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, including using a solid support, using mass spectrometry, and using quantitative polymerase chain reaction (Q-PCR).
1. A method for detecting a target molecule that may be present in a test sample, the method comprising:
(a) contacting a test sample with an aptamer having a specific affinity for a target molecule, wherein an aptamer affinity complex is formed by the interaction of an aptamer with its target molecule, if said target molecule is present in said test sample, wherein said aptamer binds to said target molecule through non-Watson Crick interactions, and wherein the aptamer has been previously selected to have binding affinity for the target molecule;
(b) after equilibration of aptamer affinity complex formation, exposing said test sample to conditions that kinetically challenge components of said test sample;
(c) at any point prior to (d), contacting said aptamer affinity complex with a labeling agent; and
(d) detecting and/or quantifying the aptamer affinity complex partitioned from the remainder of the test sample,
wherein the kinetic challenge comprises (i) introducing a competitor molecule to the test sample, 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.
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. The method of claim 1 wherein said target molecule is selected from the group consisting of a protein, a carbohydrate, a polysaccharide, a glycoprotein, a hormone, a receptor, an antigen, an antibody, a virus, a substrate, a metabolite, a cofactor, an inhibitor, a drug, a dye, a nutrient, a growth factor, and a tissue.
5. The method of claim 1 , wherein said test sample is a biological sample selected from whole blood, leukocytes, peripheral blood mononuclear cells, plasma, serum, sputum, breath, urine, semen, saliva, meningial 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.
6. A method for detecting a target molecule that may be present in a test sample, the method comprising:
(a) contacting a test sample with an aptamer having a specific affinity for a target molecule, wherein an aptamer affinity complex is formed by the interaction of an aptamer with its target molecule, if said target molecule is present in said test sample, wherein said aptamer binds to said target molecule through non-Watson Crick interactions, and wherein the aptamer affinity complex is a non-covalent complex, and wherein the aptamer has been previously selected to have binding affinity for the target molecule;
(b) after equilibration of aptamer affinity complex formation, exposing said test sample to conditions that kinetically challenge components of said test sample;
(c) at any point prior to (d), contacting said aptamer affinity complex with a labeling agent; and
(d) detecting and/or quantifying the aptamer affinity complex partitioned from the remainder of the test sample, wherein the kinetic challenge comprises (i) introducing a competitor molecule to the test sample, or (ii) the capture of aptamer affinity complexes on a solid support followed by washing with competitor molecules present in the wash solution.
7. The method of claim 6 , wherein said competitor molecule is a polyanion.
8. The method of claim 6 , wherein said conditions that kinetically challenge components of said test sample comprise introducing a competitor molecule to the test sample wherein said competitor molecule is independently selected from an oligonucleotide, a polyanion, an abasic phosphodiester polymer, a dNTP, a pyrophosphate, heparin, or a polydextran.
9. The method of claim 6 , wherein the polyanion is dextran sulphate.
10. The method of claim 6 , wherein said aptamer is a single-stranded nucleic acid, double-stranded nucleic acid, DNA or RNA.
11. The method of claim 6 wherein said target molecule is selected from the group consisting of a protein, a carbohydrate, a polysaccharide, a glycoprotein, a hormone, a receptor, an antigen, an antibody, a virus, a substrate, a metabolite, a cofactor, an inhibitor, a drug, a dye, a nutrient, a growth factor, and a tissue.
12. The method of claim 6 , wherein said test sample is a biological sample selected from whole blood, leukocytes, peripheral blood mononuclear cells, plasma, serum, sputum, breath, urine, semen, saliva, meningial 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.
13. The method of claim 1 , wherein said aptamer has been previously selected by SELEX.
14. The method of claim 6 , wherein said aptamer has been previously selected by SELEX.
15. 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 of the aptamer affinity complex.
16. The method of claim 6 , wherein said detecting and/or quantifying of the aptamer affinity complex comprises detecting and/or quantifying the aptamer and/or protein of the aptamer affinity complex.