WO1994026932A1 - Dosage immonologique marque a l'acide nucleique - Google Patents

Dosage immonologique marque a l'acide nucleique Download PDF

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Publication number
WO1994026932A1
WO1994026932A1 PCT/US1994/005407 US9405407W WO9426932A1 WO 1994026932 A1 WO1994026932 A1 WO 1994026932A1 US 9405407 W US9405407 W US 9405407W WO 9426932 A1 WO9426932 A1 WO 9426932A1
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Prior art keywords
oligonucleotide
antigen
subject
linked
amplification
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PCT/US1994/005407
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English (en)
Inventor
Howard A. Fields
Yury E. Khudyakov
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United States Of America, As Represented By The Secretary, Department Of Health And Human Services
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Priority to AU69133/94A priority Critical patent/AU6913394A/en
Publication of WO1994026932A1 publication Critical patent/WO1994026932A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • 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
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals

Definitions

  • PCR polymerase chain reaction
  • Sano describes a method, whereby a known antibody is allowed to react with a sample (e.g., an antigen bound to a solid phase) , unbound antibody is removed, then a biotinylated double stranded linear marker DNA molecule is attached to any resulting solid-phase bound antigen-IgG antibody complexes by means of a linker molecule having affinity for both biotin and the Fc of IgG molecules, resulting in the formation of an antigen-antibody-DNA conjugate.
  • This attached DNA can then be amplified by PCR with appropriate primers, and such amplified DNA is detected by agarose gel electrophoresis, resulting in enhanced sensitivity of the detection of the antigen.
  • the methods disclosed Sano by have the disadvantage that they cannot be used to detect antibodies from a sample, since the antibody must be selectively bound to a solid support.
  • the present invention provides the advantages of increased sensitivity and in addition overcomes the above disadvantages by providing an improved nucleic acid tagged immunoassay for the detection of antigens and antibodies in specimens.
  • the present invention provides a method for detecting the presence of an antigen in a specimen from a subject comprising the steps of (a) binding antigens in a specimen from the subject to a solid support; (b) contacting the bound antigens with a specifically immunoreactive ligand, wherein the ligand is linked to an oligonucleotide, having a known sequence, under conditions that permit reaction of the oligonucleotide-linked ligand with the bound antigen;
  • a method for detecting the presence of an antibody in a specimen from a subject is also provided.
  • the method can include the following steps: (a) binding antibodies in a specimen from the subject to a solid support; (b) contacting the bound antibodies with a specifically immunoreactive antigen, wherein the antigen is linked to an oligonucleotide, having a known sequence, under conditions that permit reaction of the oligonucleotide- linked antibody with the bound antibody from the subject;
  • the invention also includes a composition comprising an oligonucleotide of known sequence linked to a specifically immunoreactive antigen or ligand.
  • the ligand-oligonucleotide compositions and antigen- oligonucleotide compositions of the invention are each referred to herein as an initiator of nucleic acid amplification (INAA) .
  • INAA nucleic acid amplification
  • Nucleic acid tagged immunoassay is a technique for the detection of an antigen-antibody interaction using methods for the amplification of nucleic acids. This method can be used for the detection of very diluted antigens or antibodies because of the high efficiency and sensitivity of the nucleic acid amplification step. Sensitivity of NATIA can be 1,000 - 1,000,000 times greater than conventional enzyme immunoassays or radioimmunoassays.
  • NATIA is a method for detecting the presence of an antigen in a specimen from a subject comprising the steps of (a) binding antigens in a specimen from the subject to a solid support; (b) contacting the bound antigens with a specifically immunoreactive ligand, wherein the ligand is linked to an oligonucleotide, having a known sequence, under conditions that permit reaction of the oligonucleotide-linked ligand with the bound antigen; (c) removing unreacted oligonucleotide-linked ligand; and (d) detecting the presence of the oligonucleotide, the presence of the oligonucleotide indicating the presence of the antigen in the subject.
  • immunoreactive means capable of binding or otherwise associating nonrandomly with an antigen or antibody.
  • Specifically immunoreactive as used herein describes an antibody or other ligand or an antigen that does not cross react substantially with any antigen or antibody other than the one specified.
