Kits employing generalized target-binding e-tag probes
View Patent ↗Kits for the multiplexed detection of the binding of, or interaction between, one or more ligands and target antiligands are provided. Detection involves the release of identifying tags as a consequence of target recognition. The kits include sets of electrophoretic tag probes or e-tag probes, a capture agent and optionally a cleaving agent. The e-tag probes comprise a detection region and a mobility-defining region called the mobility modifier, both linked to a target-binding moiety. In using the kits, target antiligands are contacted with a set of e-tag probes and the contacted antiligands are treated with a selected cleaving agent resulting in a mixture of e-tag reporters and uncleaved and/or partially cleaved e-tag probes. The mixture is exposed to a capture agent effective to bind to uncleaved or partially cleaved e-tag probes, followed by electrophoretic separation. In a multiplexed assay, different released e-tag reporters may be separated and detected providing for target identification.
1. A kit for detecting the presence or absence of one or more target compounds, the kit comprising:
a plurality of electrophoretic probes each comprising target-binding moiety specific for a target compound, each target-binding moiety having one or more eTag reporters attached thereto by cleavable linkages such that upon cleavage of the cleavable linkages the eTag reporters from different electrophoretic probes form distinct peaks upon electrophoretic separation; and
a second reagent for each of the one or more target compounds, each second reagent binding to one of said one or more target compounds, and each second reagent being capable of generating an active species to cleave cleavable linkages of electrophoretic probes that bind to the same target compound as said second reagent, the active species being selected from the group consisting of singlet oxygen, hydrogen peroxide, NADH, and hydroxyl radicals.
2. The kit according to claim 1 wherein said electrophoretic probes are selected from the group defined by the formula:
[(D, M)—L] k —T
wherein:
T is a target-binding moiety specific for a target compound;
k is an integer in the range of from 1 to 20;
L is said cleavable linkage;
D is a detection group; and
M is a mobility modifier consisting of from 1 to 500 atoms selected from the group consisting of carbon, hydrogen, oxygen, sulfur, nitrogen, phosphorus, and boron.
3. The kit of claim 2 wherein said plurality is in the range of from 5 to 100, and wherein M is a mobility modifier consisting of from 1 to 300 atoms selected from the group consisting of carbon, hydrogen, oxygen, phosphorus, nitrogen, sulfur, and boron.
4. The kit of claim 3 wherein said cleavable linkage is cleavable by oxidation and is selected from the group consisting of olefins, thioethers, sulfoxides, and selenium analogs of thioethers or sulfoxides.
5. The kit of claim 4 wherein said detection group is a fluorescent label.
6. The kit of claim 5 wherein said target-binding moiety and said second reagent are each a monoclonal antibody or a polyclonal antibody; and wherein k is in the range of from 1 to 3.
7. The kit of claim 6 wherein said detection group is a fluorescein.
8. A kit of specific binding pairs for detecting the presence or absence of one or more target compounds, the kit comprising a plurality of pairs of first reagents and second reagents, the first reagent and second reagent of each pair binding to the same target compound, the first reagent of each pair being selected from the group defined by the formula:
[(D, M)—L] k —T
wherein:
T is a target-binding moiety specific for a target compound,
k is an integer in the range of from 1 to 20,
L is a cleavable linkage,
D is a detection group, and
M is a mobility modifier consisting of from 1 to 500 atoms selected from the group consisting of carbon, hydrogen, oxygen, sulfur, nitrogen, phosphorus, and boron, wherein upon cleavage of L an eTag reporter comprising a detection group, D, and a mobility modifier, M, is produced with a distinct charge/mass ratio so that eTag reporters of different electrophoretic probes form distinct peaks upon electrophoretic separation; and
the second reagent of each pair being capable of generating an active species to cleave cleavable linkages of first reagents that bind to the same target compound as said second reagent, the active species being selected from the group consisting of singlet oxygen, hydrogen peroxide, NADH, and hydrogen radicals.
9. The kit of specific binding pairs of claim 8 wherein said plurality is in the range of from 5 to 100, and wherein M is a mobility modifier consisting of from 1 to 300 atoms selected from the group consisting of carbon, hydrogen, oxygen, phosphorus, nitrogen, sulfur, and boron.
10. The kit of specific binding pairs of claim 9 wherein said cleavable linkage is selected from the group consisting of olefins, thioethers, sulfoxides, and selenium analogs of thioethers or sulfoxides.
11. The kit of specific binding pairs of claim 10 wherein said detection group is a fluorescent label, and wherein said charge/mass ratio is in the range from −0.001 to 0.5.
12. The kit of specific binding pairs of claim 11 wherein said target-binding moiety is a monoclonal antibody or a polyclonal antibody, and wherein k is in the range of from 1 to 3.
13. The kit of specific binding pairs ascording to claim 8 , 9 , 10 , 11 , or 12 wherein said second reagent is a monoclonal antibody or a polyclonal antibody, and wherein said active species is singlet oxygen.