Conjugate preparation methods and related kit
AssigneeShenzhen Mindray Bio-Medical Electronics Co., Ltd.
InventorsChungen Qian, Ke Li, Yuping Zhang
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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.
Claims22
A method for preparing a conjugate of a first substance to be conjugated and a second substance to be conjugated, the method comprising:
- 1) providing a first substance to be conjugated, wherein the first substance to be conjugated has a reactive amino group and a reactive carboxyl group and is 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) providing a second substance to be conjugated, wherein the second substance to be conjugated has a reactive amino group;
- 3) reacting a reactive amino group of the first substance to be conjugated with an amino group blocking agent that is a carboxyl compound having General Formula I, in the presence of a first cross-linking agent, to obtain a first substance to be conjugated having a blocked amino group:
- 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;
- 4) optionally quenching the first cross-linking agent with a mercapto compound;
- 5) purifying the first substance to be conjugated having a blocked amino group;
- 6) reacting a second cross-linking agent with a reactive carboxyl group of the first substance to be conjugated having a blocked amino group, to create an activated first substance to be conjugated having a blocked amino group, wherein the second cross-linking agent comprises a carbodiimide and, optionally, a hydroxysuccinimide;
- 7) cross-linking the activated first substance to be conjugated having a blocked amino group, with a reactive amino group of the second substance to be conjugated to obtain a conjugate;
- and
- 8) optionally quenching the second cross-linking agent with a mercapto compound.
A kit for preparing a conjugate between an enzyme and a substance having a reactive amino group, or a substance to which a reactive amino group is introduced through modification, 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) an amino group blocking agent for blocking reactive amino groups on the enzyme, said amino group blocking agent being 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, said cross-linking agent being a carbodiimide and, optionally, a hydroxysuccinimide;
- and
- 4) an optional cross-linking agent quenching agent, said quenching agent being a mercapto compound.
Dependencies are inferred from the claim text.