ONE-STEP IMMUNOASSAYS EXHIBITING INCREASED SENSITIVITY AND SPECIFICITY

• US 20100267055A1
• Filed: 04/19/2010
• Published: 10/21/2010
• Est. Priority Date: 10/21/2007
• Status: Abandoned Application

• Current Assignee: Abbott Laboratories Incorporated
• Original Assignee: Abbott Laboratories Incorporated
• Inventors: KONRATH, JOHN G.
• Application Number:
  US12/762,903
• Publication Number:
  US 20100267055A1
• Time in Patent Office: Days
• US Class Current: 435/7.900
• CPC Class Codes: G01N 33/54393 Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding

Abstract

The present disclosure relates to immunoassays for detecting or quantifying at least one analyte of interest in a test sample which exhibits improved sensitivity or sensitivity and specificity compared to the immunoassay formats known in the art.

Claims

1. An immunoassay comprising the steps of:

a) incubating for a first incubation period a first mixture comprising;

(1) a test sample being assessed for at least one analyte of interest, (2) a first specific binding partner that is immobilized on a solid phase, wherein the first specific binding partner binds to the at least one analyte of interest, and (3) a second specific binding partner labeled with a first detectable label, wherein the analyte, first specific binding partner and second specific binding partner form a solid phase-first specific binding partner-analyte-second specific binding partner complex;

b) capturing the solid phase-first specific binding partner-analyte-second specific binding partner complex and isolating the solid phase-first specific binding partner-analyte-second specific binding partner complex from the supernatant of the first mixture;

c) removing freely accessible unbound second specific labeled binding partner from the captured solid phase first specific binding partner-analyte-second specific binding partner complex;

d) resuspending the captured solid phase-first specific binding partner-analyte-second specific binding partner complex to form a second mixture comprising resuspended solid phase-first specific binding partner-analyte-second specific binding partner complex;

e) incubating the second mixture for a second incubation period;

f) capturing the resuspended solid phase-first specific binding partner-analyte-second specific binding partner complex and isolating the solid phase-first specific binding partner-analyte-second specific binding partner complex from the supernatant of the second mixture;

g) removing residual unbound second specific labeled binding partner from the second mixture; and

h) detecting the labeled second specific binding partner from the first specific binding partner-analyte-second specific binding partner complex in step g) as a measure of the at least one analyte of interest.

2. The immunoassay of claim 1, wherein the freely accessible unbound second specific labeled binding partner is removed from the first mixture in step c) by washing.

3. The immunoassay of claim 2, wherein the captured solid-phase first specific binding partner-analyte-second specific binding partner complex is washed with a buffer or diluent, a salt, a protein, a polymer, an organic solvent or any combinations thereof.

4. The immunoassay of claim 3, wherein the buffer or diluent contains at least one additive.

5. The immunoassay of claim 1, wherein the residual unbound second specific labeled binding partner is removed from the second mixture in step g) by washing.

6. The immunoassay of claim 1, wherein the second mixture of step d) further comprises a third specific binding partner and the first mixture to form a second mixture, wherein said third specific binding partner is labeled with a second detectable label.

7. The immunoassay of claim 1, wherein said first specific binding partner is an antibody or an antigen.

8. The immunoassay of claim 7, wherein said antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a human antibody, and an affinity maturated antibody.

9. The immunoassay of claim 1, wherein the solid phase is selected from the group consisting of a magnetic particle, bead, test tube, microtiter plate, cuvette, membrane, a scaffolding molecule, film, filter paper, disc, and chip.

10. The immunoassay of claim 1, wherein the first detectable label is selected from the group consisting of a radioactive label, an enzymatic label, a chemiluminescent label, a fluorescent label, a thermometric label, and an immuno-polymerase chain reaction label.

11. The immunoassay of claim 6, wherein the second detectable label is selected from the group consisting of a radioactive label, an enzymatic label, a chemiluminescent label, a fluorescent label, a thermometric label, and an immuno-polymerase chain reaction label.

12. The immunoassay of claim 6, wherein the first detectable label and the second detectable label are the same.

13. The immunoassay of claim 6, wherein the first detectable label and the second detectable label are different.

14. The immunoassay of claim 1, wherein the second specific binding partner is immobilized on a solid phase.

15. The immunoassay of claim 14, wherein the solid phase is selected from the group consisting of a magnetic particle, bead, test tube, microtiter plate, cuvette, membrane, a scaffolding molecule, film, filter paper, disc, and chip.

16. The immunoassay of claim 6, wherein the second specific binding partner and the third specific binding partner are the same.

17. The immunoassay of claim 6, wherein the second specific binding partner and the third specific binding partner are different.

18. The immunoassay of claim 6, wherein the third specific binding partner comprises multiple binding partners.

19. The immunoassay of claim 1, wherein the first incubation period comprises a period of from about 5 minutes to about 60 minutes.

20. The immunoassay of claim 1, wherein the second incubation period comprises a period of from about 30 seconds to about 30 minutes.

21. The immunoassay of claim 1, wherein the immunoassay relates the amount of said first specific binding partner-analyte-second specific binding partner complex in step g) to the amount of the at least one analyte of interest in the test sample either by use of a standard curve for the analyte, or by comparison to a reference standard.

22. The immunoassay of claim 6, wherein the amount of the at least one analyte of interest in the test sample is quantitated by measuring the amount of the second detectable label.