WO1997000446A1 - Immunoassay and kit with two reagents that are cross-linked if they adhere to an analyte - Google Patents

Immunoassay and kit with two reagents that are cross-linked if they adhere to an analyte Download PDF

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Publication number
WO1997000446A1
WO1997000446A1 PCT/SE1996/000779 SE9600779W WO9700446A1 WO 1997000446 A1 WO1997000446 A1 WO 1997000446A1 SE 9600779 W SE9600779 W SE 9600779W WO 9700446 A1 WO9700446 A1 WO 9700446A1
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WO
WIPO (PCT)
Prior art keywords
antigen
oligonucleotides
specific
antibody
antibodies
Prior art date
Application number
PCT/SE1996/000779
Other languages
French (fr)
Inventor
Ulf Landegren
Original Assignee
Ulf Landegren
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Ulf Landegren filed Critical Ulf Landegren
Priority to JP50299197A priority Critical patent/JP4091113B2/en
Priority to CA002224674A priority patent/CA2224674C/en
Priority to DE69614539T priority patent/DE69614539T2/en
Priority to AU61439/96A priority patent/AU702125B2/en
Priority to EP96918976A priority patent/EP0832431B1/en
Priority to US08/981,310 priority patent/US6878515B1/en
Publication of WO1997000446A1 publication Critical patent/WO1997000446A1/en
Priority to US11/011,438 priority patent/US20050233351A1/en

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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
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • 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
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/682—Signal amplification
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/81—Packaged device or kit
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/975—Kit

Definitions

  • the present invention relates to ultrasensitive immunoassays. More specifically, it relates to immunological test kits and processes for immunological detection of a specific antigen. In the present invention, the fields of immunology and molecular genetics are combined.
  • Immunoassays represent powerful tools to identify a very wide range of compounds, such as antigens and antibodies. Examples of immunoassays are ELISA (enzyme linked immunosorbent assay), EIA (enzyme immunoassay), and RIA (radio immunoassay). Common to all these immunoassays, is that detection sensitivity is limited by the affinity of typical antibodies.
  • the labeled DNA-antibody complexes are assembled in situ during the assay. This can create variable stoichiometry in the assembly of the components and in the attachment of the DNA label. Moreover, extra steps are required for addition of biotinylated reagents and binding proteins. Numerous wash steps are also needed to remove excess reagents and to free assay components of non-specifically bound reagents.
  • WO 91/17442 describes a molecular probe for use as a signal amplifier in immunoassays for detecting i.a. antigens.
  • the probe comprises an antibody, a double stranded polynucleotide functioning as a promoter for a DNA dependend RNA polymerase, and a single or double stranded template for the promoter.
  • the transcription product is quantifi ⁇ ed and correlated to the amount of present antigen in a sample.
  • oligonucleoti ⁇ des attached to antibodies having bound antigen and oligo ⁇ nucleotides attached to antibodies not having bound antigen, i.e. those being non-specifically trapped.
  • Non-specifically trapped antibodies give rise to an undesired background signal and limits the minimun number of antigen molecules that can be detected and it will not be possible to distinguish between false positive and true positive results below a certain number of antigen molecu ⁇ les.
  • solid supports such as microtiter plates, are used for the reactions. According to prior art, there will always be an excess of oligonucleotide-labeled antibody that cannot be removed from the solid support by adding background-lowering agents and by repeated wash steps.
  • the present invention enables detection of extremely low numbers of antigenic molecules, even down to a single molecule.
  • the invention provides reliable immunoassays in situations where insufficient numbers of antigens are available for conventional assays.
  • an immunological test kit comprising a first immobilized reagent having affinity for a specific macromolecule, such as a protein. Furthermore, the test kit comprises a second and a third affinity reagent specific for different determinantes of said macro ⁇ molecule, and modified with crosslinkable compounds enabling a) conjugation of said second and third affinity reagent only when both are bound to the said, same macromolecule, and b) detection by amplification.
  • the affinity reagents are antibodies and the crosslinkable compounds are oligonucleotide extensions attached to the second and third antibody, respectively.
  • the macromolecule is in this case a specific antigen.
  • an immunoassay for detection of a specific antigen comprising the following steps: a) contacting a sample suspected of containing said specific antigen with a first antibody linked to a solid support, said first antibody being specific for a first epitope on the antigen, b) washing off excess reagents, c) incubating with a solution of a second and a third antibody specific for a second and third epitope of said antigen, and modified with crosslinkable oligonucleotides enabling conjugation of said second and third antibody when both are bound to the said, same antigen, d) washing off excess reagents, e) amplifying said crosslinked oligonucleotides, and f) detecting the amplified products.
  • Products from the amplification reaction only result when two antibodies, i.e. the second and the third, have bound to the same antigen. Thus, amplification is specific for antibodies having bound to antigen. Non-specifically trapped antibodies do not give rise to any signal.
  • Fig. 1 is a schematic view of the principles of the immunoassay according to the invention.
  • Fig.2 shows chemical coupling of amino-modified oligonucleotides to macromolecules.
  • Fig. 1 there is shown an immobilized antibody to a specific antigen applied together with two other antibodies, specific for other determinants on the same antigen.
  • antibodies other specifically interacting species with a known affinity, such as lectins, receptors, single chain antibodies, cofactors, oligo ⁇ nucleotides and other non-proteins, can be used in the invention.
  • the interacting species are modified with crosslinkable compounds in the form of an interacting pair, preferably short oligo ⁇ nucleotide extentions.
  • oligonucleotides of neighbouring antibodies are conjugated to each other.
  • the conjugation may or may not necessitate an enzymatic ligation step depending on the orientation of the oligonucleotide extensions.
  • ligation is necessary, such as by T4 RNA ligase or T4 DNA ligase.
  • T4 RNA ligase T4 DNA ligase.
  • conjugation is between free 3' ends these have to be designed to be mutually complementary to achieve base pairing and initiation of DNA synthesis extending the 3' ends of the the molecules.
  • oligo ⁇ nucleotide extension there is shown a suitable way to attach the oligo ⁇ nucleotide extension to the antibodies.
  • the oligonucle ⁇ otides are terminally amino-modified and then attached to primary amines on the antibodies via disulphide bonds, e.g. according to the technique of Chue and Orgel, Nucleic Acid Research, Vol. 16, No. 9, 1988.
  • Another way is by direct covalent coupling as described by Hendrickson et el. , supra.
  • the antibodies used in the invention can be polyclonal, mono ⁇ clonal or single chain antibodies produced by bacteriophages. In the latter case, it is possible to have antibodies equipped with an oligonucleotide binding part, rendering the above coupling step between antibody and oligonucleotide unnecessary.
  • the amplification technique to obtain detectable products is, for example, PCR (Polymerase Chain Reaction), LCR (Ligase Chain Reaction), SDA (Strand Displacement Amplification) bacteriophage Q ⁇ replication, and 3SR (Self-Sustained Synthetic Reaction), of which the latter three methods do not require temperature cycling.
  • the method for detecting amplified products can, for example, be direct incorporation of a label, such as radioisotopes, fluoro ⁇ chromes, and enzymes, into the amplified products with the use of label-conjugated primers or nucleotides.
  • a label such as radioisotopes, fluoro ⁇ chromes, and enzymes
  • the accumulation of amplified products is monitored via the fluore ⁇ scence from intercalating dyes, such as propidium iodide, etidium bromide and SYBRTM green from Molecular Probes.
  • the invention is not restricted to detection of any special kind of macromolecule, such as an antigen; the only criterion it has to fulfil is that it must be able to simultaneously bind three antibodies/affinity reagents.
  • the affinity reagents are antibodies
  • the three antibodies are specific for different epitopes on the antigen.
  • macromolecules examples are human myoglobin and human growth hormone. Ultrasensitive assays for growth hormone will have significant value in clinical situations where hormone levels are undetectable by prior art assays.
  • Immunoglobulins were modified in a reaction with SPDP (3-(-pyri- dyldithio)propionic acid N-hydroxysuccinimide ester, from Pharmacia Biotech) according to the manufacturer's suggestions. Oligonucleotides were thiolated, either through the addition of a suitable phosphoramidite according to Connolly (Connolly BA, Nucl. Acid. Res. 1987 15:3131), or 3'aminomodified oligonucleo- tides were reacted with SPDP, followed by reduction of the dithiopyridyl bond, using dithiothreitol.
  • SPDP 3-(-pyri- dyldithio)propionic acid N-hydroxysuccinimide ester
  • SPDP-modified antibodies were incubated with three equivalents of SH-containing oligonucleotides at 4°C over night.
  • the reaction mixture was separated using a Zorbax HPLC gel filtration column. Residual free antibody were removed from the isolated conjugate by ion exchange MonoQ FPLC separation.
  • Oligo 1 5'Tr S C3-ATA GAC TGA GCG TGG ACA TTA ATA TGT ACG TAC GCT TAA TTG AGT 3' and Oligo 2: 5'P ATG TAC GAC CCG TAG ATA TTA TCA TAC TGG CAT GGG CAT GAT GAA CAT C-NHSPDP T3*
  • the immune test was performed by first binding l ⁇ g of biotinyla ⁇ ted antibody (#1) to individual streptavidin-coated prongs on a manifold support. [Parik et al., Anal. Biochem; (1993) 211: 144- 150B] . After washes using PBS (phosfhate buffered saline) with 0.5% Tween 20, the prongs were lowered into solutions of antigen (myoglobin) at variable concentrations. After further washes, the supports with bound antigen were incubated in a solution of two oligonucleotide-conjugated antibodies #2 and #3 at 5 ng each per reaction.
  • PBS phosfhate buffered saline
  • antigen myoglobin
  • the supports were washed, an oligonucleotide complemen ⁇ tary to the free ends of the antibody-conjugated oligonucleotides was added (4 pmol per reaction, 5'CTA CGG GTC GTA CAT ACT CAA TTA AGC GTA 3' ), and the ends of oligonucleotides on nearby anti ⁇ bodies were joined covalently by ligation at 37°C for 30 min using 1 U of T4 DNA ligase.
  • the supports were then washed in a standard PCR buffer, and the supports were added as templates in a PCR mix, including two primers specific for sequences located at either side of the ligation junction (5'TTA ATG GCG AG 3' ) and Taq polymerase. After two cycles, the supports were removed and the amplification was continued for 26 more cycles. Amplification products were examined by separation in an agarose gel and ethidium bromide staining.

