Immunoassay based on DNA replication using labeled primer
First Claim
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1. A method for determining an analyte in a sample suspected of containing said analyte comprising the steps of:
- a. combining said sample with a single stranded DNA template capable of replication and comprising a starting nucleotide sequence, a deoxyribonucleotide precursor, a DNA polymerase enzyme, a receptor capable of binding with said analyte, a compound capable of generating a detectable signal in the presence of double stranded DNA, and a primer, said primer linked to a ligand or analog of said analyte and comprising a sequence complementary to said starting sequence of said template, under conditions favorable for DNA replication, b. monitoring the generation of double stranded DNA by said enzyme by measuring the signal produced by said compound, and c. correlating the production of said signal with the presence or amount of said analyte in said sample.
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Abstract
An immunoassay method based upon inhibition of a DNA polymerase enzyme accomplished by linking a ligand of the analyte to a primer through a covalent bond. The interaction between the primer-bound ligand and a receptor specific for the ligand inhibits the DNA polymerase enzyme from generating double stranded DNA. The degree of inhibition of double stranded DNA synthesis is inversely proportional to the concentration of analyte in the sample. The analyte is determined by measuring the formation of double stranded DNA, e.g., by a fluorescence DNA intercalation technique.
29 Citations
49 Claims
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1. A method for determining an analyte in a sample suspected of containing said analyte comprising the steps of:
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a. combining said sample with a single stranded DNA template capable of replication and comprising a starting nucleotide sequence, a deoxyribonucleotide precursor, a DNA polymerase enzyme, a receptor capable of binding with said analyte, a compound capable of generating a detectable signal in the presence of double stranded DNA, and a primer, said primer linked to a ligand or analog of said analyte and comprising a sequence complementary to said starting sequence of said template, under conditions favorable for DNA replication, b. monitoring the generation of double stranded DNA by said enzyme by measuring the signal produced by said compound, and c. correlating the production of said signal with the presence or amount of said analyte in said sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
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9. A reagent for determining an analyte in a sample via a method involving inhibition of DNA replication, said reagent comprising a primer bound to a ligand of said analyte or an analog of said analyte.
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10. A method for determining an analyte in a sample suspected of containing said analyte comprising the steps of:
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a. combining said sample with a single stranded DNA template capable of replication and comprising a starting nucleotide sequence, a deoxyribonucleotide precursor, a DNA polymerase enzyme, said enzyme linked to a ligand or analog of said analyte, a receptor capable of binding with said analyte, a compound capable of generating a detectable signal in the presence of double stranded DNA, and a primer, said primer comprising a sequence complementary to said starting sequence of said template, under conditions favorable for DNA replication, b. monitoring the generation of double stranded DNA by said enzyme by measuring the signal produced by said compound, and c. correlating the production of said signal with the presence or amount of said analyte in said sample. - View Dependent Claims (11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23, 24, 25, 28, 29, 30, 31, 32, 33, 34, 36, 37, 38, 39, 40, 41, 42)
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17. A reagent for determining an analyte in a sample via a method involving inhibition of DNA replication, said reagent comprising a DNA polymerase enzyme linked to a ligand or analog of said analyte.
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18. A method for determining an analyte in a sample suspected of containing said analyte comprising the steps of:
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a. combining said sample with a single stranded DNA template capable of replication and comprising a starting nucleotide sequence, a deoxyribonucleotide precursor, a DNA polymerase enzyme, a compound capable of generating a detectable signal in the presence of double stranded DNA, and a primer, said primer linked to a receptor capable of binding with said analyte and comprising a sequence complementary to said starting sequence of said template, under conditions favorable for DNA replication, b. monitoring the generation of double stranded DNA by said enzyme by measuring the signal produced by said compound, and c. correlating the production of said signal with the presence or amount of said analyte in said sample.
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26. A reagent for determining an analyte in a sample via a method involving inhibition of DNA replication, said reagent comprising a primer bound to a receptor capable of binding with said analyte
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27. A test kit for the determination of an analyte comprising in packaged combination:
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a single stranded DNA template capable of replication and comprising a starting nucleotide sequence, a primer linked to a ligand or analog of said analyte, said primer comprising a sequence complementary to said starting sequence of said template, a DNA polymerase enzyme, a deoxyribonucleotide precursor, a receptor capable of binding with said analyte, and a compound capable of generating a detectable signal in the presence of double stranded DNA.
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35. A test kit for the determination of an analyte comprising in packaged combination:
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a single stranded DNA template capable of replication and comprising a starting nucleotide sequence, a primer linked to a receptor capable of binding with said analyte, said primer comprising a sequence complementary to said starting sequence of said template, a DNA polymerase enzyme, a deoxyribonucleotide precursor, and a compound capable of generating a detectable signal in the presence of double stranded DNA.
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43. A test kit for the determination of an analyte comprising in packaged combination:
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a single stranded DNA template capable of replication and comprising a starting nucleotide sequence, a primer comprising a sequence complementary to said starting sequence of said template, a DNA polymerase enzyme, said enzyme linked to a ligand or analog of said analyte, a deoxyribonucleotide precursor, and a compound capable of generating a detectable signal in the presence of double stranded DNA. - View Dependent Claims (44, 45, 46, 47, 48, 49)
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Specification