UNIVERSAL TANDEM SOLID-PHASES BASED IMMUNOASSAY

• US 20100009394A1
• Filed: 07/14/2008
• Published: 01/14/2010
• Est. Priority Date: 07/14/2008
• Status: Active Grant

• Current Assignee: Ribo Guo
• Original Assignee: Ribo Guo
• Inventors: Guo, Ribo
• Granted Patent: US 8,021,850 B2
• Time in Patent Office: Days
• US Class Current: 435/7.940
• CPC Class Codes: G01N 33/5082 Supracellular entities, e.g. tissue, organisms
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms

Abstract

Universal tandem solid-phases based immunoassay (UTSIA) is a sandwich-ELISA equivalent assay for low abundance antigen determination that overcomes limitations of sandwich-ELISA (antibody inactivation by solid phase and strict requirement of a pair of primary and secondary antibodies) by using an affinity binding solid phase to capture antigen specifically from a fluid sample, sequentially dissociating the antigen, transferring, and coating the antigen to a non-affinity binding solid phase for specific antigen determination. Cell-based UTSIA is a cell-based ELISA equivalent assay that overcomes limitations of image method for determining an antigen in the cells or tissue immobilized on a solid phase by dissociating and transferring the detection antibody bound on the antigen of the cells or tissue immobilized on the solid phase to a second solid phase and immobilizing the detection antibody there for specific detection of the antigen via the detection of the detection antibody.

Claims

1. An alternative method of sandwich-ELISA for detecting and quantifying an analyte in a fluid sample, the universal tandem solid-phases based immunoassay (UTSIA), which comprises:

(a) an affinity binding solid phase based specific analyte capture process to capture the analyte from the fluid sample via contacting the fluid sample with the affinity binding solid phase;

(b) a dissociation buffer based process to release the analyte captured on the affinity binding solid phase into a liquid phase via incubating the affinity binding solid phase bound with the analyte with the dissociation buffer;

(c) a non-affinity binding solid phase based analyte physical adsorption process to coat the analyte released in the liquid phase of the dissociation buffer on the non-affinity binding solid phase for further specific analyte detection and quantitation via incubating the dissociation buffer containing the analyte released with the non-affinity binding solid phase; and

(d) a specific analyte determining process via incubating the analyte coated on the non-affinity binding solid phase with a labeled detection molecule capable of specific binding with the analyte coated, or with an unlabeled detection molecule capable of specific binding with the analyte coated and with a labeled molecule capable of specific binding with the unlabeled detection molecule bound.

2. The method of claim 1(a), wherein said affinity binding solid phase comprises (i) a solid phase immobilized with an adaptor molecule, wherein the adaptor molecule immobilized is capable of binding with a capture molecule pre-added in said fluid sample which is capable of specifically binding with said analyte, (ii) a solid phase immobilized with a capture molecule, wherein the capture molecule immobilized on the solid phase is capable of binding with said analyte in said fluid sample specifically, or (iii) a solid phase immobilized with a capture molecule via an adaptor molecule wherein the capture molecule is capable of binding with said analyte in said fluid sample specifically.

3. The method of claim 1, wherein said solid phase comprises polystyrene support.

4. The method of claim 2, wherein said adaptor molecule comprises molecules capable of protecting the capture molecule from inactivation by a solid phase and immobilizing the capture molecule on the solid phase.

5. The method of claim 2, wherein said adaptor molecule comprises Protein G.

6. The method of claim 2, wherein said capture molecule comprises antibody.

7. The method of claim 1, wherein said analyte comprises protein.

8. The method of claim 1, wherein said fluid sample comprises cell lysate, blood, serum, plasma, salvia, urine, spinal fluid, and other biological fluids or fluids made from biological materials.

9. The method of claim 1(b), wherein said dissociation buffer comprises acidic or basic buffer which is capable of breaking the affinity binding between the capture molecule and the analyte and releasing the analyte into liquid phase of the buffer from solid phase, while the buffer is not capable of releasing the adaptor molecule immobilized on solid phase into the liquid phase, and not capable of blocking the analyte'"'"'s physical adsorption on non-affinity binding solid phase.

10. The method of claim 1(d), wherein said labeled means that a molecule is tagged with another molecule which is detectable directly, or indirectly by adding additional components.

11. The method of claim 1, wherein said detection molecule comprises antibody.

12. The method of claim 1(d), wherein said labeled molecule comprises labeled antibody.

13. An alternative method of image method for detecting and quantifying an analyte in a tissue or cells immobilized on a solid phase, the cell-based UTSIA, which comprises:

(a) a specific analyte detection process via incubating the tissue or cells immobilized on said solid phase with a labeled detection molecule capable of specific binding with the analyte, or with an unlabeled detection molecule capable of specific binding with the analyte;

(b) a dissociation buffer based process to release the labeled detection molecule or unlabeled detection molecule bound on the analyte in the tissue or cells immobilized on the solid phase into a liquid phase of the dissociation buffer via incubating the solid phase bound with the labeled detection molecule or unlabeled detection molecule with the dissociation buffer;

(c) (1) a non-affinity binding solid phase based physical adsorption process that the labeled detection molecule or unlabeled detection molecule released in the liquid phase of the dissociation buffer is coated on the non-affinity binding solid phase for further specific detection and quantitation, via incubating the liquid phase of the dissociation buffer containing the labeled detection molecule or unlabeled detection molecule released with the non-affinity binding solid phase;

or (2) an affinity binding solid phase based labeled detection antibody capture process, in which the solid phase is pre-immobilized with a molecule which is capable of binding and capturing the labeled detection molecule in the dissociation buffer, and the labeled detection molecule released in the liquid phase of the dissociation buffer is immobilized on the affinity binding solid phase for further detection and quantitation, via incubating the liquid phase of the dissociation buffer containing the labeled detection molecule released with the affinity binding solid phase;

(d) a specific analyte determining process via detecting and quantifying the labeled detection molecule on the non-affinity binding solid phase or on the affinity binding solid phase directly or indirectly;

or via incubating the unlabeled detection molecule coated on the non-affinity binding solid phase with a labeled molecule capable of specific binding with the unlabeled detection molecule coated and then detecting and quantifying the labeled molecule bound on the non-affinity binding solid phase directly or indirectly;

wherein the amount of analyte is correlated with the amount of labeled or unlabeled detection molecule measured.

14. The method of claim 13(c)(1), wherein said molecule which is capable of binding and capturing the detection molecule in the dissociation buffer comprises protein G.

15. The method of claim 13, wherein said analyte comprises protein.

16. The method of claim 13, wherein said solid phase comprises glass and plastic support.

17. The method of claim 13, wherein said detection molecule comprises antibody.

18. The method of claim 13(b), wherein said dissociation buffer comprises acidic or basic buffer which is capable of breaking the affinity binding between the labeled detection molecule or unlabeled detection molecule and the analyte, and releasing the labeled detection molecule or unlabeled detection molecule that bound on the analyte in tissue or cells immobilized on solid phase into liquid phase of the buffer, while the buffer is not capable of releasing the tissue or cells immobilized on the solid phase into the liquid phase of the buffer.

19. The method of claim 13(c), wherein said solid phase comprises hydrophobic solid phase such as polystyrene support.

20. The method of claim 13(d), wherein said labeled molecule comprises labeled antibody.

21. The method of claim 13, wherein said labeled means that a molecule is tagged with another molecule which is detectable directly, or indirectly by adding additional components.