Abstract

The present invention relates to an immunological test kit and immunoassay using a first immobilized antibody having affinity for a specific antigen. The invention is characterized by a second and third antibody being specific for different determinants of the antigen and modified with cross-linkable oligonucleotides. For detection, the oligonucleotides are amplified, whereby only such oligonucleotides will be amplified which have been cross-linked to each other. In this way unspecific background is avoided and detection is possible down to single molecules.

Description

IMMUNOASSAY AND KIT WITH TWO REAGENTS THAT ARE CROSS-LINKED IF THEY
ADHERETOANANALYTE
Technical field
The present invention relates to ultrasensitive immunoassays. More specifically, it relates to immunological test kits and processes for immunological detection of a specific antigen. In the present invention, the fields of immunology and molecular genetics are combined.
Background of the invention
Immunoassays represent powerful tools to identify a very wide range of compounds, such as antigens and antibodies. Examples of immunoassays are ELISA (enzyme linked immunosorbent assay), EIA (enzyme immunoassay), and RIA (radio immunoassay). Common to all these immunoassays, is that detection sensitivity is limited by the affinity of typical antibodies.
With the prior art immunoassays, detection is not possible below a certain number of molecules, because the background, i.e. unspecifically bound material, interferes with the results. Detection of very low numbers of antigen is becoming increasingly important, especially for diagnostic applications. Therefore, further developments in sensitivity as well as specificity of immunological assays are desired.
Cantor et al, Science, Vol. 258, 2 Oct. 1992, have previously reported the attachment of oligonucleotides to antibodies in order to permit detection of such antibodies having bound antigen in immune reactions. A streptavidin-protein A chimera that posseses tight and specific binding affinity for both biotin and immunoglobulin G was used to attach biotinylated DNA specifically to antigen-monoclonal antibody complexes that had been immobili¬ zed on microtiter plate wells. Then, a segment of the attached DNA was amplified by PCR (Polymerase Chain Reaction). Analysis of the PCR products by agarose gel electrophoresis after staining with ethidium bromide allowed detection of 580 antigen molecules (9,6 x IO'22 moles) which is a significant improvement compared to, for example, conventional ELISA.
However, in Cantor et al., the labeled DNA-antibody complexes are assembled in situ during the assay. This can create variable stoichiometry in the assembly of the components and in the attachment of the DNA label. Moreover, extra steps are required for addition of biotinylated reagents and binding proteins. Numerous wash steps are also needed to remove excess reagents and to free assay components of non-specifically bound reagents.
Hendrickson et al., Nucleic Acids Research, 1995, Vol 23, No.3, report an advancement of the Cantor et al. assay that reduces complexity. This is achieved through labeling antibody with DNA by direct covalent linkage of the DNA to the antibody. In this approach, the analyte specific antibody and the 5' amino modified DNA oligonucleotide are independently activated by means of separate heterobifunctional cross-linking agents. The activated antibody and DNA label are then coupled in a single spontaneous reaction.
International patent publication no. WO 91/17442 describes a molecular probe for use as a signal amplifier in immunoassays for detecting i.a. antigens. The probe comprises an antibody, a double stranded polynucleotide functioning as a promoter for a DNA dependend RNA polymerase, and a single or double stranded template for the promoter. The transcription product is quantifi¬ ed and correlated to the amount of present antigen in a sample.
However, in all three of the above described immunoassays the attached DNA is only used as a marker by being amplified to detectable levels. There is no distinction between oligonucleoti¬ des attached to antibodies having bound antigen and oligo¬ nucleotides attached to antibodies not having bound antigen, i.e. those being non-specifically trapped. Non-specifically trapped antibodies give rise to an undesired background signal and limits the minimun number of antigen molecules that can be detected and it will not be possible to distinguish between false positive and true positive results below a certain number of antigen molecu¬ les. Commonly, solid supports such as microtiter plates, are used for the reactions. According to prior art, there will always be an excess of oligonucleotide-labeled antibody that cannot be removed from the solid support by adding background-lowering agents and by repeated wash steps.
Suπtmary of the invention
The present invention enables detection of extremely low numbers of antigenic molecules, even down to a single molecule. The invention provides reliable immunoassays in situations where insufficient numbers of antigens are available for conventional assays.
According to a first aspect of the invention, there is provided an immunological test kit comprising a first immobilized reagent having affinity for a specific macromolecule, such as a protein. Furthermore, the test kit comprises a second and a third affinity reagent specific for different determinantes of said macro¬ molecule, and modified with crosslinkable compounds enabling a) conjugation of said second and third affinity reagent only when both are bound to the said, same macromolecule, and b) detection by amplification.
According to a preferred embodiment of the invention, the affinity reagents are antibodies and the crosslinkable compounds are oligonucleotide extensions attached to the second and third antibody, respectively. The macromolecule is in this case a specific antigen.
According to a second aspect of the invention there is provided an immunoassay for detection of a specific antigen, comprising the following steps: a) contacting a sample suspected of containing said specific antigen with a first antibody linked to a solid support, said first antibody being specific for a first epitope on the antigen, b) washing off excess reagents, c) incubating with a solution of a second and a third antibody specific for a second and third epitope of said antigen, and modified with crosslinkable oligonucleotides enabling conjugation of said second and third antibody when both are bound to the said, same antigen, d) washing off excess reagents, e) amplifying said crosslinked oligonucleotides, and f) detecting the amplified products.
