Rapid homogeneous immunoassay using electrophoresis

摘要

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.

主权项

1. A method for detecting the presence of a target molecule in a sample performed entirely in solution phase, comprising:
a) detecting the presence of a target molecule in a sample by subjecting the sample, to determine if the sample contains the target molecule, to electrophoresis entirely in a conductive aqueous solution phase, further comprising
(i) a first binding agent conjugated to a detection molecule and(ii) a second binding agent conjugated to a capture molecule, wherein the target molecule, when present in the sample, binds to the first and second binding agents to form a target complex, wherein the electrophoresis is performed
(iii) in stacked layers comprising at least one of each of a sample layer that is mixed with, a capture layer and a stacking layer of entirely conductive aqueous solutions; wherein the sample layer comprises the target molecule, detection conjugate and capture conjugate; wherein the electrophoresis moves (i) non-complexed and (ii) complexed molecules comprising said target complex through the capture layer, wherein the capture layer comprises uncharged liquid polymers that bind a cognate binding agent provided in the capture molecule; wherein the electrophoresis moves said target complex through the capture layer and separates non-complexed molecules from the complexed molecules comprising the target complex that are bound to the uncharged liquid polymers through the cognate binding agent of the capture molecule; wherein the electrophoresis moves said complex through the stacking layer, wherein the stacking layer comprises uncharged liquid polymers that impede the mobility of complexes bound to the uncharged liquid polymers that becomes concentrated and immobile in the stacking layer in order to separate the non-complexed molecules from the complexed molecules comprising the target complex that are bound to the uncharged liquid polymers; and wherein the presence of the target complex is detected by measuring or detecting the amount or presence of the detection molecule provided in the target complex.