Conjugate preparation methods and related kit
AssigneeShenzhen Mindray Bio-Medical Electronics Co., Ltd.
InventorsChungen Qian, Ke Li, Yuping Zhang
Life of this patent
Estimating the term…
Abstract
Methods for preparing conjugates including enzyme conjugates and especially alkaline phosphatase (ALP) conjugates, and a kit are provided. The methods include: blocking an amino group on a molecule surface of a first substance to be conjugated containing an amino group and a carboxyl group (for example, an enzyme) with a carboxyl compound; adding a carbodiimide to activate the first substance to be conjugated with the amino group blocked; inactivating or removing the carbodiimide; and adding a second substance containing an amino group (for example, a substance to be labeled). Conjugates (for example, enzyme conjugates) are obtained.
Claims24
A method for preparing a conjugate with a first substance to be conjugated and a second substance to be conjugated, wherein the first substance to be conjugated contains an amino group and a carboxyl group, and the second substance to be conjugated contains an amino group, the method comprising:
- 1) blocking the amino group of the first substance to be conjugated with a carboxyl compound having General Formula I, in the presence of a first cross-linking agent, to obtain a first substance to be conjugated with the amino group blocked:
- wherein R1 is selected from at least one of following:
- H, C1-6 alkyl, CnH2nCOOH, phenyl, and phenylC1-6 alkyl, wherein n is an integer of 0-4, and phenyl or phenylC1-6 alkyl is optionally and independently substituted with a substitution group selected from at least one of the following:
- halogen, hydroxyl, mercapto, cyano, nitro, alkyl, aryl, alkoxy, heterocyclyl, haloalkyl, and carboxyl;
- and R2 is a group which is capable of dissociating in water;
- and wherein
- the first cross-linking agent comprises a carbodiimide and, optionally, a hydroxysuccinimide;
- 2) purifying the first substance to be conjugated with the amino group blocked;
- 3) adding a second cross-linking agent to activate the carboxyl group of the first substance to be conjugated with the amino group blocked, wherein the cross-linking agent comprises a carbodiimide and, optionally, a hydroxysuccinimide;
- and
- 4) cross-linking the activated first substance to be conjugated with the amino group blocked and the second substance to be conjugated.
A kit for preparing an enzyme conjugate, comprising:
- 1) an enzyme for labeling, selected from at least one of the following:
- alkaline phosphatase (ALP), horseradish peroxidase, β-galactosidase, urease, glucose-6-phosphate dehydrogenase, glucose oxidase, and malate dehydrogenase;
- 2) a carboxyl compound having General Formula I:
- where R1 is selected from at least one of the following:
- H, C1-6 alkyl, CnH2nCOOH, phenyl, and phenylC1-6 alkyl, wherein n is an integer of 0-4, and phenyl or phenylC1-6 alkyl is optionally and independently substituted with a substitution group selected from at least one of the following:
- halogen, hydroxyl, mercapto, cyano, nitro, alkyl, aryl, alkoxy, heterocyclyl, haloalkyl, and carboxyl;
- and R2 is a group which is capable of dissociating in water;
- 3) a cross-linking agent, being a carbodiimide and, optionally, a hydroxysuccinimide;
- and
- 4) an optional mercapto compound.
Dependencies are inferred from the claim text.