Products from the amplification reaction only result when two antibodies, i.e. the second and the third, have bound to the same antigen. Thus, amplification is specific for antibodies having bound to antigen. Non-specifically trapped antibodies do not give rise to any signal.
Detailed description of the invention
The present invention will be described more detailed below with reference to the accompanying drawings, in which
Fig. 1 is a schematic view of the principles of the immunoassay according to the invention, and
Fig.2 shows chemical coupling of amino-modified oligonucleotides to macromolecules.
In Fig. 1 there is shown an immobilized antibody to a specific antigen applied together with two other antibodies, specific for other determinants on the same antigen. Besides antibodies other specifically interacting species with a known affinity, such as lectins, receptors, single chain antibodies, cofactors, oligo¬ nucleotides and other non-proteins, can be used in the invention.
The interacting species are modified with crosslinkable compounds in the form of an interacting pair, preferably short oligo¬ nucleotide extentions. Upon the coordinated binding of several so modified antibodies, oligonucleotides of neighbouring antibodies are conjugated to each other. The conjugation may or may not necessitate an enzymatic ligation step depending on the orientation of the oligonucleotide extensions.
If the conjugation is between free 3' and 5' ends ligation is necessary, such as by T4 RNA ligase or T4 DNA ligase. To facilitate the conjugation, it is convenient to use a stretch of oligonucleotides base pairing to and, thereby, juxtaposing the free ends of the oligonucleotides and permitting their joining through ligation.
If the conjugation is between free 3' ends these have to be designed to be mutually complementary to achieve base pairing and initiation of DNA synthesis extending the 3' ends of the the molecules.
Thus, only in those cases where the antibodies are brought close enough through binding to the same antigen molecule can the oligonucleotides be ligated. Ligated molecules subsequently serve as templates for nucleic acid amplification reactions.
In Fig. 2, there is shown a suitable way to attach the oligo¬ nucleotide extension to the antibodies. First, the oligonucle¬ otides are terminally amino-modified and then attached to primary amines on the antibodies via disulphide bonds, e.g. according to the technique of Chue and Orgel, Nucleic Acid Research, Vol. 16, No. 9, 1988. Another way is by direct covalent coupling as described by Hendrickson et el. , supra.
The antibodies used in the invention can be polyclonal, mono¬ clonal or single chain antibodies produced by bacteriophages. In the latter case, it is possible to have antibodies equipped with an oligonucleotide binding part, rendering the above coupling step between antibody and oligonucleotide unnecessary.
The amplification technique to obtain detectable products is, for example, PCR (Polymerase Chain Reaction), LCR (Ligase Chain Reaction), SDA (Strand Displacement Amplification) bacteriophage Qβ replication, and 3SR (Self-Sustained Synthetic Reaction), of which the latter three methods do not require temperature cycling.
The method for detecting amplified products can, for example, be direct incorporation of a label, such as radioisotopes, fluoro¬ chromes, and enzymes, into the amplified products with the use of label-conjugated primers or nucleotides. Preferably, the accumulation of amplified products is monitored via the fluore¬ scence from intercalating dyes, such as propidium iodide, etidium bromide and SYBR™ green from Molecular Probes.
The invention is not restricted to detection of any special kind of macromolecule, such as an antigen; the only criterion it has to fulfil is that it must be able to simultaneously bind three antibodies/affinity reagents. In the case where the affinity reagents are antibodies, the three antibodies are specific for different epitopes on the antigen. By biosensor analysis, it is possible to assure that the antibodies do not bind to overlapping epitopes on the antigen.
Examples of macromolecules are human myoglobin and human growth hormone. Ultrasensitive assays for growth hormone will have significant value in clinical situations where hormone levels are undetectable by prior art assays.
The invention will now be described below in a non-limiting Example.
EXAMPLE
Immunoglobulins were modified in a reaction with SPDP (3-(-pyri- dyldithio)propionic acid N-hydroxysuccinimide ester, from Pharmacia Biotech) according to the manufacturer's suggestions. Oligonucleotides were thiolated, either through the addition of a suitable phosphoramidite according to Connolly (Connolly BA, Nucl. Acid. Res. 1987 15:3131), or 3'aminomodified oligonucleo- tides were reacted with SPDP, followed by reduction of the dithiopyridyl bond, using dithiothreitol.
SPDP-modified antibodies were incubated with three equivalents of SH-containing oligonucleotides at 4°C over night. The reaction mixture was separated using a Zorbax HPLC gel filtration column. Residual free antibody were removed from the isolated conjugate by ion exchange MonoQ FPLC separation.
The two oligonucleotides used to conjugate the antibodies were Oligo 1: 5'Tr S C3-ATA GAC TGA GCG TGG ACA TTA ATA TGT ACG TAC GCT TAA TTG AGT 3' and Oligo 2: 5'P ATG TAC GAC CCG TAG ATA TTA TCA TAC TGG CAT GGG CAT GAT GAA CAT C-NHSPDP T3*
The immune test was performed by first binding lμg of biotinyla¬ ted antibody (#1) to individual streptavidin-coated prongs on a manifold support. [Parik et al., Anal. Biochem; (1993) 211: 144- 150B] . After washes using PBS (phosfhate buffered saline) with 0.5% Tween 20, the prongs were lowered into solutions of antigen (myoglobin) at variable concentrations. After further washes, the supports with bound antigen were incubated in a solution of two oligonucleotide-conjugated antibodies #2 and #3 at 5 ng each per reaction. The supports were washed, an oligonucleotide complemen¬ tary to the free ends of the antibody-conjugated oligonucleotides was added (4 pmol per reaction, 5'CTA CGG GTC GTA CAT ACT CAA TTA AGC GTA 3' ), and the ends of oligonucleotides on nearby anti¬ bodies were joined covalently by ligation at 37°C for 30 min using 1 U of T4 DNA ligase. The supports were then washed in a standard PCR buffer, and the supports were added as templates in a PCR mix, including two primers specific for sequences located at either side of the ligation junction (5'TTA ATG GCG AG 3' ) and Taq polymerase. After two cycles, the supports were removed and the amplification was continued for 26 more cycles. Amplification products were examined by separation in an agarose gel and ethidium bromide staining.