  • ligand includes, for example, antibodies (monoclonal or polyclonal) , immunoreactive fragments of such antibodies and other molecules capable of binding or otherwise associating nonrandomly with an antigen or antibody.
  • Antigen refers to immunoreactive proteins and immunoreactive polypeptide fragments thereof, and includes disease markers.
  • a method for detecting the presence of an antibody in a specimen from a subject is also provided.
  • the method can include the following steps: (a) binding antibodies in a specimen from the subject to a solid support; (b) contacting the bound antibodies with a specifically immunoreactive antigen, wherein the antigen is linked to an oligonucleotide, having a known sequence, under conditions that permit reaction of the oligonucleotide- linked antibody with the bound antibody from the subject; (c) removing unreacted oligonucleotide-linked antigen; and (d) detecting the presence of the oligonucleotide, the presence of the oligonucleotide indicating the presence of the antibody in the subject.
  • the step of binding antigens or antibodies in a specimen to a solid support can comprise any of the known methods for attaching an antigen to a solid surface. Examples of such methods are provided in the Examples.
  • the solid support used in the methods can, for example, include microtiter plates or other labware, beads, magnetic particles or other solid material.
  • the specimen can comprise any body fluid which would contain the antigen, a cell containing the antigen or the antibody, such as blood, plasma, serum, saliva and urine sputum, mucus, gastric juice and the like.
  • the specimen can also be stool or tissue from the subject.
  • the ligand in the method for detecting an antigen in a specimen from the subject, can be linked to an oligonucleotide by biotin-streptavidin-biotin as known in the art and described below in the Examples.
  • the oligonucleotide and the antigen can be linked covalently by chemical reaction or non-covalently linked as by biotin-streptavidin. Methods for generating the covalent linkage of the oligonucleotide to the antigen are well known in the art and exemplified in the Examples.
  • the antigens bound to the solid support are contacted with a specifically immunoreactive ligand linked to an oligonucleotide as detailed below in the Examples.
  • the bound antibodies are contacted with a specifically immunoreactive antigen, wherein the antigen is linked to an oligonucleotide.
  • the conditions of the contacting step that permit reaction of the oligonucleotide-linked ligand with the bound antigen are exemplified below.
  • the oligonucleotide used in the method has a known sequence, which permits detection of the presence of the oligonucleotide linked to the specifically immunoreactive ligand or antigen.
  • the unreacted oligonucleotide-linked ligand is removed by washing the solid support to which the antigen is attached.
  • the oligonucleotide is amplified prior to detection.
  • the amplification can be by polymerase chain reaction, ligase chain reaction or other method for amplifying nucleic acids.
  • Other amplification methods that can be used in the present methods are well known in the art and include 3SR amplification (Guatelli et al. "Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication" Pros. Natl. Acad. Sci. USA 87:1874-1878, 1990); amplification with Q-beta replicase (Wolcott, M. J.
  • the oligonucleotide can then be detected by detecting the product of amplification.
  • the methods that can be used to detect the amplified product will depend on the amplification method generally and the primers used.
  • the presence of the detectable moiety indicates the presence of amplification product.
  • the detectable moiety can be directly detectable (e.g., a radioactive or fluorescent marker) or indirectly detectable, such as by an enzymatic reaction producing a detectable product (e.g., horseradish peroxidase) as described in the Examples.
  • RNA transcripts generated from the oligonucleotide can then be detected using standard methods, such as by gel electrophoresis followed by northern blotting or by the methods used to detect DNA, such as those described in the Examples. Because the oligonucleotide is linked to a specifically immunoreactive ligand or antigen, the presence of the oligonucleotide indicates the presence in the subject of the respective antigen or ligand specifically reactive therewith.
  • HCV hepatitis C virus
  • the method can comprise the steps of binding antibodies in a specimen from the subject to a solid support and contacting the bound antibodies with an oligonucleotide covalently linked to the polypeptide consisting essentially of the amino acid sequence defined in the Sequence Listing by SEQ ID N0:1 under conditions that permit the reaction of that composition with an antibody from the subject.
  • "Consisting essentially of” can include minor amino acid substitutions, insertions and deletions which do not substantially diminish the polypeptides antigenicity.
  • the presence of the oligonucleotide is detected, the presence of the oligonucleotide indicates the reaction of the polypeptide and an antibody from the subject.
