|Title:||Method for detecting a target compound in a substance using a nucleic acid ligand|
|Abstract:||This invention discloses novel detection methods for determining the presence of a target compound in a substance using nucleic acid ligands as detection molecules. Specifically, the substance is bound to a solid support matrix, such as those used in blot procedures, and detection of the target molecule is accomplished using the affinity and specificity of nucleic acid ligands to the target molecule. The method utilized herein for identifying and preparing said nucleic acid ligands is called SELEX. The method of the present invention is additionally useful to isolate the target compounds from the various substances.|
|Inventor(s):||Drolet; Dan (Boulder, CO), Jayasena; Sumedha D. (Boulder, CO), Gold; Larry (Boulder, CO)|
|Assignee:||NeXstar Pharmaceuticals, Inc. (Boulder, CO)|
|Filing Date:||Apr 05, 1996|
|Claims:||1. A method for detecting the presence of a target compound in a complex mixture which may contain said target compound comprising: |
a) identifying a nucleic acid ligand from a candidate mixture of nucleic acids, said nucleic acid ligand being a ligand of said target compound, by the method comprising:
i) contacting the candidate mixture with said target compound, wherein nucleic acids having an increased affinity to said target relative to the candidate mixture may be partitioned from the remainder of the candidate mixture;
ii) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture;
iii) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids; and
iv) identifying said nucleic acid ligand;
b) immobilizing complex mixture which may contain said target compound to a solid support;
c) exposing said mixture to a nucleic acid ligand identified by the method of step (a) to said target compound wherein said nucleic acid ligand binds to said target compound forming a nucleic acid ligand:target compound complex; and
d) detecting said nucleic acid ligand:target compound complex.
2. The method of claim 1 wherein said nucleic acid ligand comprises an enzyme linked to said nucleic acid ligand.
3. The method of claim 2 wherein detection is accomplished by addition of a substrate which said enzyme can hydrolyze and produce a measurable color.
4. The method of claim 2 wherein said enzyme is selected from the group consisting of horseradish peroxidase, alkaline phosphatase, and b-galactosidase.
5. The method of claim 1 wherein said solid support is selected from the group consisting of nitrocellulose, nylon, and charged paper.
6. The method of claim 1 wherein said target compound is a protein.
7. The method of claim 6 wherein said protein is selected from the group consisting of VEGF, hCG and hTSH.
8. The method of claim 1 wherein said complex mixture is a biological fluid.
9. The method of claim 8 wherein said biological fluid is selected from the group consisting of blood, plasma, serum, sputum, urine, semen, cerebrospinal fluid, bronchial aspirate, and macerated tissue.
10. The method of claim 1 wherein said detection is achieved by PCR amplification of said nucleic acid ligand.
11. The method of claim 10 wherein primers used for PCR amplification further comprise detection moieties.
12. The method of claim 11 wherein said detection moieties are selected from the group consisting of biotin and enzymes.