Claims

1. An immunological test kit comprising a first immobilized reagent having affinity to a specific macromolecule, charac¬ terized in a second and a third affinity reagent specific for different determinantes of said macromolecule, and modified with crosslinkable oligonucleotides.
2. An immunological test kit according to claim 1, charac¬ terized in that the affinity reagents are antibodies, and that the macromolecule is a specific antigen.
3. An immunological test kit according to claim 1, charac¬ terized in that the affinity reagents are lectins, receptors, single chain antibodies, cofactors and nucleic acids.
4. An immunological test kit according to any one of the claims 1-3, characterized in that the oligonucleotides are complementary to each other.
5. An immunological test kit according to any of the claims 1- 4, characterized in that it further comprises a ligase.
6. An immunoassay for detection of a specific antigen, characterized in: a) contacting a sample suspected of containing• said specific antigen with a first antibody linked to a solid support, said first antibody being specific for a first epitope on the antigen, b) washing off excess reagents, c) incubating with a solution of a second and a third antibody specific for a second and third epitope of said antigen, and modifiedwithcrosslinkable oligonucleotides enabling conjugation of said second and third antibody when both are bound to the said, same antigen, d) washing off excess reagents, e) amplifying said crosslinked oligonucleotides, and f) detecting the amplified products.
7. An immunoassay according to claim 6, characterized in that a ligase is added before step d).
8. An immunoassay according to claim 6 or 7, characterized in that an oligonucleotide complementary to the crosslinkable oligonucleotides is added before step d).
PCT/SE1996/000779 1995-06-16 1996-06-14 Immunoassay and kit with two reagents that are cross-linked if they adhere to an analyte WO1997000446A1 (en)

Priority Applications (7)

Application Number Priority Date Filing Date Title
JP50299197A JP4091113B2 (en) 1995-06-16 1996-06-14 Immunoassay and kit with two reagents that crosslink when attached to an analyte
CA002224674A CA2224674C (en) 1995-06-16 1996-06-14 Immunoassay and kit with two reagents that are cross-linked if they adhere to an analyte
DE69614539T DE69614539T2 (en) 1995-06-16 1996-06-14 IMMUNOASSAY AND KIT WITH TWO REAGENTS TO BE NETWORKED WHEN ADHESIVE TO ANALYT
AU61439/96A AU702125B2 (en) 1995-06-16 1996-06-14 Immunoassay and kit with two reagents that are cross-linked if they adhere to an analyte
EP96918976A EP0832431B1 (en) 1995-06-16 1996-06-14 Immunoassay and kit with two reagents that are cross-linked if they adhere to an analyte
US08/981,310 US6878515B1 (en) 1995-06-16 1996-06-14 Ultrasensitive immunoassays
US11/011,438 US20050233351A1 (en) 1995-06-16 2004-12-15 Ultrasensitive immunoassays

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE9502196A SE504798C2 (en) 1995-06-16 1995-06-16 Immunoassay and test kits with two reagents that can be cross-linked if adsorbed to the analyte
SE9502196-0 1995-06-16

Related Child Applications (1)

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US11/011,438 Continuation US20050233351A1 (en) 1995-06-16 2004-12-15 Ultrasensitive immunoassays

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EP (1) EP0832431B1 (en)
JP (1) JP4091113B2 (en)
AU (1) AU702125B2 (en)
CA (1) CA2224674C (en)
DE (1) DE69614539T2 (en)
SE (1) SE504798C2 (en)
WO (1) WO1997000446A1 (en)