  • the reaction indicates current or previous hepatitis C virus infection.
  • the binding, contacting, removing and detecting steps in the NATIA for detecting HCV infection are carried out as described above and in the Examples.
  • the invention provides methods and compositions capable of detecting very small quantities of antigen, antibody or other marker of disease
  • the invention also provides a method for the early detection of malignancies in a manner according to the methods described above and in the Examples with the antigen or ligand selected based on the disease to be detected.
  • Existing methods do not allow for the routine detection of cancer tumors at the stage when it may be easily cured.
  • NATIA is a very sensitive method for the detection of antigen-antibody binding. This method has wide applications in the development of diagnostics for many diseases, especially those with low levels of circulating markers of infection.
  • Increased sensitivity of the method improves the specificity of many immunodiagnostic reactions by allowing these tests to be run at very high specimen dilutions, which result in decreased detection of non-specific reactions.
  • the increased sensitivity will compensate for any signal lost as the result of diluting the test specimen.
  • the invention also includes a composition comprising an oligonucleotide of known sequence linked to a specifically immunoreactive antigen or ligand.
  • the ligand-oligonucleotide compositions and antigen- oligonucleotide compositions of the invention are each referred to herein as an initiator of nucleic acid amplification (INAA) .
  • INAA nucleic acid amplification
  • an example of the composition comprises an oligonucleotide covalently linked to the polypeptide consisting essentially of the amino acid sequence defined in the Sequence Listing by SEQ ID NO:l.
  • This polypeptide is a specifically immunoreactive antigen of hepatitis C virus (HCV) and can be used in the method of detecting current or previous HCV infection.
  • HCV hepatitis C virus
  • composition of the present invention comprises an oligonucleotide covalently linked to the polypeptide consisting essentially of the amino acid sequence defined in the Sequence Listing by SEQ ID NO:2.
  • This polypeptide is a specifically immunoreactive antigen of hepatitis E virus (HEV) and can be used in a method of detecting current or previous HEV infection, comprising the same general steps as described above for HCV.
  • HEV hepatitis E virus
  • nucleic acid comprising essentially the nucleotide sequence defined in the Sequence Listing by SEQ ID NO:3 is also provided.
  • the nucleic acid can be used as the oligonucleotide in the NATIA methods of the present invention.
  • the nucleic acid can be linked to a number of antigens or antibodies to form the INAA.
  • a composition of the invention can comprise an immunoreactive polypeptide covalently linked to the above nucleic acid. More specifically, the immunoreactive polypeptide of the composition can consist essentially of the amino acid sequence defined in the Sequence Listing by SEQ ID N0:1. Alternatively, the polypeptide can consist essentially of the amino acid sequence defined in the Sequence Listing by SEQ ID NO:2.
  • the main advantage of the NATIA is the extremely efficient method of enhancing a signal initiated by an antigen-antibody binding.
  • Another advantage is a shift from protein-protein interactions (antigen-antibody recognition) to nucleic acid synthesis. This shift significantly diminishes the "noise” often associated with antigen-antibody interactions. Non-specific protein- protein interaction will not be detected because of the use of nucleic acid amplification.
  • any protein such as viral or bacterial antigens or antigenic fragments of antigens, or antibodies, can be conjugated to a nucleic acid tag.
  • oligonucleotide (1) short synthetic oligonucleotides result in better efficiency of amplification; (2) the structure of the oligonucleotide can be modified in any way to facilitate amplification (weak secondary structure) or detection, particularly for the solid-phase detection of amplification products; (3) simplicity of introducing specific chemical modifications for chemical attachment of oligonucleotides to immunoreactive components of NATIA; (4) greater ability to be specifically designed for the use in amplification reactions other than PCR.
  • Lack of secondary structure allows for the use of room temperature to initiate efficient isothermal amplification reactions such as Q-beta polymerase specific amplification; and (5) with the use of synthetic peptides, the linkage of oligonucleotide and peptide can be chemical, the resulting composition can be easily purified and characterized and can be obtained in homogeneous form without significant contamination with unlinked oligonucleotide or antigen.
  • a synthetic immunoreactive peptide of the sequence ANPPDHSAPLGVTRPSA (SEQ ID NO:l) containing an antigenically active region from the protein encoded by the open reading frame 3 (ORF3) of the hepatitis E virus (HEV)
