Catalytic amplification of multiplexed assay signals
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.
1. A method of determining the presence or absence of each of a plurality of analytes in a sample, the method comprising the steps of:
contacting with the sample a plurality of a first binding reagents and a plurality of electrophoretic probes; each of the first binding reagent comprising a cleavage-inducing moiety attached to a first binding agent specific for at least one analyte, and each of the electrophoretic probe comprising a second binding agent specific for one of said analyte and an electrophoretic tag containing a signal amplification moiety, wherein each of said electrophotecic tag has a unique electrophoretic mobility and is attached to the second binding agent by a cleavable linkage; such that when the first binding reagent and an electrophoretic probe bind to the analyte, the cleavable linkage of the electrophoretic probes is within an effective proximity to a cleavage-inducing moiety such that the elcetrophoretic tag is released form its second binding agent;
electrophoretically separating the released electrophoretic tags in an electrophoretic separation medium that contains or to which is added a substrate capable of reacting with each tag's signal amplification moiety to produce a signal; and
determining the presence or absence of one or more analytes based on the presence or absence, for each analyte, of a signal corresponding to the electrophoretic mobility of a tag associated with that analyte, by reactions between said substrate and the signal amplification moiety of each released electrophoretic tags.
2. The method of claim 1 wherein said determining includes determining the presence of at least two different analytes, based on different analyte-specific electrophoretic mobilities of the at least two different electrophoretic tags.
3. The method of claim 2 wherein said cleavage-inducing moiety is a sensitizer capable of producing an active species.
4. The method of claim 3 wherein said sensitizer is a photosensitizer, said cleavable linkage is an oxidation-labile linkage, and 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 photosensitizer.
8. The method of claim 7 wherein said signal amplification moiety that is a 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 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, L is an electrophoretic tag, and k is an integer greater than 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 L, when released from its second binding agent, has a molecular weight in the range of from about 150 to 2500 daltons.
15. The method of claim 13 wherein L 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.
16. A kit for detecting the presence or absence of each of a plurality of analytes in a sample, the kit comprising:
a plurality of a first binding reagents, each comprising a cleavage-inducing moiety attached to a first binding agent specific for at least one analyte; and
a plurality of electrophoretic probes, each comprising a second binding agent specific for one of said analyte and an electrophoretic tag containing a signal amplification moiety, wherein each of said electrophotecic tag has a unique electrophoretic mobility and is attached to the second binding agent by a cleavable linkage; such that when the first binding reagent and an electrophoretic probe bind to the analyte, the cleavable linkage of the electrophoretic probes is within an effective proximity to a cleavage-inducing moiety such that the elcetrophoretic tag is released form its second binding agent.
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 in said one or more electrophoretic probes is an antibody.
20. The kit of claim 19 which includes between 2 to 50 electrophoretic probes.
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 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, when released from its second binding agent, has a molecular weight in the range of from about 150 to 2500 daltons.
25. The kit of claim 23 wherein L 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.