Cited By (33)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999063109A1 (en) * 1998-06-04 1999-12-09 Baxter Aktiengesellschaft Method for determining antigens
WO2001061037A1 (en) * 2000-02-18 2001-08-23 Ulf Landegren Methods and kits for proximity probing
WO2003029489A1 (en) * 2001-10-03 2003-04-10 Iseao Technologies Limited Methods for detection of target molecules and molecular interactions
WO2005123963A3 (en) * 2004-06-14 2006-06-08 Univ Leland Stanford Junior Methods and compositions for use in analyte detection using proximity probes
US7074564B2 (en) * 1998-03-25 2006-07-11 Ulf Landegren Rolling circle replication of padlock probes
EP1704256A2 (en) * 2004-01-13 2006-09-27 U.S. Genomics, Inc. Detection and quantification of analytes in solution using polymers
WO2007107743A1 (en) * 2006-03-20 2007-09-27 Olink Ab Method for analyte detection using proximity probes
EP1842226A2 (en) * 2004-11-03 2007-10-10 Iris Molecular Diagnostics, Inc. Homogeneous analyte detection
US7306904B2 (en) 2000-02-18 2007-12-11 Olink Ab Methods and kits for proximity probing
US7402399B2 (en) 2003-10-14 2008-07-22 Monogram Biosciences, Inc. Receptor tyrosine kinase signaling pathway analysis for diagnosis and therapy
US7595160B2 (en) 2004-01-13 2009-09-29 U.S. Genomics, Inc. Analyte detection using barcoded polymers
US7648828B2 (en) 2003-04-01 2010-01-19 Monogram Biosciences, Inc. Methods for detecting receptor complexes comprising PI3K
US7790388B2 (en) 2001-08-03 2010-09-07 Olink Ab Nucleic acid amplification method
US7795009B2 (en) * 2005-06-15 2010-09-14 Saint Louis University Three-component biosensors for detecting macromolecules and other analytes
US7811809B2 (en) 2005-06-15 2010-10-12 Saint Louis University Molecular biosensors for use in competition assays
US7932060B2 (en) 2003-04-18 2011-04-26 Becton, Dickinson And Company Immuno-amplification
US7939313B2 (en) 2003-12-12 2011-05-10 Saint Louis University Biosensors for detecting macromolecules and other analytes
EP2369015A1 (en) 2010-03-15 2011-09-28 Olink AB Assay for localised detection of analytes
WO2012007511A1 (en) 2010-07-15 2012-01-19 Olink Ab Blocking reagent and methods for the use thereof
WO2012049316A1 (en) 2010-10-15 2012-04-19 Olink Ab Dynamic range methods
WO2012152942A1 (en) 2011-05-11 2012-11-15 Olink Ab Unfolding proximity probes and methods for the use thereof
US8513032B2 (en) 2004-11-03 2013-08-20 Iris International, Inc. Microbubbles for affinity separation
US8632994B2 (en) 2003-12-01 2014-01-21 Dako Denmark A/S Methods and compositions for immuno-histochemical detection
WO2014076214A1 (en) 2012-11-14 2014-05-22 Olink Ab Rca reporter probes and their use in detecting nucleic acid molecules
US8945840B2 (en) 2005-06-10 2015-02-03 Saint Louis University Methods for the selection of aptamers
US8956857B2 (en) * 2005-06-06 2015-02-17 Mediomics, Llc Three-component biosensors for detecting macromolecules and other analytes
US8993245B2 (en) 2008-11-21 2015-03-31 Mediomics, Llc Biosensor for detecting multiple epitopes on a target
US9040287B2 (en) 2010-02-12 2015-05-26 Mediomics, Llc Molecular biosensors capable of signal amplification
US9777315B2 (en) 2011-01-31 2017-10-03 Olink Proteomics Ab Exonuclease enabled proximity extension assays
US10274484B2 (en) 2014-09-12 2019-04-30 Mediomics Llc Molecular biosensors with a modular design
US10597701B2 (en) 2011-05-11 2020-03-24 Navinci Diagnostics Ab Unfolding proximity probes and methods for the use thereof
US11034995B2 (en) 2014-02-04 2021-06-15 Navinci Diagnostics Ab Proximity assay with detection based on hybridisation chain reaction (HCR)
US11649486B2 (en) 2016-08-16 2023-05-16 Navinci Diagnostics Ab Double-stranded circle probes