  • a synthetic peptide of the sequence AFASRGNHVSPTHYVPESDA (SEQ ID NO:2) containing an antigenic epitope encoded by the hepatitis C virus (HCV)
  • the sequence of the oligonucleotide may vary depending on the method of amplification chosen for detection.
  • An example of oligonucleotide for use in the present invention has the following primary structure: 5'-
  • This oligonucleotide can be covalently attached by the 5'-terminus to the N- or C-terminal sequence of a synthetic peptide using well-known methods of conjugation. For example, using an amino-group attached to an oligonucleotide during the synthesis of the oligonucleotide, this oligonucleotide may be covalently linked to a peptide by amino-(such as lysine, or N- terminal amino-group) or carboxy-group (such amino acids as glutamic or aspartic acid, or C-terminal group) .
  • the 1,3-diisopropylcarbodiimide can be used in combination with H-hydroxybenzotriazole for conjugation of the 5' - terminal amino group of an oligonucleotide to the C- terminal carboxy-group of a peptide.
  • Other carbodiimide derivatives may be used to obtain this product. (see catalog of Pierce Company for additional examples of suitable coupling agents) .
  • Other reactions well known for protein-protein coupling can also be used with routine modifications to link an oligonucleotide and a peptide.
  • the covalent complex composed of the oligonucleotide and a synthetic peptide is called an initiator of nucleic acid amplification (INAA) .
  • INAA initiator of nucleic acid amplification
  • Plastic tubes or microtiter wells are adsorbed with serum from a subject suspected of containing anti-HCV or anti-HEV antibodies reactive with the synthetic HCV and
  • HEV peptides were prepared 1:500 by phosphate buffered saline (PBS). 100 ⁇ l of the diluted specimen was applied to each microtiter plate well and incubated at room temperature overnight. Next day, the plates were washed thoroughly with PBS containing 1% bovine serum albumin, 0,05% Tween 20.
  • PBS phosphate buffered saline
  • the INAA is dissolved in 0.01 M PBS, pH 7.2, containing 1% bovine serum albumin, and 10% normal goat serum. Tubes or wells adsorbed with the specimen are incubated with this solution for 30 min to 1 h to allow the synthetic peptides to specifically react with antibody in the specimen. After thorough washing of the tubes or wells to remove unbound INAA, the bound INAA containing an available oligonucleotide having the specifically designed sequence remains in the tube or in the well attached to the solid support.
  • the INAA When the immunosorption reaction is carried out in microtiter wells, the INAA must be dissociated from the surface by treatment with 0.2 M glycine, pH 2.5, for approximately 2 min, and transferred into plastic tubes for amplification.
  • the oligonucleotide can be used for the initiation of the amplification by several different methods, such as the polymerase chain reaction (PCR) or ligase chain reaction (LCR) .
  • oligonucleotide primers with the sequences: primer 1 5'- uTJOTJUUUUUUUUUUUUUUUUUU -3' (SEQ ID NO:4) primer 2 5'-Biotin-CCTCCCCTATCTTCTCCT (SEQ ID NO:5) can be used.
  • Primers were synthesized with an automatic synthesizer (Applied Biosystem, Inc. (Foster City, CA) Model 480A) and purified by electrophoresis in 10% PAGE containing 7 M urea with TBE buffer (0.045M Tris-borate, 0.001 EDTA, pH8.3). Oligonucleotides were recovered from the gel by electroelution.
  • PCR is conducted, briefly as follows: 20-50 pmol of each primer was added to the reaction mixture. PCR consisted of 30 cycles as follows: 94"C for 45 sec, 65° for 20 sec, and 72°C for 1 min. This protocol was designed especially for this particular sequence. However, other PCR protocols can yield satisfactory results for the present methods (see, for example Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989) .
  • oligonucleotides with the sequences: Oligo 1 5'-Biotin-CCTCCCCTATCTTCTCCTCCCCA (SEQ ID NO:6) Oligo 2 5'-ACCCCAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:7) Oligo 3 5'-UUuTJUUUUUUUUUUUUUUUGGGGT (SEQ ID NO:8) oligo 4 5'-TGGGGACCAGAAGATAGGGGAGG (SEQ ID NO:9) can be used. These oligonucleotides can be generated as described above.
  • thermostable ligase As provided in Barany, F. "Genetic disease detection and DNA amplification using cloned thermostable ligase" (Proc. Natl. Acad. Sci. USA 88:189-193, 1991): The reaction was incubated with thermostable DNA ligase at 94°C for 1 min followed by 65°C for 4 min, and this cycle was repeated 20 times.
  • Other protocols for LCR are well known in the art and can be applied to the present methods with routine modifications.
  • the PCR or LCR product will have the following structure: 5'-Biotin-CCTCCCCTTTCTTCTCCTCCCCAACCCCAAAAAAAAAAAAAAAAAAAA- 3'
  • NATIA The sensitivity of NATIA is based on the efficiency of amplification using PCR and LCR to amplify nucleic acids.
  • the inconvenience of special thermal conditions associated with both PCR and LCR amplification steps complicates the application of the present method to routine diagnostics.
