Rapid Homogeneous Immunoassay Using Electrophoresis

• US 20080230388A1
• Filed: 01/31/2008
• Published: 09/25/2008
• Est. Priority Date: 02/07/2007
• Status: Active Grant

• Current Assignee: Lyzer Diagnostics, Inc.
• Original Assignee: Lyzer Diagnostics, Inc.
• Inventors: Tyrrell, Steven Patrick, Vant-Hull, Barry
• Granted Patent: US 9,081,006 B2
• Time in Patent Office: Days
• US Class Current: 204/451
• CPC Class Codes: B01D 57/02 by electrophoresis treatment of water, waste water, sewage or sludge by electrophoresis C02F1/469; electrophoretic production of compounds or non-metals C25B7/00; investigating or analysing materials by using electrophoresis G01N27/26
• CPC Class Codes: G01N 27/447 using electrophoresis
• CPC Class Codes: G01N 33/561 Immunoelectrophoresis

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.

Claims

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.

2. ) The method of claim 1, further comprising:

i) performing the electrophoresis in the presence of an essentially uncharged polymer that is capable of binding to the capture molecule to become part of the complex.

3. ) The method of claim 1, wherein the binding agents are antibodies or antigen-binding fragments thereof.

4. ) The method of claim 1, wherein one or both of the binding agents are biological receptors or fragments thereof.

5. ) The method of claim 3, wherein the antibodies are monoclonal antibodies.

6. ) The method of claim 1, wherein the target molecule is a protein or peptide.

7. ) The method of claim 1, wherein the electrophoresis is performed in a tube, channel or container with a minimum hydrodynamic radius, equal to twice the cross-sectional area divided by the circumference, of 0.5 mm.

8. ) The method of claim 7, wherein sample is loaded at the top of the tube, channel or container and the electrophoresis is performed in a gradient of increasing density and/or viscosity from top to bottom of the tube, channel or container, the gradient comprising at least two phases of different density or viscosity.

9. ) The method of claim 7, wherein sample is loaded at or near the bottom of the tube, channel or container and the electrophoresis is performed in a gradient of increasing density and/or viscosity from top to bottom of the tube, channel or container, the gradient comprising at least two phases of different density or viscosity.

10. ) The method of claims 8, wherein the gradient comprises a sample layer, a capture layer and a stacking layer.

11. ) The method of claim 10, wherein the gradient comprises a sample layer mixed with the capture layer and a stacking layer.

12. ) The method of claim 10, wherein the capture molecule is biotin and the electrophoresis is performed in the presence of a biotin binding protein.

13. ) The method of claim 12, wherein the biotin binding protein is selected from the group consisting of avidin, streptavidin and neutravidin.

14. ) The method of claims 12, wherein the electrophoresis is performed in the presence of a neutral polymer conjugated to biotin.

15. ) The method of claim 14, wherein the complex comprises target molecule, first binding agent, second binding agent, a multivalent biotin binding agent selected from avidin, streptavidin or neutravidin and one or more polymers conjugated to biotin.

16. ) The method of claim 15, wherein formation of the complex results in an increase of the mass to charge ratio and a decrease in electrophoretic mobility of the target molecule and complex.

17. ) The method of claim 15, wherein formation of the complex comprising target molecule, first binding agent, second binding agent, multivalent biotin binding agent and one or more polymers conjugated to biotin self-assemble in solution to form a detectable low mobility complex.

18. ) The method of claim 15, wherein the complex is concentrated at the stacking layer to improve detection.

19. ) The method of claim 18, wherein the complex becomes essentially immobile at the stacking layer.

20. ) The method of claim 18, wherein first binding agent that is not part of a complex migrates through the stacking layer and is separated from the complex.

21. ) The method of claim 1, further comprising:

ii) measuring the amount of detection molecule in the complex to determine the concentration of the target molecule in the sample.

22. ) The method of claim 1, wherein the detection molecule is fluorescent, luminescent, chemiluminescent, a radionuclide or an enzyme that produces a fluorescent, luminescent, chemiluminescent or colored product.

23. ) An apparatus for detecting a target molecule comprising:

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.

24. ) The apparatus of claim 23, further comprising:

i) target molecule, first binding agent conjugated to detection molecule and second binding molecule conjugated to capture molecule, wherein the target molecule, first and second binding molecules are capable of forming a complex in the sample layer;

ii) an essentially uncharged polymer in the capture layer that is capable of binding to the capture molecule to become part of the complex.