Families Citing this family (49)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SE504798C2 (en) * 1995-06-16 1997-04-28 Ulf Landegren Immunoassay and test kits with two reagents that can be cross-linked if adsorbed to the analyte
US20030109067A1 (en) 2001-12-06 2003-06-12 Immunetech, Inc. Homogeneous immunoassays for multiple allergens
US20040018577A1 (en) * 2002-07-29 2004-01-29 Emerson Campbell John Lewis Multiple hybrid immunoassay
WO2005074417A2 (en) * 2003-09-03 2005-08-18 Salk Institute For Biological Studies Multiple antigen detection assays and reagents
EP1756307A1 (en) * 2004-05-20 2007-02-28 Trillion Genomics Limited Use of mass labelled probes to detect target nucleic acids using mass spectrometry
US20060204999A1 (en) * 2005-03-14 2006-09-14 Stephen Macevicz Detecting molecular complexes
US7883848B2 (en) * 2005-07-08 2011-02-08 Olink Ab Regulation analysis by cis reactivity, RACR
US8778846B2 (en) * 2006-12-04 2014-07-15 General Electric Company Composition, device and associated method
US8133701B2 (en) * 2006-12-05 2012-03-13 Sequenom, Inc. Detection and quantification of biomolecules using mass spectrometry
US7902345B2 (en) 2006-12-05 2011-03-08 Sequenom, Inc. Detection and quantification of biomolecules using mass spectrometry
WO2009032781A2 (en) 2007-08-29 2009-03-12 Sequenom, Inc. Methods and compositions for universal size-specific polymerase chain reaction
EP2408936A4 (en) * 2009-03-18 2013-01-30 Sequenom Inc Use of thermostable endonucleases for generating reporter molecules
WO2010127186A1 (en) 2009-04-30 2010-11-04 Prognosys Biosciences, Inc. Nucleic acid constructs and methods of use
US10787701B2 (en) 2010-04-05 2020-09-29 Prognosys Biosciences, Inc. Spatially encoded biological assays
US20190300945A1 (en) 2010-04-05 2019-10-03 Prognosys Biosciences, Inc. Spatially Encoded Biological Assays
CA2794522C (en) 2010-04-05 2019-11-26 Prognosys Biosciences, Inc. Spatially encoded biological assays
US20130323729A1 (en) * 2010-10-29 2013-12-05 Olink Ab Proximity Ligation Technology for Western Blot Applications
JP5766296B2 (en) 2010-12-23 2015-08-19 エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ Polypeptide-polynucleotide complexes and their use in targeted delivery of effector components
EP2694709B1 (en) 2011-04-08 2016-09-14 Prognosys Biosciences, Inc. Peptide constructs and assay systems
GB201106254D0 (en) 2011-04-13 2011-05-25 Frisen Jonas Method and product
GB201108678D0 (en) 2011-05-24 2011-07-06 Olink Ab Multiplexed proximity ligation assay
CN103454427A (en) * 2012-06-03 2013-12-18 河北省健海生物芯片技术有限责任公司 Method for detecting trace protein in blood by utilizing antibody-nucleic acid combined amplification technology
CA2871386A1 (en) * 2012-06-27 2014-01-03 F. Hoffmann-La Roche Ag Method for the selection and production of tailor-made, selective and multi-specific therapeutic molecules comprising at least two different targeting entities and uses thereof
CA2871880A1 (en) 2012-06-27 2014-01-03 F. Hoffmann-La Roche Ag Method for selection and production of tailor-made highly selective and multi-specific targeting entities containing at least two different binding entities and uses thereof
BR112014028368A2 (en) 2012-06-27 2017-11-14 Hoffmann La Roche method of producing antibody fc region conjugate, antibody fc region conjugate and pharmaceutical formulation
RU2646159C2 (en) 2012-09-14 2018-03-01 Ф. Хоффманн-Ля Рош Аг Method for production and selection of molecules including, at least two different groups, and application thereof
JP6408552B2 (en) * 2013-03-13 2018-10-17 メソ スケール テクノロジーズ エルエルシー Improved assay method
US10114015B2 (en) 2013-03-13 2018-10-30 Meso Scale Technologies, Llc. Assay methods
WO2014160036A1 (en) 2013-03-14 2014-10-02 The Regents Of The University Of California Nanopipette device and method for subcellular analysis
US10202500B2 (en) 2013-03-15 2019-02-12 Lubrizol Advanced Materials, Inc. Heavy metal free CPVC compounds
CN105358984B (en) 2013-03-15 2020-02-18 普罗格诺西斯生物科学公司 Methods for detecting peptide/MHC/TCR binding
US9593364B2 (en) 2013-05-21 2017-03-14 Src, Inc. Detecting a target molecule in a sample using a dual-antibody quantitative fluorescence-based detection method
US9879313B2 (en) 2013-06-25 2018-01-30 Prognosys Biosciences, Inc. Methods and systems for determining spatial patterns of biological targets in a sample
WO2014210376A1 (en) * 2013-06-27 2014-12-31 The Board Of Trustees Of The Leland Stanford Junior University Dna-conjugated antibodies for improved antibody affinity and reduced antibody cross reactivity
WO2015070037A2 (en) 2013-11-08 2015-05-14 Prognosys Biosciences, Inc. Polynucleotide conjugates and methods for analyte detection
US9957554B1 (en) 2013-12-19 2018-05-01 National Technology & Engineering Solutions Of Sandia, Llc Microfluidic platform for multiplexed detection in single cells and methods thereof
WO2015175856A1 (en) 2014-05-15 2015-11-19 Meso Scale Technologies, Llc. Improved assay methods
PL3227332T3 (en) 2014-12-03 2020-06-15 F. Hoffmann-La Roche Ag Multispecific antibodies
EP4321627A3 (en) 2015-04-10 2024-04-17 10x Genomics Sweden AB Spatially distinguished, multiplex nucleic acid analysis of biological specimens
BR112017022329A2 (en) * 2015-04-17 2018-07-17 Univ California methods for detecting agglutination and compositions for use in practice thereof
CA2999888C (en) 2015-09-24 2024-04-09 Abvitro Llc Affinity-oligonucleotide conjugates and uses thereof
JP6929354B2 (en) 2016-09-24 2021-09-01 アブビトロ， エルエルシー Affinity-oligonucleotide conjugates and their use
GB201700567D0 (en) 2017-01-12 2017-03-01 Genagon Therapeutics Ab Therapeutic agents
WO2019238944A1 (en) 2018-06-15 2019-12-19 Olink Proteomics Ab Biomarker panel for ovarian cancer
CN114144422A (en) * 2019-03-01 2022-03-04 中尺度技术有限责任公司 Electrochemiluminescence labeled probes for use in immunization methods, methods of using such probes, and kits comprising such probes
EP4025692A2 (en) 2020-06-02 2022-07-13 10X Genomics, Inc. Nucleic acid library methods
CN116249785A (en) 2020-06-02 2023-06-09 10X基因组学有限公司 Space transcriptomics for antigen-receptor
EP4162074B1 (en) 2020-06-08 2024-04-24 10X Genomics, Inc. Methods of determining a surgical margin and methods of use thereof
EP4347865A1 (en) 2021-05-25 2024-04-10 Cavidi AB Method for sensitive analyte detection assays and kits therefor