  • new methods of isothermal amplification have been described (see, for example, Wolcott, M. J. "Advances in nucleic acid-based detection methods", Clinical Microbiology Reviews, 5(4) :370-386, 1992) .
  • reverse transcriptase and RNA polymerase work together to amplify a DNA or RNA template without thermal cycler.
  • the reaction is carried out at constant temperature 37°C. Guatelli et al. "Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication" Pros. Natl. Acad. Sci. USA 87:1874-1878, 1990.
  • amplification with Q-beta replicase is based on the use of the extraordinary efficiency of the Q- beta polymerase to replicate in vitro Q-beta phage DNA.
  • Sequences specific to the oligonucleotide linked to the immunoreactive component of the NATIA has to be inserted in the genome of the Q-beta phage.
  • the phage DNA will be bound to the oligonucleotide moiety adsorbed to a solid-phase. After thorough washing of unbound phage DNA, this DNA may be replicated by Q-beta polymerase (see, for example, Wolcott, M.J. "DNA-based rapid methods for the detection of foodborne pathogens" J. Food Prot. 54:387-401. 1991).
  • Strand displacement amplification based on the creation of a nick at specific place in a double-stranded DNA by a restriction endonuclease and use of the nicked DNA as a template for a DNA polymerase possessing DNA strand displacement activity.
  • a specific primer is annealed to the oligonucleotide-linked polypeptide (ligand or antigen) .
  • ligand or antigen oligonucleotide-linked polypeptide
  • the product When treated with uracil DNA glycosylase to remove uracils from the nucleic acid, the product is converted into a partially double-stranded DNA fragment with an oligo(dA) 3'-protrusion:
  • This final product is captured with oligo(dT) attached to a solid-phase (e.g. microtiter wells) .
  • the method for attaching the oligo(dT) to the solid support is similar to the method used for linking an oligonucleotide to a synthetic peptide.
  • Special CovaLink NH (Nunc) plates are used to attach an oligonucleotide to the surface of microtiter plates. Rasmussen et al. "Covalent immobilization of DNA onto polystyrene microwells: the molecules are only bound to the 5' -end" Analytical Biochemistry 198:138-142, 1991.
  • plates are coated with biotinylated bovine serum albumin 9B-BSA.
  • streptavidin is added to the wells to be bound to the biotin linked to BSA.
  • INAA is used to capture biotinylated oligo(dT).
  • a complex B-BSA- streptavidin-oligo(dT) is attached to the solid-phase and used for the solid-phase detection of reaction amplification products as described elsewhere herein.
  • the captured product is detected by adding the reporter enzyme, horseradish peroxidase, conjugated to streptavidin to the captured product.
  • one primer for PCR has an oligo(dU)-tail.
  • the second oligonucleotide is labeled with biotin (or e.g., digoxigenin) .
  • a PCR product obtained with the use of these primers will contain on one end biotin (or another moiety for detection) and oligo(dU)-oligo(dA)-tail.
  • oligo(dU) After treatment with Uracil DNA glycosilase (UDG) , oligo(dU) will be removed allowing exposure of the oligo(dA) in single- stranded form for interaction with oligo(dT) linked to a solid-phase (solid support) .
  • UDG Uracil DNA glycosilase
  • the oligonucleotide component of the INAA composition can be designed to include a promoter of the T7 phage ligated to a short specifically designed sequence that can be transcribed using T7 RNA polymerase.
  • the promoter of the T7 phage or SP6 phage is a very short sequence of about 20 - 30 nucleotides long. This sequence can be designed in the synthetic oligonucleotide to be recognized with T7 or SP6 RNA polymerase.
  • RNA transcript When recognized by RNA polymerase this promoter will induce synthesis of RNA transcript from the oligonucleotide.
  • This transcript may be detected by different methods including gel electrophoresis, or solid-phase detection as described above but with one modification: there is no need to treat the transcript with UDG.
  • This transcript may be designed to contain oligo(A) sequence for the capture with oligo(dT) on solid-phase. Transcript can be labeled for the detection with radioactive nucleotides. Krupp, G. "RNA synthesis: strategy for the use of bacteriophage RNA polymerase" Gene 72:75-89, 1988.
  • Anti-HCV (IgM) antibodies are captured by anti-mu antibodies attached to the surface of the microtiter wells. Briefly, anti-mu antibodies are diluted in PBS 1:1000 and applied to the microtiter plates wells, incubated overnight at room temperature. After washing the wells to remove unbound anti-mu, specimens are added to the wells at different dilutions. The serum specimen may be diluted 1000 times greater than conventional methods of IgM detection. This extreme dilution should decrease the "noise signal" without any deletion effect on sensitivity of the NATIA.
  • the captured IgM antibody is incubated with an HCV-specific antigen conjugated to a specifically designed oligonucleotide to form the INAA as described above.
  • the INAA also can be added to the reaction in a very diluted form.
