Methods and reagents for signal amplification
View Patent ↗The present invention provides reagents containing binding moieties conjugated to dextran moieties, methods of making such reagents, and use of such reagents in a variety of molecular and cellular assays.
1. A detectable reagent comprising:
a binding moiety conjugated to a dextran component, wherein the dextran component has a total of about 4 to about 8 dextrans per binding moiety and the binding moiety is not a dextran or a target entity in a sample, and wherein a detectable entity is conjugated to the dextran component.
2. The detectable reagent of claim 1 , wherein each dextran is from about 30 kDa to about 100 kDa molecular weight.
3. The detectable reagent of claim 1 , wherein each dextran is about 70 kDa molecular weight.
4. The detectable reagent of claim 1 , wherein each dextran has substantially the same molecular weight.
5. The detectable reagent of claim 1 , wherein at least one dextran has a molecular weight different from another dextran.
6. The detectable reagent of claim 1 , wherein the detectable entity is a fluorophore.
7. The detectable reagent of claim 6 , wherein the fluorophore is selected from a fluorophore with green fluorescence, orange fluorescence, red fluorescence, and far red fluorescence.
8. The detectable reagent of claim 6 , wherein the fluorophore is selected from a fluorophore with excitation and emission spectra in the range of about 350 nm to 775 nm.
9. The detectable reagent of claim 8 , wherein the fluorophore is selected from a fluorophore with excitation and emission spectra of about 346 nm/446 nm, about 494 nm/519 nm, about 554 nm/570 nm, about 555 nm/572 nm, about 590 nm/617 nm, about 651 nm/672 nm, about 679 nm/702 nm and about 749 nm/775 nm.
10. The detectable reagent of claim 1 , wherein the binding moiety is selected from the group consisting of avidin, streptavidin, biotin, digoxigenin, immunoreagent, oligonucleotide, peptide, protein, nucleic acid, peptide nucleic acid, protein A, protein G, a derivative thereof, and a ligand-binding portion thereof.
11. A method of making a detectable reagent comprising:
providing a dextran component comprising a plurality of inter-connected dextran;
conjugating the dextran component with a binding moiety to form a dextran-binding moiety complex, wherein the binding moiety is not a dextran or a target entity in a sample, and wherein the dextran-binding moiety complex has a total of about 4 to about 8 dextrans per binding moiety; and
attaching a detectable entity to the dextran-binding moiety complex.
12. A method of making a detectable reagent comprising:
conjugating a binding moiety to a dextran to form a core dextran-binding moiety, wherein the binding moiety is not a dextran or a target entity in a sample;
reacting the core dextran-binding moiety with one or more dextrans to form a dextran-binding moiety complex which contains a total of about 4 to about 8 dextrans per binding moiety; and
attaching a detectable entity to the dextran-binding moiety complex.
13. The method of claim 12 , wherein the binding moiety is avidin or strepavidin.
14. A method for detecting or quantifying a target molecule comprising:
contacting the detectable reagent of claim 1 with a sample suspected of containing a target molecule, wherein the binding moiety is capable of binding to the target molecule; and
detecting a signal of the detectable entity thereby detecting or quantifying the target molecule.
15. The detectable reagent of claim 1 , wherein the total molecular weight of dextrans is at least 500 kDa.
16. The detectable reagent of claim 1 , wherein the dextrans within the dextran component are inter-connected and configured in a layered configuration or branched configuration.
17. The detectable reagent of claim 1 , wherein each dextran is from 30 kDa to 100 kDa molecular weight.
18. The detectable reagent of claim 1 , wherein each dextran is from about 50 kDa to about 70 kDa molecular weight.
19. The detectable reagent of claim 1 , wherein the detectable reagent contains a total of about 6 dextrans per binding moiety.
20. The detectable reagent of claim 1 , wherein the detectable reagent contains a total of 6 dextrans per binding moiety.
21. The detectable reagent of claim 1 , wherein the detectable reagent provides an enhanced, increased or amplified detection when compared to a detection achieved from a binding moiety conjugated to a detectable entity in the absence of dextrans or a binding moiety conjugated to a single dextran containing a detectable entity.
22. The detectable reagent of claim 21 , wherein the enhanced, increased or amplified detection is at least about 2 fold more signal compared to detection achieved from a binding moiety conjugated to a detectable entity in the absence of dextrans or a binding moiety conjugated to a single dextran containing a detectable entity.
23. A method of making a detectable reagent of claim 1 comprising:
derivatizing a dextran-amine to generate maleimide functional groups;
derivatizing a binding moiety to generate thiol functional groups, wherein the binding moiety is not a dextran or a target entity in a sample;
reacting the derivatized binding moiety with a molar ratio of one or more derivatized dextran-amine to form a dextran-binding moiety;
derivatizing the dextran-binding moiety to generate one or more thiol groups per dextran;
reacting the derivatized dextran-binding moiety with a molar ratio of one or more derivatized dextran-amine to form an inter-connected dextran-binding moiety complex, wherein the dextran-binding moiety complex has a total of about 4 to about 8 dextrans per binding moiety; and
reacting the inter-connected dextran-binding moiety complex with a molar ratio of one or more detectable entities to form a detectable reagent.
24. The detectable reagent of claim 1 , wherein the binding moiety is avidin or streptavidin.
25. The detectable reagent of claim 1 , wherein the detectable reagent has a total of 4 to 8 dextrans per binding moiety.