Rapid Homogeneous Immunoassay Using Electrophoresis
First Claim
1. ) A method for detecting a target molecule comprising:
- a) obtaining a sample that may contain the target molecule;
b) subjecting the sample to vertical stacked electrophoresis in the presence of a first binding agent conjugated to a detection molecule and a second binding agent conjugated to a capture molecule, wherein the target molecule binds to the first and second binding agents to form a complex; and
c) detecting the presence of the complex formed by the target molecule and first and second binding agents.
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Abstract
The present invention concerns methods, compositions and apparatus for detection and/or determination of concentration of target molecules. In preferred embodiments the methods comprise allowing target molecules to form a complex with a first binding agent conjugated to a detection molecule and a second binding agent conjugated to a capture molecule, allowing the complex to further bind to an essentially uncharged polymer capable of binding to the capture agent, and performing a vertical gradient electrophoresis to separate the complex from unbound target, first and second binding agents and polymer. The intact complex is concentrated by electrophoresis at a stacking layer and the detection molecule is detected and/or quantified. Because the complex contains a very high mass to charge ratio, it becomes essentially immobile at the stacking layer, while unbound components migrate through the stacking layer and are separated from the complex. This provides a very rapid and sensitive assay that can detect very low concentrations of target molecules in short time.
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Citations
24 Claims
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1. ) A method for detecting a target molecule comprising:
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a) obtaining a sample that may contain the target molecule; b) subjecting the sample to vertical stacked electrophoresis in the presence of a first binding agent conjugated to a detection molecule and a second binding agent conjugated to a capture molecule, wherein the target molecule binds to the first and second binding agents to form a complex; and c) detecting the presence of the complex formed by the target molecule and first and second binding agents. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)
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23. ) An apparatus for detecting a target molecule comprising:
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a) a vertically arranged tube, channel or container with a hydrodynamic radius of at least 0.5 mm; b) a gradient of increasing density within the tube, channel or container, said gradient comprising a sample layer, a capture layer and a stacking layer. - View Dependent Claims (24)
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Specification