  • the INAA is allowed to react with any captured anti-HCV antibody and the unreacted INAA removed as described abov .
  • the oligonucleotide tag is then amplified and the product detected or otherwise detected as described above.
  • a synthetic peptide from the NS4 protein of the hepatitis C virus with structure IIPDREVLYREFDEMEECSQ (SEQ ID NO:12) is biotinylated using a commercially available kit (Pierce, Rockford, Illinois) . This peptide was selected because biotinylation does not affect the immunoreactivity of the peptide.
  • a biotinylated oligonucleotide with structure ⁇ '-biotin- TTTTTTTTTTCAGAAAGCGTCTAGCCATGGCGTTCACTTGTGGTACTGCCTGATA GGG-3' (SEQ ID NO:13) was prepared as described above.
  • This oligonucleotide is composed of sequences of two PCR primers, which are known to work well in PCR, separated by a short additional sequence. The shorter the region to be amplified the better the efficiency of amplification obtained.
  • the biotinylated oligonucleotide is preincubated with streptavidin (Pierce, Rockford, Illinois) . Then, this complex is incubated with the biotinylated peptide resulting in formation of an oligonucleotide-peptide complex linked by biotin- streptavidin binding. This INAA complex is then used in place of chemically prepared oligonucleotide-peptide conjugates mentioned above.
  • Anti-human antibodies (anti-IgM, -IgG etc.) are adsorbed on the surface of microcentrifuge tubes and used to capture antibodies from human sera specimens as described above. Then, the tubes are incubated with the peptide-streptavidin -oligonucleotide complex as described above.
  • the primer labeled with digoxigenin could be labeled with another moiety, for example, biotin.
  • the other primer contains a poly(U)-tail.
  • PCR product is treated with Uracil-DNA glycosylase as described above to produce an oligo(A)- protrusion on one side and digoxigenin (or biotin) on the other and can be detected by PSSPD (primer specific solid- phase detection.
  • PSSPD primary specific solid- phase detection.
  • different aliquots of the amplification reaction mixture usually 4 ⁇ l, were treated with 1 unit of UDG (Gibco BRL, Great Island, New York) and 1 ⁇ l of MRP-SA (Gibco BRL, Great Island, New York) in lxUDG buffer containing 30 mM tris-HCl, pH8.0, 50 mM KC1, 5 mM magnesium chloride for 10 min at 37 C.
  • Sera diluted with phosphate buffered saline are directly adsorbed on the surface of the microcentrifuge tubes.
  • the adsorption step is as described above and as routinely practiced in the art.
  • the immunoreaction, amplification and detection steps are as described above.
  • NATIA Another application of the NATIA is the direct detection of HC virions (undetectable by existing immunodiagriostic methods) in sera specimens.
  • Serum preparation to detect virions is similar to the specimen preparation for detection of antigens.
  • the virion can be captured by HCV specific antibodies preadsorbed on the solid-phase as was described above for capture of IgM. After this step, an HCV specific antibody linked with an oligonucleotide by any of described above approaches may be added.
  • an antibody reactive with an HCV envelope protein is covalently linked (conjugated) to the nucleic acid tag (oligonucleotide) .
  • Conjugation of the antibody to the oligonucleotide is accomplished in the same manner as described above for the covalent linking of peptides and oligonucleotides. Immunoreaction and detection of reacted INAA.
  • the above-described methods can be applied to detect other viruses, micro-organisms or disease markers, simply by utilizing an antigenic synthetic peptide, antigen, antibody or ligand specifically reactive with the substance to be detected.
  • MOLECULE TYPE oligonucleotide
  • oligonucleotide SEQUENCE DESCRIPTION: SEQ ID N0:3: CCTCCCCTAT CTTCTCCTCC CCAACCCCAA AAAAAAAAAA AAAAAA 46 (2) INFORMATION FOR SEQ ID N0:4:
  • MOLECULE TYPE oligonucleotide
  • oligonucleotide SEQUENCE DESCRIPTION: SEQ ID N0:4: UUUUUUUUUU UUUUUUU 18
  • MOLECULE TYPE oligonucleotide
  • SEQUENCE DESCRIPTION SEQ ID NO:5: CCTCCCCTAT CTTCTCCT 18
  • MOLECULE TYPE oligonucleotide
  • xi SEQUENCE DESCRIPTION: SEQ ID N0:6: CCTCCCCTAT CTTCTCCTCC CCA 23
  • MOLECULE TYPE oligonucleotide
  • xi SEQUENCE DESCRIPTION: SEQ ID N0:7: ACCCCAAAAA AAAAAAAAAA AAA 23
  • MOLECULE TYPE oligonucleotide
  • oligonucleotide SEQUENCE DESCRIPTION: SEQ ID N0:8: UUUUUUUUUUU UUUUUUUGG GGT 23
  • MOLECULE TYPE oligonucleotide
  • oligonucleotide SEQUENCE DESCRIPTION: SEQ ID NO:9: TGGGGACCAG AAGATAGGGG AGG 23
  • MOLECULE TYPE double stranded oligonucleotide
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:10: CCTCCCCTTT CTTCTCCTCC CCAACCCCAA AAAAAAAAAA AAAAAA 46
  • MOLECULE TYPE oligonucleotide
  • SEQUENCE DESCRIPTION SEQ ID NO:12: TTTTTTTTTT CAGAAAGCGT CTAGCCATGG CGTTCACTTG TGGTACTGCC TGATAGGG 58
  • MOLECULE TYPE oligonucleotide
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:13: CCCTATCAGG CAGTACCACA A 21
  • MOLECULE TYPE oligonucleotide
  • oligonucleotide SEQUENCE DESCRIPTION: SEQ ID NO:14: UUUUUUUUUUU UUUCAGAAAG CGTCTAGCCA TGGCGTT 37

