CONTROL OF CHEMICAL MODIFICATION

• US 20100009342A1
• Filed: 07/10/2009
• Published: 01/14/2010
• Est. Priority Date: 09/06/2005
• Status: Abandoned Application

• Current Assignee: Resmed Limited (ResMed Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: STEINBERG, Thomas Harry, GREENFIELD, Lawrence I., MAURO, John Matthew, LEONG, Louis
• Application Number:
  US12/501,253
• Publication Number:
  US 20100009342A1
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: C07K 1/13 Labelling of peptides
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Provided is a method for controlling the degree of labeling (DOL) of a carrier molecule or solid support by the addition of a reactive label competitor to the labeling reaction. When the reactive label competitor is added to the labeling solution the competitor competes with the carrier molecule or solid support for the label, reducing the number of labels available to conjugates to the carrier molecule or solid support. This provides for a facile method that predictably alters the DOL of a carrier molecule or solid support.

Claims

1. A method for controlling the degree of labeling (DOL) of a carrier molecule or solid support, wherein the method comprises:

a) contacting the carrier molecule or solid support with a reactive label to form a labeling solution;

b) contacting the labeling solution with a reactive label competitor to form a controlled labeling solution; and

c) incubating the controlled labeling solution for an appropriate amount of time whereby the degree of labeling of the carrier molecule or solid support is controlled.

2. The method according to claim 1, wherein the carrier molecule comprises a amino acid, a peptide, a protein, a polysaccharide, a nucleotide, a nucleoside, an oligonucleotide, a nucleic acid, a hapten, a psoralen, a drug, a hormone, a lipid, a lipid assembly, a synthetic polymer, a polymeric microparticle, a biological cell or a virus.

3. The method according to claim 1, wherein the carrier molecule comprises an antibody or fragment thereof, an avidin or streptavidin, a biotin, a blood component protein, a dextran, an enzyme, an enzyme inhibitor, a hormone, an IgG binding protein, a fluorescent protein, a growth factor, a lectin, a lipopolysaccharide, a microorganism, a metal binding protein, a metal chelating moiety, a non-biological microparticle, a peptide toxin, a phosphotidylserine-binding protein, a structural protein, a small-molecule drug, or a tyramide.

4. The method according to claim 1, wherein the solid support comprises a microfluidic chip, a silicon chip, a microscope slide, a microplate well, silica gels, polymeric membranes, particles, derivatized plastic films, glass beads, cotton, plastic beads, alumina gels, polysaccharides, polyvinylchloride, polypropylene, polyethylene, nylon, latex bead, magnetic bead, paramagnetic bead, and superparamagnetic bead.

5. The compound according to claim 1, wherein the solid support comprises Sepharose, poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose and starch.

6. The method according to claim 1, wherein the reactive label comprises a fluorophore, a phosphorescent dye, a tandem dye, a particle, an electron transfer agent, biotin or a radioisotope.

7. The method according to claim 6, wherein the fluorophore is dansyl, xanthene, naphthalene, borapolyazaindacene, coumarin, cyanine, pyrene, or derivatives thereof.

8. The method according to claim 6, wherein the fluorophore has an emission spectra greater than about 600 nm.

9. The method according to claim 6, wherein the fluorophore has an emission spectra greater than about 620 nm.

10. The method according to claim 6, wherein the fluorophore has an emission spectra greater than about 650 nm.

11. The method according to claim 6, wherein the fluorophore has an emission spectra great than about 700 nm.

12. The method according to claim 6, wherein the fluorophore has an emission spectra greater than about 750 nm.

13. The method according to claim 6, wherein the fluorophore has an emission spectra greater than about 800 nm.

14. The method according to claim 6, wherein the particle label comprises a nanocrystal or a resonance light scattering particle.

15. The method according to claim 1, wherein the reactive label comprises a reactive group.

16. The method according to claim 15, wherein the reactive group comprises an acrylamide, an activated ester of a carboxylic acid, a carboxylic ester, an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, an anhydride, an aniline, an amine, an aryl halide, an azide, an aziridine, a boronate, a diazoalkane, a haloacetamide, a haloalkyl, a halotriazine, a hydrazine, an imido ester, an isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a reactive platinum complex, a silyl halide, a sulfonyl halide, a silanol, or a thiol.

17. The compound according to claim 15, wherein the reactive group comprises a carboxylic acid, succinimidyl ester of a carboxylic acid, hydrazide, amine and a maleimide.

18. The method according to claim 1, wherein the reactive label competitor comprises an amino or thiol group.

19. The method according to claim 1, wherein the reactive label comprises α

-amino acids, β

-amino acids, amino alcohols, ε

-amino acids, primary amine containing compounds or reactive secondary amine-containing compounds.

20. The method according to claim 1, wherein the reactive label competitor comprises D-lysine, L-lysine, D,L-lysine, ethanolamine, 5-amino caproic acid, or ammonia (NH₃).

21. The method according to claim 1, wherein the reactive label competitor is L-Lysine Hydrochloride.

22. The method according to claim 1, wherein the reactive label competitor comprises α

-mercapto acids, β

-mercapto acids, mercapto alcohols, ε

-mercapto acids, primary mercaptan compounds or reactive secondary mercaptan compounds.

23. The method according to claim 1, wherein the reactive label competitor comprises D-cysteine, L-cysteine, D,L-cysteine, mercaptoethanol, 5-mercapto caproic acid, or H₂S.

24. The method according to claim 20, wherein the DOL is about 4 when the concentration of lysine is about 0.3 mM.

30. The method according to claim 1, further comprising a buffer.

25. A method of modulating the amount of reactive label present in a solution, said method comprising:

a) contacting a solution comprising a carrier molecule or solid support with a reactive label to form a labeled carrier molecule or labeled solid support; and

b) contacting the solution with a reactive label competitor to form a labeled competitor;

wherein the amount of reactive label in the solution is attenuated or eliminated after contacting the reactive label with the reactive label competitor.

26. The method of claim 25, further comprising a step of separating labeled competitor from the labeled carrier molecule or labeled solid support.

27. The method of claim 25, wherein the amount of labeled carrier molecule or labeled solid support is essentially unaffected by the concentration of carrier molecule or solid support in solution.

28. The method of claim 25, wherein the pH of the solution is between 3 and 10.

29. The method of claim 25, wherein the pH of the solution is between 7 and 9.

31. The method according to claim 25, further comprising a buffer.

32. A method for monitoring the degree of labeling (DOL) of a carrier molecule or solid support, said method comprising:

a) contacting a solution comprising a carrier molecule or solid support with a reactive label to form a labeled carrier molecule or labeled solid support; and

b) contacting the solution with a reactive label competitor to form a labeled competitor, wherein the reactive label competitor quenches or is capable of FRET interaction with the reactive label;

wherein the degree of labeling (DOL) is monitored by the amount of quenching or FRET by the reactive label competitor.

33. A kit for controlling the degree of labeling (DOL) of a carrier molecule or solid support, wherein the kit comprises:

a) carrier molecule or solid support;

b) a reactive label;

c) a reactive label competitor; and

d) instructions for performing a method resulting in the controlled degree of labeling of the carrier molecule or solid support.

34. The kit of claim 33, further comprising a buffer.