-09-06 uBiome, Inc. Procédé et système de manipulation de particules à haut débit en utilisant des champs magnétiques et dispositif
US11634703B2 (en) 2018-03-02 2023-04-25 Psomagen, Inc. Method and system for high-throughput particle handling by use of magnetic fields and device

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