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US 5,843,680 AGranted
Differential separation assay methods and test kits
AssigneeBiometric Imaging, Inc.
InventorsBala S. Manian, Vartan E. Ghazarossian, Paul G. Hayter
Life of this patent
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Abstract
Analytes can be detected from among closely related substances by reacting the analyte in a test sample with a labeled binding agent which specifically binds to the analyte to form a complex. The labeled binding agent is supplied in excess, and the complex is identified through a time window relative to the detection of the excess unbound agent. The complex and labeled binding agent are isolated on a separation media and identified by the differential rate of migration.
Claims15
A method for detecting an analyte in a test sample comprising:
- a) labeling a specific binding agent with a detectable label, wherein the labeled binding agent specifically binds to the analyte to form a complex;
- b) providing a separation medium on which the labeled binding agent and complex migrate at different velocities during electrophoresis;
- c) contacting the test sample with an excess amount of labeled binding agent to produce a reaction mixture containing the labeled binding agent and the complex;
- d) separating the labeled binding agent and complex by applying the reaction mixture to the separation medium to separate the complex from the labeled specific binding agent by electrophoresis;
- e) measuring and recording the migration of the labeled binding agent and the complex as a function of time between at least two reference points on the separation medium;
- f) defining a time window using predetermined migration values of the labeled binding agent and complex between at least two reference points on the separation medium;
- and
- g) comparing the recorded migration of the complex with the time window as a time domain filter to determine the presence of the analyte, wherein finding the complex within the time window indicates presence of the analyte.
5 more elements
A method for measuring the concentration of an analyte which forms a complex with a specific binding agent in a test sample comprising:
- (a) labeling the specific binding agent with a detectable label;
- (b) contacting the test sample with an amount of the labeled binding agent in excess of what will react with the analyte so as to form a complex with analyte, and produce a reaction mixture containing the labeled binding agent and the complex;
- (c) providing a separation medium on which the labeled binding agent and complex migrate at different velocities;
- (d) separating the labeled binding agent and complex by applying said reaction mixture to the separation medium for separation by electrophoresis;
- (e) measuring and recording the migration of the labeled binding agent and the complex as a function of time between at least two reference points on the separation medium by detecting the label wherein the recorded migrations are characterized as peaks;
- (f) defining a time window statistically using predetermined migration times for the labeled specific binding agent and complex between at least two reference points on the separation medium;
- and
- (g) comparing the migration of the complex with the time window as a time domain filter to determine the presence of the analyte, wherein detecting the labeled complex within the time window indicates presence of the analyte;
- (h) measuring the area under each peak of the detected label within the time window;
- and
- (i) normalizing the areas under each peak of the labeled complex to determine the concentration of the analyte.
8 more elements
A method for assaying the concentration of an analyte which forms an analyte complex with a binding agent in a competition reaction, comprising the steps of:
- labeling the binding agent with a detectable first label;
- labeling an antigen with a detectable second label, wherein the antigen is capable of forming an antigen complex with the specific binding agent;
- contacting the analyte and the labeled antigen with an amount of the labeled binding agent to produce a reaction mixture including the labeled binding agent, the analyte complex, the labeled antigen, and the antigen complex;
- providing a separation medium upon which the labeled binding agent and the antigen and analyte complexes migrate at different velocities;
- separating the labeled binding agent and the antigen and analyte complexes by applying said reaction mixture to the separation medium for separation by electrophoresis;
- measuring and recording the migration of the labeled binding agent, the analyte complex, and the antigen complex between at least two reference points on the separation medium by detecting the first label, wherein the recorded migration of the labeled binding agent, the analyte complex, and the antigen complex are characterized as peaks;
- measuring and recording the migration of the labeled antigen and the antigen complex between at least two reference points on the separation medium by detecting the second label, wherein the recorded migration of the antigen and the antigen complex are characterized as peaks;
- measuring the area under the peaks of the measured labeled binding agent, analyte complex, and antigen complex according to the detected first label;
- measuring the area under the peaks of the measured labeled antigen, and antigen complex according to the detected second label;
- determining a ratio between the area under the peak of the measured labeled antigen and the area under the peaks of the measured antigen and analyte complexes;
- and
- determining the concentration of analyte by relating the determined ratio to an assay standard curve representing the competition reaction between the analyte and antigen for the binding agent.
9 more elements
Dependencies are inferred from the claim text.