Conjugation reactions
We describe methods that allow either carbodiimides or other carboxyl-reactive substances to be mixed with solutions of carboxylic acids or phosphates or amines or combinations thereof, so as to form a homogeneous mixture which is then dried, preferably in a freeze drying process. The mixture is then contacted with an entity, which preferably involves the dissolution of the mixture with a buffered solution of the entity, so as to initiate a conjugation reaction between the entity and a component in the mixture.
1. A method of producing a reagent for use in a conjugation reaction comprising
a) forming a homogeneous solution or suspension of
i) a carboxyl-activating or phosphate-activating non-enzymatic substance comprising 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-Cyclohexyl-N′-(β-[N-methylmorpholino]ethyl)carbodiimide (CMC), 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide (EAC), 2,2-dichloro-5-(2-phenylethyl)-4-(trimethylsilyl)-3-furanone (DPTF), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM), or N-ethyl-5-phenylisoxazolium-3′-sulphonate (Woodwards reagent K (WRK)), wherein the substance is capable of acting on a carboxyl group- or phosphate group-containing moiety to facilitate formation of (1) an amide bond between a carboxyl group and an amine group or (2) a phosphoramidate bond between a phosphate group and an amine group, and
ii) a first reactant containing a carboxyl group or a phosphate group, or a second reactant containing an amine group; and
b) drying the solution or suspension to produce the reagent comprising a homogeneous mixture of i) and ii).
2. The method according to claim 1 , wherein the carboxyl-activating or phosphate-activating substance and the first or second reactant are mixed in the form of a solution.
3. The method according to claim 2 , wherein the solution is an aqueous solution.
4. The method according to claim 1 , wherein the drying comprises freeze drying.
5. The method according to claim 1 , wherein the solution or suspension comprises a first reactant containing a carboxyl group or phosphate group.
6. The method according to claim 1 , wherein first or second reactant in the solution or suspension comprises a coloured, fluorescent, enzyme, magnetic or particle label.
7. The method according to claim 1 , wherein the first or second reactant in the solution or suspension comprises a microparticle.
8. The method according to claim 7 , wherein the microparticle has a polymer coating.
9. The method according to claim 1 , wherein the solution or suspension includes a polyhydroxylic material.
10. The method according to claim 1 , wherein the solution or suspension includes a buffer.
11. The method according to claim 1 , wherein the pH of the solution or suspension is greater than 4.
12. The method according to claim 11 , wherein the pH is greater than 6.
13. The method according to claim 11 , wherein the pH is greater than 7.
14. The method according to claim 11 , wherein the pH is greater than 8.
15. A conjugation kit comprising the reagent produced by claim 1 and instructions for use.
16. A method of performing a conjugation reaction comprising using the conjugation kit of claim 15 in accordance with the instructions.
17. The method of claim 1 further comprising reconstituting the reagent in a liquid.
18. The method of claim 17 further comprising contacting the reconstituted reagent with a molecule to be conjugated.
19. The method of claim 1 , wherein the solution or suspension excludes N-hydroxysuccinimide.
20. A method of producing a reagent for use in a conjugation reaction between a first reactant containing a carboxyl group or a phosphate group and a second reactant containing an amine group, comprising:
a) forming a solution or suspension of
i) a carboxyl-activating or phosphate-activating non-enzymatic substance capable of acting on a carboxyl group- or phosphate group-containing moiety to facilitate the formation of an amide bond between a carboxyl group and an amine group; or a phosphoramidate bond between a phosphate group and an amine group, and
ii) the first reactant or the second reactant; and
b) drying the solution or suspension to produce the reagent comprising a mixture of i) and ii),
wherein said reagent excludes N-hydroxysuccinimide, and wherein the carboxyl-activating or phosphate-activating substance comprises 1 ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-Cyclohexyl-N′-(β-[N-methylmorpholino]ethyl)carbodiimide (CMC), 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide (EAC), 2,2-dichloro-5-(2-phenylethyl)-4-(trimethylsilyl)-3-furanone (DPTF), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM), or N-ethyl-5-phenylisoxazolium-3′-sulphonate (Woodwards reagent K (WRK)).