Catalytic amplification of multiplexed assay signals

• US 20020197649A1
• Filed: 05/24/2002
• Published: 12/26/2002
• Est. Priority Date: 05/26/2001
• Status: Active Grant

• Current Assignee: Monogram Biosciences, Inc. (Laboratory Corporation Of America Holdings)
• Original Assignee: Monogram Biosciences, Inc. (Laboratory Corporation Of America Holdings)
• Inventors: Singh, Sharat
• Granted Patent: US 7,358,052 B2
• Time in Patent Office: Days
• US Class Current: 435/7.1
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2565/125 Electrophoretic separation
• CPC Class Codes: G01N 2458/10 Oligonucleotides as tagging agents for labelling antibodies
• CPC Class Codes: G01N 33/561 Immunoelectrophoresis

Abstract

Methods, compositions, kits, and a system are disclosed for detecting one or more analytes in a sample. A mixture comprising the (i) sample, (ii) a first binding reagent comprising a cleavage-inducing moiety and a first binding agent specific for an analyte, and (ii) one or more electrophoretic probes each having a second binding agent is subjected to conditions under which binding of respective binding agents occurs. The interaction between the binding agents and the analyte brings the cleavage-inducing moiety within a proximity effective for cleaving a cleavable linkage tethering an electrophoretic tag to the second binding agent, thereby releasing the tag for electrophoretic separation. Separation of different tags occurs by virtue of their distinct electrophoretic mobilities. After separation, a signal amplification moiety on each tag is activated to generate a signal to indicate the presence of a particular analyte in the sample.

Claims

1. A method for determining the presence or absence of one or more analytes in a sample, the method comprising the steps of:

providing a first binding reagent and one or more electrophoretic probes, the first binding reagent having a cleavage-inducing moiety with an effective proximity and a first binding agent specific for at least one analyte, and the one or more electrophoretic probes each having a second binding agent specific for at least one analyte and each having one or more electrophoretic tags each such tag attached thereto by a cleavable linkage and each such tag having a signal amplification moiety;

mixing the sample, the first binding reagent, and the one or more electrophoretic probes such that the first binding reagent and the one or more electrophoretic probes bind to the one or more analytes and at least one cleavable linkage of the one or more electrophoretic probes is within the effective proximity of a cleavage-inducing moiety so that one or more electrophoretic tags are released;

electrophoretically separating the released electrophoretic tags in an electrophoretic separation medium that contains a substrate capable of reacting with at least one signal amplification moiety to produce a signal; and

determining the presence or absence of the one or more analytes based on the presence or absence of signals produced by reactions between a substrate and a signal amplification moiety of the released electrophoretic tags.

2. The method of claim 1 wherein said one or more analytes is a plurality of analytes.

3. The method of claim 2 wherein said cleavage-inducing moiety is a sensitizer that produces an active species.

4. The method of claim 3 wherein said sensitizer is a first photosensitizer, said cleavable linkage is an oxidation-labile linkage, and said said active species is selected from the group consisting of hydrogen peroxide, hydroxyl radical, superoxide anion, phenoxy radical, and singlet oxygen.

5. The method of claim 4 wherein said active species is singlet oxygen.

6. The method of claim 5 wherein said first binding agent of said first binding reagent is selected from the group consisting of antibodies, protein receptors, ligands of protein receptors, lectins, biotin-containing moieties, boronic acid-containing moieties, aptamers, enzyme substrates, enzyme cofactors, and enzyme subunits, and wherein said second binding agent of said electrophoretic probe is an antibody or an antibody binding composition.

7. The method of claim 5 wherein said signal amplification moiety is a second photosensitizer.

8. The method of claim 7 wherein said second photosensitizer is in inactivated form prior to cleavage of said cleavable linkage.

9. The method of claim 5 wherein said substrate is a leuco dye.

10. The method of claim 5 wherein said first photosensitizer is selected from the group consisting of porphyrins, phthalocyanines, halogenated derivatives of a fluorescein dye, halogenated derivatives of a rhodamine dye, and naphthalocyanines.

11. The method of claim 5 wherein said cleavable linkage comprises an olefin, a thioether, a selenoether, a thiazole, an oxazole, or an imidazole.

12. The method of claim 5 wherein said electrophoretic probe is defined by the following formula:

T-(L-E)_k

wherein;

T is a second binding agent specific for a target polypeptide, L is an oxidation-labile linkage, E is an electrophoretic tag, and k is an integer greater than or equal to 1.

