US 2005/0239108 A1Published application

Immuno-PCR method for the detection of a biomolecule in a test sample

AssigneeUniversity of Maryland, Baltimore

InventorsJanet Barletta, Niel Constantine, Daniel Edelman, Mark Manak, Jay Ji, Chang-Chih Tai, William Highsmith

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Abstract

The invention relates to methods and kits for detecting and/or monitoring biological molecules in a test sample. For example, the invention relates to methods and kits for detecting and/or monitoring HIV p24 antigen in human body fluid, biological toxins such as ricin or botulism in an environmental or biological sample, and prion protein from human, deer or bovine, such as PrPSC, in a biological sample. The antigen detection signal is boosted by amplification of a polynucleotide linked to a detector molecule using methods for nucleic acid amplification technology.

Claims89

Dependent claims

A method for detecting a biomolecule in a sample, said method comprising:

(a) incubating a sample with a capture molecule that specifically binds a selected biomolecule under conditions such that said selected biomolecule in said sample is bound by said capture molecule, wherein said capture molecule comprises a molecule that specifically binds to said biomolecule and said capture molecule is attached to a solid support,
(b) incubating the resulting product of step (a) with a detector molecule that specifically binds said selected biomolecule under conditions such that said selected biomolecule bound by said capture molecule is also bound by said detector molecule, wherein said detector molecule comprises (i) a molecule that specifically binds to said biomolecule and (ii) at least one biotin moiety,
(c) incubating the resulting product of step (b) with a linker molecule under conditions such that said linker molecule binds to a biotin moiety of said detector molecule, wherein said linker molecule comprises at least one avidin moiety or at least one streptavidin moiety, or a combination thereof,
(d) incubating the resulting product of step (c) with an amplification reagent under conditions such that said amplification reagent binds to said linker molecule, wherein said amplification reagent comprises (i) a polynucleotide molecule and (ii) at least one biotin moiety,
(e) performing polymerase chain reaction (PCR) on the resulting product of step (d) in the presence of a signal molecule, and
(f) detecting a PCR product produced in step (e) by detecting a signal from the signal molecule, thereby detecting a biomolecule in said sample.

3 more elements

A method for detecting a biomolecule in a sample, said method comprising:

(a) incubating a sample with a capture molecule that specifically binds a selected biomolecule under conditions such that said selected biomolecule in said sample is bound by said capture molecule, wherein said capture molecule comprises a molecule that specifically binds to said biomolecule and said capture molecule is attached to a solid support,
(b) incubating the resulting product of step (a) with a detector molecule that specifically binds said selected biomolecule under conditions such that said selected biomolecule bound by said capture molecule is also bound by said detector molecule, wherein said detector molecule comprises (i) a molecule that specifically binds to said biomolecule and (ii) at least one avidin moiety or at least one streptavidin moiety, or a combination thereof,
(c) incubating the resulting product of step (b) with an amplification reagent under conditions such that said amplification reagent binds to said avidin or said streptavidin, or both, of said detector molecule, wherein said amplification reagent comprises (i) a polynucleotide molecule and (ii) at least one biotin moiety,
(d) performing polymerase chain reaction (PCR) on the resulting product of step (c) in the presence of a signal molecule, and
(e) detecting a PCR product produced in step (d) by detecting a signal from the signal molecule, thereby detecting a biomolecule in said sample.

2 more elements

A method for detecting bovine PrPSC in a sample, said method comprising:

(a) incubating a sample with a capture molecule that specifically binds bovine PrPSC under conditions such that bovine PrPSC in said sample is bound by said capture molecule, wherein said capture molecule comprises a molecule that specifically binds to bovine PrPSC and said capture molecule is attached to a solid support,
(b) incubating the resulting product of step (a) with a detector molecule that specifically binds bovine PrPSC under conditions such that bovine PrPSC bound by said capture molecule is also bound by said detector molecule, wherein said detector molecule comprises (i) a molecule that specifically binds to bovine PrPSC and (ii) at least one biotin moiety,
(c) incubating the resulting product of step (b) with a linker molecule under conditions such that said linker molecule binds to a biotin moiety of said detector molecule, wherein said linker molecule comprises at least one avidin moiety or at least one streptavidin moiety, or a combination thereof,
(d) incubating the resulting product of step (c) with an amplification reagent under conditions such that said amplification reagent binds to said linker molecule, wherein said amplification reagent comprises (i) a polynucleotide molecule and (ii) at least one biotin moiety,
(e) performing polymerase chain reaction (PCR) on the resulting product of step (d) in the presence of a signal molecule, and
(f) detecting a PCR product produced in step (e) by detecting a signal from the signal molecule, thereby detecting bovine PrPSC in said sample.

3 more elements

A kit comprising:

(a) a first capture molecule attached to a solid support, wherein said first capture molecule comprises a molecule that specifically binds to a pre-selected biomolecule,
(b) a second capture molecule attached to said solid support, wherein said second capture molecule comprises a molecule that specifically binds to an internal control molecule,
(c) an internal control molecule,
(d) a first detector molecule-amplification reagent conjugate, said conjugate comprising a first detector molecule attached to a first polynucleotide molecule, wherein said first detector molecule comprises a molecule that specifically binds to said pre-selected biomolecule,
(e) a second detector molecule-amplification reagent conjugate, said conjugate comprising a second detector molecule attached to a second polynucleotide molecule, wherein said second detector molecule comprises a molecule that specifically binds to said internal control molecule, and wherein said second polynucleotide molecule is different from said first polynucleotide molecule,
(f) a first oligonucleotide primer that specifically binds to said first polynucleotide molecule,
(g) a second oligonucleotide primer that specifically binds to said second polynucleotide molecule,
(h) a first signal molecule comprising (i) an oligonucleotide molecule that specifically binds the first polynucleotide molecule, (ii) a first fluorophore and (iii) a quencher dye,
(i) a second signal molecule comprising (i) an oligonucleotide molecule that specifically binds the second polynucleotide molecule, (ii) a second fluorophore that can be distinguish from said first fluorophore and (iii) a quencher dye,
(j) a first set of standards, wherein said first set of standards allows an approximation of concentration of said pre-selected biomolecule in a sample, and
(k) a second set of standards, wherein said second set of standards allows an approximation of concentration of said internal control molecule in a sample.

8 more elements

Dependencies are inferred from the claim text.