Family Cites Families (40)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2079172A (en) * 1934-02-28 1937-05-04 Chandler & Price Co Platen printing press
US4748111A (en) * 1984-03-12 1988-05-31 Molecular Diagnostics, Inc. Nucleic acid-protein conjugate used in immunoassay
US4690890A (en) * 1984-04-04 1987-09-01 Cetus Corporation Process for simultaneously detecting multiple antigens using dual sandwich immunometric assay
US5011771A (en) * 1984-04-12 1991-04-30 The General Hospital Corporation Multiepitopic immunometric assay
US4563417A (en) * 1984-08-31 1986-01-07 Miles Laboratories, Inc. Nucleic acid hybridization assay employing antibodies to intercalation complexes
US5242794A (en) * 1984-12-13 1993-09-07 Applied Biosystems, Inc. Detection of specific sequences in nucleic acids
US4824775A (en) * 1985-01-03 1989-04-25 Molecular Diagnostics, Inc. Cells labeled with multiple Fluorophores bound to a nucleic acid carrier
US5026653A (en) * 1985-04-02 1991-06-25 Leeco Diagnostic, Inc. Scavenger antibody mixture and its use for immunometric assay
US5656731A (en) * 1987-10-15 1997-08-12 Chiron Corporation Nucleic acid-amplified immunoassay probes
US4988617A (en) * 1988-03-25 1991-01-29 California Institute Of Technology Method of detecting a nucleotide change in nucleic acids
US5079172A (en) * 1988-11-04 1992-01-07 Board Of Trustees Operating Michigan State University Method for detecting the presence of antibodies using gold-labeled antibodies and test kit
US5948634A (en) * 1988-12-21 1999-09-07 The General Hospital Coporation Neural thread protein gene expression and detection of alzheimer's disease
US6143508A (en) * 1989-06-29 2000-11-07 Rhone-Poulenc Rorer Pharmaceuticals Inc. Device and process for cell capture and recovery
HU218095B (en) * 1990-05-01 2000-05-28 Amgen Inc. Process for reducing transitional contaminations in amplification processes
AU659798B2 (en) 1990-05-04 1995-06-01 Chiron Corporation Protein-nucleic acid probes and immunoassays using same
AU3942993A (en) * 1992-03-31 1993-11-08 Abbott Laboratories Method of multiplex ligase chain reaction
WO1994016105A1 (en) * 1993-01-15 1994-07-21 The Public Health Research Institute Of The City Of New York, Inc. Rna assays using rna binary probes and ribozyme ligase
AU6031094A (en) 1993-01-15 1994-08-15 Public Health Research Institute Of The City Of New York, Inc., The Sensitive nucleic acid sandwich hybridization assays and kits
US5985548A (en) * 1993-02-04 1999-11-16 E. I. Du Pont De Nemours And Company Amplification of assay reporters by nucleic acid replication
US5384255A (en) * 1993-06-21 1995-01-24 Rappaport Family Institute For Research In The Medical Sciences Ubiquitin carrier enzyme E2-F1, purification, production, and use
US5635602A (en) * 1993-08-13 1997-06-03 The Regents Of The University Of California Design and synthesis of bispecific DNA-antibody conjugates
US5415839A (en) * 1993-10-21 1995-05-16 Abbott Laboratories Apparatus and method for amplifying and detecting target nucleic acids
SE9400522D0 (en) * 1994-02-16 1994-02-16 Ulf Landegren Method and reagent for detecting specific nucleotide sequences
US5596081A (en) * 1994-03-11 1997-01-21 University Of Kentucky Research Foundation Nucleotide or nucleoside photoaffinity compound modified antibodies, methods for their manufacture and use thereof as diagnostics and therapeutics
JPH11506605A (en) * 1995-06-07 1999-06-15 アボツト・ラボラトリーズ Method of masking probes to reduce background in amplification reactions
US5667974A (en) * 1995-06-07 1997-09-16 Abbott Laboratories Method for detecting nucleic acid sequences using competitive amplification
SE504798C2 (en) * 1995-06-16 1997-04-28 Ulf Landegren Immunoassay and test kits with two reagents that can be cross-linked if adsorbed to the analyte
US5780231A (en) * 1995-11-17 1998-07-14 Lynx Therapeutics, Inc. DNA extension and analysis with rolling primers
US5804384A (en) * 1996-12-06 1998-09-08 Vysis, Inc. Devices and methods for detecting multiple analytes in samples
US5812272A (en) * 1997-01-30 1998-09-22 Hewlett-Packard Company Apparatus and method with tiled light source array for integrated assay sensing
US5919626A (en) * 1997-06-06 1999-07-06 Orchid Bio Computer, Inc. Attachment of unmodified nucleic acids to silanized solid phase surfaces
EP1985714B1 (en) * 1998-03-25 2012-02-29 Olink AB Method and kit for detecting a target molecule employing at least two affinity probes and rolling circle replication of padlock probes
US7306904B2 (en) * 2000-02-18 2007-12-11 Olink Ab Methods and kits for proximity probing
US20030207300A1 (en) * 2000-04-28 2003-11-06 Matray Tracy J. Multiplex analytical platform using molecular tags
US6511809B2 (en) 2000-06-13 2003-01-28 E. I. Du Pont De Nemours And Company Method for the detection of an analyte by means of a nucleic acid reporter
GB0016953D0 (en) * 2000-07-11 2000-08-30 Wilson Stuart M A method for distinguishing aggregated or polymerised forms of a given molecule from the unaggregated or unpolymerised form
WO2003031591A2 (en) * 2001-10-10 2003-04-17 Superarray Bioscience Corporation Detecting targets by unique identifier nucleotide tags
US20050287528A1 (en) * 2002-04-04 2005-12-29 Mount Sinai Hospital Methods for detecting ovarian cancer
JP5117719B2 (en) * 2003-04-18 2013-01-16 ベクトン・ディキンソン・アンド・カンパニー Immune amplification
US7572409B2 (en) * 2003-05-23 2009-08-11 Applied Biosystems, Llc Ionic liquid apparatus and method for biological samples

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CHEMTECH, Volume 24, January 1995, TAKESHI SANO et al., "Detecting Minute Amounts of Antigen". *