13. The method of claim 12 wherein L is selected from the group consisting of olefins, thioethers, selenoethers, thiazoles, oxazoles, and imidazoles.

14. The method of claim 13 wherein E is a water-soluble organic compound having a molecular weight in the range of from about 150 to 2500 daltons.

15. The method of claim 13 wherein E has the form (M,I), wherein I is said signal amplification moiety and wherein M is a mobility modifying moiety that is a bond or an organic molecule having up to 100 atoms other than hydrogen selected from the group consisting of carbon, oxygen, nitrogen, phosphorus, boron, and sulfur.

17. The kit of claim 16 wherein said cleavage-inducing moiety is a first photosensitizer and said cleavable linkage is an oxidation-labile linkage.

18. The kit of claim 17 wherein said first binding agent is selected from the group consisting of antibody binding compositions, protein receptors, ligands of protein receptors, lectins, biotin-containing moieties, boronic acid-containing moieties, aptamers, enzyme substrates, enzyme cofactors, and enzyme subunits.

19. The kit of claim 17 wherein said second binding agents of said one or more electrophoretic probes are antibody binding compositions.

20. The kit of claim 19 wherein said one or more electrophoretic probes are a plurality in the range of from 2 to 50.

21. The kit of claim 20 wherein each different electrophoretic probe is specific for a different analyte.

22. The kit of claim 20 wherein each of said one or more electrophoretic probes is defined by the formula:

T-(L-E)_k

wherein;

T is said second binding agent specific for an analyte, L is said oxidation-labile linkage, E is an electrophoretic tag, and k is an integer greater than or equal to 1.

23. The kit of claim 22 wherein L is selected from the group consisting of olefins, thioethers, selenoethers, thiazoles, oxazoles, and imidazoles.

24. The kit of claim 23 wherein E is a water-soluble organic compound having a molecular weight in the range of from about 150 to 2500 daltons.

25. The kit of claim 23 wherein E has the form (M,I), wherein I is said signal amplification moiety and wherein M is a mobility modifying moiety that is a bond or an organic molecule having up to 100 atoms other than hydrogen selected from the group consisting of carbon, oxygen, nitrogen, phosphorus, boron, and sulfur.

27. The composition of claim 26 wherein L is an oxidation-labile linkage, said plurality is in the range of from 2 to 500, and E has a molecular weight in the range of from 150 to 10,000 daltons.

28. The composition of claim 27 wherein said electrophoretic probe is defined by the formula:

T-(L-(M,I)_k

wherein T is an antibody binding composition or an oligonucleotide;

L, and k are defined as above;

I is a signal amplification moiety; and

M is a bond or a water soluble organic compound consisting of from 1 to 100 atoms, not including hydrogen, that are selected from the group consisting of carbon, oxygen, nitrogen, phosphorus, boron, and sulfur.

29. The composition of claim 28 wherein M is a polymer selected from any one of polyethers, polyesters, polypeptides, oligosaccharides, polyurethanes, polyamides, polysulfonamides, polysulfoxides, polyphosphonates, and block copolymers thereof.

30. The composition of claim 28 wherein L is selected from the group consisting of olefins, thioethers, selenoethers, thiazoles, oxazoles, and imidazoles.

31. The composition of claim 28 wherein M is made by coupling a plurality of from 2 to 10 of the compounds selected from the group consisting of dimethoxytrityl-protected hexaethylene glycol phosphoramidite, 6-(4-Monomethoxytritylamino)hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite, 12-(4-Monomethoxytritylamino)dodecyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite, 2-[2-(4-Monomethoxytrityl)aminoethoxy]ethyl-(2-cyanoethyl), N,N-diisopropyl)-phosphoramidite, (S-Trityl-6-mercaptohexyl)-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite, 9-O-Dimethoxytrityl-triethylene glycol, 1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 3(4,4Dimethoxytrityloxy)propyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5′

-O-Dimethoxytrityl-1′

,2′

-Dideoxyribose-3′

-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 18-0 Dimethoxytritylhexaethyleneglycol,1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 12-(4,4′

-Dimethoxytrityloxy)dodecyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 1,3-bis-[5-(4,4′

-dimethoxytrityloxy)pentylamido]propyl-2-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 1-[5-(4,4′

-dimethoxytrityloxy)pentylamido]-3-[5-fluorenomethoxycarbonyloxy pentylamido]-propyl-2-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, Tris-2,2,2-[3-(4,4′

-dimethoxytrityloxy)propyloxymethyl]ethyl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, succinimidyl trans-4-(maleimidylmethyl) cyclohexane-1-carboxylate, succinimidyl 3-(2-pyridyldithio)propionate, succinimidyl acetylthioacetate, succinimidyl 4-(p-maleimidophenyl)butyrate;

N-γ

-maleimidobutyryl-oxysuccinimide ester;

p-nitrophenyl iodoacetate; and

4-(4-N-maleimidophenyl)butyric acid hydrazide.