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  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de détection de la présence d'un antigène dans un spécimen provenant d'un sujet. Ledit procédé consiste (a) à fixer les antigènes d'un spécimen dudit sujet sur un support solide; (b) à mettre les antigènes fixés en contact avec un ligand spécifiquement immunoréactif, ledit ligand étant fixé à un oligonucléotide comprenant une séquence connue, dans des conditions permettant la réaction du ligand fixé à l'oligonucléotide avec l'antigène fixé; (c) à enlever le ligand fixé à l'oligonucléotide n'ayant pas réagi; et (d) à détecter la présence de l'oligonucléotide, sa présence signalant la présence de l'antigène dans le sujet. L'invention porte également sur un procédé de détection de la présence d'un anticorps dans un spécimen ainsi que sur une composition contenant un polypeptide immunoréactif fixé à un oligonucléotide.
PCT/US1994/005407 1993-05-13 1994-05-13 Dosage immonologique marque a l'acide nucleique WO1994026932A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU69133/94A AU6913394A (en) 1993-05-13 1994-05-13 Nucleic acid tagged immunoassay

Applications Claiming Priority (2)

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US6169493A 1993-05-13 1993-05-13
US08/061,694 1993-05-13

Publications (1)

Publication Number Publication Date
WO1994026932A1 true WO1994026932A1 (fr) 1994-11-24

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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996004300A1 (fr) * 1994-07-29 1996-02-15 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Procedes et compositions servant a effectuer un diagnostic differentiel d'infection aigue et chronique par le virus de l'hepatite c
EP0851228A1 (fr) * 1996-06-10 1998-07-01 Laboratory of Molecular Biophotonics Immunodosage a fluorescence a haute sensibilite
WO1999015898A1 (fr) * 1997-09-22 1999-04-01 Chiron Corporation Procede pour detecter des anticorps dans un echantillon
EP0951297A1 (fr) * 1996-10-29 1999-10-27 Polyprobe, Inc. Detection d'antigenes par le biais de conjugues anticorps - oligonucleotides
EP0960213A1 (fr) * 1996-11-21 1999-12-01 The Trustees Of The University Of Pennsylvania METHODE POUR DETECTER UNE PROTEINE PAR IMMUNO-aRNA
US6261764B1 (en) 1997-09-22 2001-07-17 Chiron Corporation Buffers for stabilizing antigens
US6465193B2 (en) * 1998-12-11 2002-10-15 The Regents Of The University Of California Targeted molecular bar codes and methods for using the same
GB2375822A (en) * 2000-12-05 2002-11-27 Norchip As Immunoassay using nucleic acid amplification and real-time measurement
EP1305625A1 (fr) * 2000-07-25 2003-05-02 The Trustees Of The University Of Pennsylvania Procede d'immunodetection d'epitopes
WO2003048388A2 (fr) * 2001-12-07 2003-06-12 The University Of Liverpool Immuno-dosage utilisant l'amplification en chaine de par polymerase
US7045286B2 (en) 2000-07-25 2006-05-16 The Trustees Of The University Of Pennsylvania Methods of detecting molecules expressing selected epitopes via fluorescent dyes
US7341831B2 (en) 2001-07-18 2008-03-11 The Trustees Of The University Of Pennsylvania Method for immuno-detection of epitopes
US7524628B2 (en) 2000-07-25 2009-04-28 The Trustees Of The University Of Pennsylvania Method for detecting molecules expressing a selected epitope via fluorescent dyes
EP2284284A1 (fr) * 2009-07-30 2011-02-16 Korea Institute of Science and Technology Kit de détection d'antigènes et procédé
US7932060B2 (en) 2003-04-18 2011-04-26 Becton, Dickinson And Company Immuno-amplification

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03167474A (ja) * 1989-11-27 1991-07-19 Hitachi Ltd 免疫学的測定法
WO1991017442A1 (fr) * 1990-05-04 1991-11-14 Chiron Corporation Sondes de proteine-acide nucleique et dosages immunologiques les utilisant
WO1992000989A1 (fr) * 1990-07-10 1992-01-23 Imperial Chemical Industries Plc Procede de marquage non-isotopique d'acide nucleique
WO1993006488A1 (fr) * 1991-09-16 1993-04-01 Genelabs Technologies, Inc. Dosages immunologiques virus de l'hepatite c, a base de peptides
EP0544212A1 (fr) * 1991-11-26 1993-06-02 Nisshin Flour Milling Co., Ltd. Procédé pour la détection ou quantification de substances de trace
WO1993015229A2 (fr) * 1992-02-04 1993-08-05 E.I. Du Pont De Nemours And Company Amplification de rapporteurs d'analyse par replication d'une sequence d'acide nucleique

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03167474A (ja) * 1989-11-27 1991-07-19 Hitachi Ltd 免疫学的測定法
WO1991017442A1 (fr) * 1990-05-04 1991-11-14 Chiron Corporation Sondes de proteine-acide nucleique et dosages immunologiques les utilisant
WO1992000989A1 (fr) * 1990-07-10 1992-01-23 Imperial Chemical Industries Plc Procede de marquage non-isotopique d'acide nucleique
WO1993006488A1 (fr) * 1991-09-16 1993-04-01 Genelabs Technologies, Inc. Dosages immunologiques virus de l'hepatite c, a base de peptides
EP0544212A1 (fr) * 1991-11-26 1993-06-02 Nisshin Flour Milling Co., Ltd. Procédé pour la détection ou quantification de substances de trace
WO1993015229A2 (fr) * 1992-02-04 1993-08-05 E.I. Du Pont De Nemours And Company Amplification de rapporteurs d'analyse par replication d'une sequence d'acide nucleique