Cited By (60)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2012210213A (en) * 1998-03-25 2012-11-01 Olink Ab Rolling circle replication of padlock probe
US7074564B2 (en) * 1998-03-25 2006-07-11 Ulf Landegren Rolling circle replication of padlock probes
WO1999063109A1 (en) * 1998-06-04 1999-12-09 Baxter Aktiengesellschaft Method for determining antigens
US7306904B2 (en) 2000-02-18 2007-12-11 Olink Ab Methods and kits for proximity probing
WO2001061037A1 (en) * 2000-02-18 2001-08-23 Ulf Landegren Methods and kits for proximity probing
USRE44265E1 (en) 2001-08-03 2013-06-04 Olink Ab Nucleic acid amplification method
US7790388B2 (en) 2001-08-03 2010-09-07 Olink Ab Nucleic acid amplification method
WO2003029489A1 (en) * 2001-10-03 2003-04-10 Iseao Technologies Limited Methods for detection of target molecules and molecular interactions
US7648828B2 (en) 2003-04-01 2010-01-19 Monogram Biosciences, Inc. Methods for detecting receptor complexes comprising PI3K
USRE44437E1 (en) 2003-04-01 2013-08-13 Monogram Biosciences, Inc. Methods for detecting receptor complexes comprising PI3K
US9499858B2 (en) 2003-04-18 2016-11-22 Becton, Dickinson And Company Immuno-amplification
US8372605B2 (en) 2003-04-18 2013-02-12 Becton, Dickinson And Company Immuno-amplification
US7932060B2 (en) 2003-04-18 2011-04-26 Becton, Dickinson And Company Immuno-amplification
US7402399B2 (en) 2003-10-14 2008-07-22 Monogram Biosciences, Inc. Receptor tyrosine kinase signaling pathway analysis for diagnosis and therapy
US8632994B2 (en) 2003-12-01 2014-01-21 Dako Denmark A/S Methods and compositions for immuno-histochemical detection
US7939313B2 (en) 2003-12-12 2011-05-10 Saint Louis University Biosensors for detecting macromolecules and other analytes
US7595160B2 (en) 2004-01-13 2009-09-29 U.S. Genomics, Inc. Analyte detection using barcoded polymers
EP1704256A4 (en) * 2004-01-13 2008-01-16 Us Genomics Inc Detection and quantification of analytes in solution using polymers
EP1704256A2 (en) * 2004-01-13 2006-09-27 U.S. Genomics, Inc. Detection and quantification of analytes in solution using polymers
US7977048B2 (en) 2004-01-13 2011-07-12 Pathogenetix, Inc. Detection and quantification of analytes in solution using polymers
US8580504B2 (en) 2004-06-14 2013-11-12 The Board Of Trustees Of The Leland Stanford Junior University Methods and compositions for use in analyte detection using proximity probes
US7914987B2 (en) 2004-06-14 2011-03-29 The Board Of Trustees Of The Leland Stanford Junior University Methods and compositions for use in analyte detection using proximity probes
WO2005123963A3 (en) * 2004-06-14 2006-06-08 Univ Leland Stanford Junior Methods and compositions for use in analyte detection using proximity probes
US8338579B2 (en) * 2004-11-03 2012-12-25 Iris Molecular Diagnostics, Inc. Homogeneous analyte detection
CN103146808A (en) * 2004-11-03 2013-06-12 意力速分子诊断股份有限公司 Homogeneous analyte detection
US9234890B2 (en) 2004-11-03 2016-01-12 Iris International, Inc. Homogeneous analyte detection
CN101137758B (en) * 2004-11-03 2012-10-10 意力速分子诊断股份有限公司 Homogeneous analyte detection
EP1842226A2 (en) * 2004-11-03 2007-10-10 Iris Molecular Diagnostics, Inc. Homogeneous analyte detection
EP1842226A4 (en) * 2004-11-03 2009-08-05 Iris Molecular Diagnostics Inc Homogeneous analyte detection
AU2005333156B2 (en) * 2004-11-03 2011-05-26 Iris Molecular Diagnostics, Inc. Homogeneous analyte detection
US8513032B2 (en) 2004-11-03 2013-08-20 Iris International, Inc. Microbubbles for affinity separation
US8956857B2 (en) * 2005-06-06 2015-02-17 Mediomics, Llc Three-component biosensors for detecting macromolecules and other analytes
US9951376B2 (en) 2005-06-10 2018-04-24 Saint Louis University Methods for the selection of aptamers
US8945840B2 (en) 2005-06-10 2015-02-03 Saint Louis University Methods for the selection of aptamers
US7795009B2 (en) * 2005-06-15 2010-09-14 Saint Louis University Three-component biosensors for detecting macromolecules and other analytes
US7811809B2 (en) 2005-06-15 2010-10-12 Saint Louis University Molecular biosensors for use in competition assays
US9618505B2 (en) 2005-06-15 2017-04-11 Mediomics, Llc Biosensors for detecting macromolecules and other analytes
JP2009529904A (en) * 2006-03-20 2009-08-27 オーリンク・アクチボラゲット Specimen detection method using proximity probe
US8268554B2 (en) 2006-03-20 2012-09-18 Olink Ab Method for analyte detection using proximity probes
WO2007107743A1 (en) * 2006-03-20 2007-09-27 Olink Ab Method for analyte detection using proximity probes
US9671403B2 (en) 2008-11-21 2017-06-06 Mediomics, Llc Biosensor for detecting multiple epitopes on a target
US10416157B2 (en) 2008-11-21 2019-09-17 Saint Louis University Biosensor for detecting multiple epitopes on a target
US8993245B2 (en) 2008-11-21 2015-03-31 Mediomics, Llc Biosensor for detecting multiple epitopes on a target
US10416154B2 (en) 2010-02-12 2019-09-17 Mediomics Llc Molecular biosensors capable of signal amplification
US9040287B2 (en) 2010-02-12 2015-05-26 Mediomics, Llc Molecular biosensors capable of signal amplification
US9797892B2 (en) 2010-02-12 2017-10-24 Saint Louis University Molecular biosensors capable of signal amplification
EP2369015A1 (en) 2010-03-15 2011-09-28 Olink AB Assay for localised detection of analytes
WO2012007511A1 (en) 2010-07-15 2012-01-19 Olink Ab Blocking reagent and methods for the use thereof
US9677131B2 (en) 2010-07-15 2017-06-13 Olink Proteomics Ab Blocking reagent and methods for the use thereof
WO2012049316A1 (en) 2010-10-15 2012-04-19 Olink Ab Dynamic range methods
US10669569B2 (en) 2010-10-15 2020-06-02 Navinci Diagnostics Ab Dynamic range methods
US9777315B2 (en) 2011-01-31 2017-10-03 Olink Proteomics Ab Exonuclease enabled proximity extension assays
US10731206B2 (en) 2011-01-31 2020-08-04 Olink Proteomics Ab Exonuclease enabled proximity extension assays
US9551032B2 (en) 2011-05-11 2017-01-24 Olink Bioscience Ab Unfolding proximity probes and methods for the use thereof
WO2012152942A1 (en) 2011-05-11 2012-11-15 Olink Ab Unfolding proximity probes and methods for the use thereof
US10597701B2 (en) 2011-05-11 2020-03-24 Navinci Diagnostics Ab Unfolding proximity probes and methods for the use thereof
WO2014076214A1 (en) 2012-11-14 2014-05-22 Olink Ab Rca reporter probes and their use in detecting nucleic acid molecules
US11034995B2 (en) 2014-02-04 2021-06-15 Navinci Diagnostics Ab Proximity assay with detection based on hybridisation chain reaction (HCR)
US10274484B2 (en) 2014-09-12 2019-04-30 Mediomics Llc Molecular biosensors with a modular design
US11649486B2 (en) 2016-08-16 2023-05-16 Navinci Diagnostics Ab Double-stranded circle probes

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