32. The composition of claim 28 wherein said plurality of said electrophoretic probes is in the range of from 5 to 100.

33. The composition of claim 32 wherein said plurality of said electrophoretic probes is in the range of from 10 to 30.

35. The composition of claim 34 wherein each electrophoretic tag of said plurality has a distinct charge-mass ratio or distinct optical characteristics so that each electrophoretic tag forms a detectable peak upon electrophoretic separation.

36. The composition of claim 34 wherein I is the same for each electrophoretic tag of said plurality and wherein each electrophoretic tag of said plurality has a distinct charge-mass ratio so that each electrophoretic tag forms a distinct peak upon electrophoretic separation.

37. The composition of claim 36 wherein M is a polymer selected from any one of polyethers, polyesters, polypeptides, oligosaccharides, polyurethanes, polyamides, polysulfonamides, polysulfoxides, polyphosphonates, and block copolymers thereof.

39. The composition of claim 38 wherein said cleavable linkage contains an olefin group and one or more electron donating substituents in conjugation with said olefin group.

40. The composition of claim 38 wherein said signal amplification moiety is a second photosensitizer in an inactivated state.

41. The composition of claim 40 wherein said second photosensitizer is activated in the presence of singlet oxygen.

42. The composition of claim 41 wherein said second photosensitizer is capable of converting a leuco dye into a detectable product.

43. The composition of claim 38 wherein said plurality of electrophoretic probes is defined by the formula:

T-(L-E)_k

wherein;

T is a binding moiety specific for an analyte, L is a cleavable linkage, E is an electrophoretic tag, and k is an integer greater than or equal to 1, such that each different T of said plurality is attached to a different E, and such that each E of the plurality has a signal amplification moiety and an electrophoretic mobility that is distinct from those of every other E of said plurality.

44. The composition of claim 43 wherein said signal amplification moiety is a second photosensitizer in an inactivated state.

16. A kit for detecting the presence or absence of a plurality of analytes in a sample, the kit comprising:

a first binding reagent having a cleavage-inducing moiety and a first binding agent specific for at least one analyte; and

one or more electrophoretic probes each having a second binding agent specific for at least one analyte and each having one or more electrophoretic tags each such tag attached thereto by a cleavable linkage and each such tag having a signal amplification moiety.

26. A composition of matter for detecting one or more target polypeptides, the composition comprising a plurality of electrophoretic probes defined by the formula:

T-(L-E)_k

wherein;

T is a binding moiety specific for an analyte, L is a cleavable linkage, E is an electrophoretic tag, and k is an integer greater than or equal to 1, such that each different T of the plurality is attached to a different E, and such that each E of the plurality has a signal amplification moiety and an electrophoretic mobility that is distinct from that of every other E of the plurality.

34. A composition of matter for detecting the presence or absence of a target molecule in a sample, the compositon comprising a plurality of electrophoretic tags defined by the formula:

A-M-I

wherein;

A is —

C(═

O)R, where R is aliphatic, aromatic, alicyclic or heterocyclic having from 1 to 8 carbon atoms and 0 to 4 heteroatoms selected from the group consisting of O, S. and N;

—

CH₂—

C(═

O)—

NH—

CHO;

—

SO₂H;

—

CH₂—

C(═

O)O—

CHO;

—

C(═

O)NH—

(CH₂)_n—

NH—

C(═

O)C(═

O)—

(C₆H₅), where n is in the range of from 2 to 12;

I is a signal amplification moiety; and

M is a bond or an organic molecule having up to 100 atoms other than hydrogen selected from the group consisting of carbon, oxygen, nitrogen, phosphorus, boron, and sulfur, with the proviso that the total molecular weight of A-M-I be within the range of from about 150 to about 5000 daltons.

38. A composition comprising:

(a) a plurality of electrophoretic probes each comprising an electrophoretic tag attached thereto by a cleavable linkage capable of reacting with singlet oxygen, the electrophoretic tag further including a signal amplification moiety; and

(b) a first binding reagent comprising a first photosensitizer, wherein the first photosensitizer is capable, in its excited state, of activating oxygen to its singlet state.