RAPID HOMOGENEOUS DIAGNOSTIC TESTING PLATFORM BASED ON LANTHANIDE FLUORESCENT RESONANCE ENERGY TRANSFER

• US 20100151591A1
• Filed: 11/02/2009
• Published: 06/17/2010
• Est. Priority Date: 10/31/2008
• Status: Abandoned Application

• Current Assignee: Lumiphore Inc.
• Original Assignee: Lumiphore Inc.
• Inventors: Mimms, Larry, Butlin, Nathaniel G.
• Application Number:
  US12/611,035
• Publication Number:
  US 20100151591A1
• Time in Patent Office: Days
• US Class Current: 436/501
• CPC Class Codes: G01N 2458/40 Rare earth chelates
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching

Abstract

The present invention provides compositions and methods for detecting the presence of an analyte in a sample. Exemplary compositions comprise macrocyclic lanthanide complexes that are useful for transferring energy to an energy transfer acceptor to indicate the presence of the analyte. The compositions and methods are particularly suited for detecting antibodies in a sample.

Claims

1. A composition comprising:

(a) a first probe comprising an energy transfer donor and a first targeting moiety;

(b) a second probe comprising an energy transfer acceptor and a second targeting moiety; and

(c) an antibody;

wherein the energy transfer donor comprises a macrocyclic moiety complexed to a lanthanide ion, the macrocyclic moiety having a structure according to Formula I;

2. The composition of claim 1 wherein the lanthanide ion is selected from the group consisting of samarium, dysprosium, europium and terbium.

3. The composition of claim 2 wherein the linker moieties L¹, L², L³, L⁴, L⁵, L⁶, L⁷, L⁸, L⁹ and L¹⁰ are members independently selected from substituted or unsubstituted C₁ to C₆ alkyl.

4. The composition of claim 3 wherein the linker moieties L¹, L², L³, L⁴, L⁵, L⁶, L⁷, L⁸, L⁹ and L¹⁰ are members independently selected from substituted or unsubstituted ethyl.

5. The composition of claim 4 wherein at least one of the linker moieties L¹, L², L³, L⁴, L⁵, L⁶, L⁷, L⁸, L⁹ and L¹⁰ is substituted by —

(CH₂)₄R, wherein R is selected from a bond, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl; and

wherein R links the donor to the first targeting compound.

6. The composition of claim 5 wherein the energy transfer donor has the structure:

7. The composition of claim 1 wherein the energy transfer acceptor is a fluorophore.

8. The composition of claim 1 wherein the energy transfer acceptor is a dark quencher.

9. The composition of claim 1 wherein the first targeting moiety and the second targeting moiety have substantially the same structure.

10. The composition of claim 9 wherein the first targeting moiety and the second targeting moiety are each epitopes to the antibody.

11. The composition of claim 1 wherein the first probe and the second probe are independently selected from the group consisting of a polypeptide, a nucleic acid, a lipid, a polysaccharide and a small molecule.

12. The composition of claim 11 wherein at least one of the first probe and the second probe is a polypeptide.

13. The composition of claim 12 wherein the polypeptide comprises a sequence of a viral peptide.

14. The composition of claim 13 wherein the viral peptide is derived from the HIV I or II proteome.

15. The composition of claim 1 wherein the antibody is not bound to a solid support.

16. The composition of claim 1 wherein the first probe, the second probe or both are bound to the antibody.

25. A method of detecting an analyte in a sample, comprising:

(a) contacting the sample with (i) a first probe comprising an energy transfer donor and a first targeting moiety and (ii) a second probe comprising an energy transfer acceptor and a second targeting moiety to form the composition of claim 16 wherein the analyte is the antibody; and

both the first probe and the second probe are bound to the antibody; and

(b) detecting the presence of the composition.

17. A kit comprising:

(a) a first probe comprising an energy transfer donor and a first targeting moiety;

(b) a second probe comprising an energy transfer acceptor and a second targeting moiety, wherein the first targeting moiety and the second targeting moiety have substantially the same structure; and

(c) instructions for using the first probe and the second probe to detect an analyte in a sample.

18. The kit of claim 17 further comprising a reaction vessel comprising the first probe and the second probe.

19. The kit of claim 17 wherein the first probe and the second probe are lyophilized.

20. The kit of claim 17 wherein the energy transfer donor is a fluorophore.

21. The kit of claim 17 wherein the energy transfer donor has a structure according to Formula I above.

22. The kit of claim 17 wherein the energy transfer acceptor is a fluorophore.

23. The kit of claim 17 wherein the energy transfer acceptor is a dark quencher.

24. The kit of claim 17 wherein the first targeting moiety and the second targeting moiety are each epitopes to a same antibody.

29. A method of detecting an analyte in a sample comprising:

(a) using the kit of claim 17 to form an assay complex by contacting the sample with the first probe and the second probe, wherein the assay complex comprises the first probe, the second probe and the analyte, wherein both the first probe and the second probe are bound to the analyte; and

(b) detecting the presence of the assay complex.

30. The method of claim 29 wherein the detecting step comprises detecting fluorescence resonance energy transfer (FRET) between the energy transfer donor and the energy transfer acceptor.

26. A method of detecting a first analyte and a second analyte in a sample, comprising:

(a) contacting the sample with (i) a first probe comprising a first energy transfer donor and a first targeting moiety;

(ii) a second probe comprising a first energy transfer acceptor and a second targeting moiety;

(iii) a third probe comprising a second energy transfer donor and a third targeting moiety; and

(iv) a fourth probe comprising a second energy transfer acceptor and a fourth targeting moiety to form a first assay complex and a second assay complex

wherein the first assay complex comprises a first multivalent biomolecule bound to the first probe and to the second probe;

the second assay complex comprises a second multivalent biomolecule bound to the third probe and to the fourth probe;

the first targeting moiety and the second targeting moiety have substantially the same structure; and

the third targeting moiety and the fourth targeting moiety have substantially the same structure; and

the first analyte is the first multivalent biomolecule and the second analyte is the second multivalent biomolecule; and

(b) detecting the presence of the first assay complex and the second assay complex.

27. The method of claim 26 wherein the first targeting moiety and the second targeting moiety are each epitopes to a first antibody;

and the third targeting moiety and the fourth targeting moiety are each epitopes to a second antibody.

28. The method of claim 26 wherein the first analyte is a first antibody and the second analyte is a second antibody.