Multiplexed analyses of test samples
The present disclosure describes methods, devices, reagents, and kits for the detection of one or more target molecules that may be present in a test sample. In one embodiment, a test sample is contacted with an aptamer that includes a tag and has a specific affinity for a target molecule. An aptamer affinity complex that includes an aptamer bound to its target molecule is allowed to form. If the test sample contains the target molecule, an aptamer affinity complex will generally form in the test sample. The aptamer affinity complex is optionally converted to an aptamer covalent complex that includes an aptamer covalently bound to its target molecule. The aptamer affinity complex (or optional aptamer covalent complex) can then be detected and/or quantified using any of a variety of methods known to one skilled in the art.
1. A method for detecting a protein or a peptide that may be present in a test sample, the method comprising:
(a) contacting a test sample with an aptamer, wherein an aptamer affinity complex is formed by the interaction of the aptamer with the protein or peptide if the protein or peptide is present in the test sample;
(b) incubating the test sample to allow aptamer affinity complex formation;
(c) after (b), (i) introducing a competitor molecule to the test sample, wherein the competitor molecule is non-specific for the protein or peptide, or (ii) the capture of aptamer affinity complexes on a solid support followed by washing with competitor molecules present in the wash solution, and wherein the competitor molecule is selected from the group consisting of an oligonucleotide, a polyanion, an abasic phosphodiester polymer, a dNTP, and a polydextran; and
(d) detecting and/or quantifying the aptamer of the aptamer affinity complex or the aptamer affinity complex partitioned from the remainder of the test sample;
wherein the test sample is a biological sample selected from the group consisting of blood, plasma, serum, sputum, breath, urine, semen, saliva, meningeal fluid, amniotic fluid, glandular fluid, lymph fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, cells, a cellular extract, stool, tissue, a tissue biopsy, and cerebrospinal fluid.
2. The method of claim 1 , wherein the polyanion is dextran sulphate.
3. The method of claim 1 , wherein said aptamer is a single-stranded nucleic acid, double-stranded nucleic acid, DNA or RNA.
4. A method for detecting a protein or peptide that may be present in a test sample, the method comprising:
(a) contacting a test sample with an aptamer, wherein an aptamer affinity complex is formed by the interaction of the aptamer with the protein or peptide if the protein or peptide is present in the test sample;
(b) incubating the test sample to allow aptamer affinity complex formation;
(c) after (b), (i) introducing a competitor molecule to the test sample, wherein the competitor molecule is non-specific for the protein or peptide, or (ii) the capture of aptamer affinity complexes on a solid support followed by washing with competitor molecules present in the wash solution; and
(d) detecting and/or quantifying the aptamer of the aptamer affinity complex or the aptamer affinity complex partitioned from the remainder of the test sample;
wherein the test sample is a biological sample selected from the group consisting of blood, plasma, serum, sputum, breath, urine, semen, saliva, meningeal fluid, amniotic fluid, glandular fluid, lymph fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, cells, a cellular extract, stool, tissue, a tissue biopsy, and cerebrospinal fluid.
5. The method of claim 4 , wherein said competitor molecule is a polyanion.
6. The method of claim 4 , wherein the competitor molecule is independently selected from an oligonucleotide, a polyanion, an abasic phosphodiester polymer, a dNTP, a pyrophosphate, heparin, and a polydextran.
7. The method of claim 5 , wherein the polyanion is dextran sulphate.
8. The method of claim 4 , wherein the aptamer is a single-stranded nucleic acid, double-stranded nucleic acid, DNA or RNA.
9. The method of claim 1 , wherein said aptamer has been previously selected by SELEX.
10. The method of claim 4 , wherein said aptamer has been previously selected by SELEX.
11. The method of claim 1 , wherein said detecting and/or quantifying of the aptamer affinity complex comprises detecting and/or quantifying the aptamer and/or protein or peptide of the aptamer affinity complex.
12. The method of claim 4 , wherein said detecting and/or quantifying of the aptamer affinity complex comprises detecting and/or quantifying the aptamer and/or protein or peptide of the aptamer affinity complex.
13. The method of claim 1 , wherein the aptamer comprises 5-(N-benzylcarboxyamide)-2′-deoxyuridine.
14. The method of claim 4 , wherein the aptamer comprises 5-(N-benzylcarboxyamide)-2′-deoxyuridine.
15. The method of claim 1 , wherein the aptamer comprises 5-(N-[2-(1H-indole-3yl)ethyl]carboxyamide)-2′-deoxyuridine.
16. The method of claim 4 , wherein the aptamer comprises 5-(N-[2-(1H-indole-3yl)ethyl]carboxyamide)-2′-deoxyuridine.
17. The method of claim 1 , wherein the aptamer comprises 5-(N-naphthylcarboxyamide)-2′-deoxyuridine.
18. The method of claim 4 , wherein the aptamer comprises 5-(N-naphthylcarboxyamide)-2′-deoxyuridine.