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE WPI Week 9135, Derwent World Patents Index; AN 91-256837 *
RUZICKA V. ET AL: "Immuno-PCR with a commercially available avidin system", SCIENCE., vol. 26O, 30 April 1993 (1993-04-30), LANCASTER, PA US, pages 698 - 99 *
SANO, T. ET AL: "Immuno-PCR: Very sensitive antigen detection by means of specific antibody-DNA conjugates", SCIENCE., vol. 258, 2 October 1992 (1992-10-02), LANCASTER, PA US, pages 120 - 122 *

Cited By (30)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5670310A (en) * 1994-07-29 1997-09-23 The United States Of America As Represented By The Department Of Health And Human Services Methods and compositions for differential diagnosis of acute and chronic hepatitis c virus infection
WO1996004300A1 (fr) * 1994-07-29 1996-02-15 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Procedes et compositions servant a effectuer un diagnostic differentiel d'infection aigue et chronique par le virus de l'hepatite c
EP0851228A1 (fr) * 1996-06-10 1998-07-01 Laboratory of Molecular Biophotonics Immunodosage a fluorescence a haute sensibilite
EP0851228A4 (fr) * 1996-06-10 2000-07-26 Lab Molecular Biophotonics Immunodosage a fluorescence a haute sensibilite
US6255048B1 (en) 1996-06-10 2001-07-03 Laboratory Of Molecular Biophotonics Highly sensitive fluoroassay
EP0951297A4 (fr) * 1996-10-29 2002-01-02 Polyprobe Inc Detection d'antigenes par le biais de conjugues anticorps - oligonucleotides
EP0951297A1 (fr) * 1996-10-29 1999-10-27 Polyprobe, Inc. Detection d'antigenes par le biais de conjugues anticorps - oligonucleotides
EP0960213A1 (fr) * 1996-11-21 1999-12-01 The Trustees Of The University Of Pennsylvania METHODE POUR DETECTER UNE PROTEINE PAR IMMUNO-aRNA
EP0960213A4 (fr) * 1996-11-21 2002-03-06 Univ Pennsylvania METHODE POUR DETECTER UNE PROTEINE PAR IMMUNO-aRNA
US6537745B2 (en) 1997-09-22 2003-03-25 Chiron Corporation Buffers for stabilizing antigens
US6261764B1 (en) 1997-09-22 2001-07-17 Chiron Corporation Buffers for stabilizing antigens
US6391540B1 (en) 1997-09-22 2002-05-21 Chiron Corporation Method for detecting antibodies in a sample
WO1999015898A1 (fr) * 1997-09-22 1999-04-01 Chiron Corporation Procede pour detecter des anticorps dans un echantillon
US6465193B2 (en) * 1998-12-11 2002-10-15 The Regents Of The University Of California Targeted molecular bar codes and methods for using the same
US7060507B2 (en) * 1998-12-11 2006-06-13 The Regents Of The University Of California Targeted molecular bar codes and methods for using the same
US7045286B2 (en) 2000-07-25 2006-05-16 The Trustees Of The University Of Pennsylvania Methods of detecting molecules expressing selected epitopes via fluorescent dyes
US7361464B2 (en) 2000-07-25 2008-04-22 The Trustees Of The University Of Pennsylvania Method, systems and kits for immuno-detection of epitopes expressed on molecules
EP1305625A4 (fr) * 2000-07-25 2004-08-18 Univ Pennsylvania Procede d'immunodetection d'epitopes
EP1305625A1 (fr) * 2000-07-25 2003-05-02 The Trustees Of The University Of Pennsylvania Procede d'immunodetection d'epitopes
US7524628B2 (en) 2000-07-25 2009-04-28 The Trustees Of The University Of Pennsylvania Method for detecting molecules expressing a selected epitope via fluorescent dyes
US7910294B2 (en) 2000-12-05 2011-03-22 Norchip A/S Ligand detection method
GB2375822B (en) * 2000-12-05 2005-04-06 Norchip As Real time ligand detection comprising isothermal amplification
GB2375822A (en) * 2000-12-05 2002-11-27 Norchip As Immunoassay using nucleic acid amplification and real-time measurement
US7341831B2 (en) 2001-07-18 2008-03-11 The Trustees Of The University Of Pennsylvania Method for immuno-detection of epitopes
WO2003048388A2 (fr) * 2001-12-07 2003-06-12 The University Of Liverpool Immuno-dosage utilisant l'amplification en chaine de par polymerase
WO2003048388A3 (fr) * 2001-12-07 2003-09-25 Univ Liverpool Immuno-dosage utilisant l'amplification en chaine de par polymerase
US7932060B2 (en) 2003-04-18 2011-04-26 Becton, Dickinson And Company Immuno-amplification
US8372605B2 (en) 2003-04-18 2013-02-12 Becton, Dickinson And Company Immuno-amplification
US9499858B2 (en) 2003-04-18 2016-11-22 Becton, Dickinson And Company Immuno-amplification
EP2284284A1 (fr) * 2009-07-30 2011-02-16 Korea Institute of Science and Technology Kit de détection d'antigènes et procédé

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