Protecting groups for biological labeling
AssigneeDade Behring Marburg GmbH
InventorsSteve de Keczer, Yen Ping Liu, Dariush Davalian, Nurith Kurn, Edwin F. Ullman
Life of this patent
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Abstract
Alpha-haloketones are useful alkylating agents for coupling to sulfhydryl-containing biomolecules. However, they react spontaneously with water, alkali and organic bases and therefore cannot be stored for extended periods of time in aqueous solutions, particularly in the presence of proteins at physiological pH. The present invention provides novel solutions to these problems, however, as novel compounds and compositions comprising protected haloketones are disclosed herein. Methods of preparing and using protected haloketones which are useful in a variety of applications—e.g., in assays and conjugation reactions—are also disclosed herein.
Claims20
A kit for use in a method for detecting and determining the amount of homocysteine in a sample, comprising in a packaged combination:
- a first reagent comprising an alkylating reagent having a haloketone or alpha haloaldehyde functional group, the carbonyl of said haloketone or alpha haloaldehyde functional group derivatized with a protected functional group said protected functional group capable of reacting with the sulfhydryl group of homocysteine to form modified homocysteine when said protected functional group is deprotected, a second reagent comprising an activating reagent capable of deprotecting said alkylating reagent by removal of the protected functional group, and a third reagent capable of specifically binding to said modified homocysteine, each in an amount sufficient to conduct at least one assay.
A method of determining the amount of homocysteine in a sample suspected of containing said homocysteine, comprising the steps of:
- (a) bringing together in an aqueous medium:
- (1) said sample,
- (2) a first reagent comprising an alkylating reagent having a haloketone or alpha haloaldehyde functional group, the carbonyl of said haloketone or alpha haloaldehyde functional group derivatized with a protected functional group capable of being activated to chemically modify the sulfhydryl groups of homocysteine to form modified homocysteine, and
- (3) a second reagent comprising an antibody capable of specifically binding to said modified homocysteine to from an immunocomplex;
- and
- (4) a third reagent capable of activating said protected alkylating reagent.
- (b) measuring the amount of said immunocomplex, the amount thereof being related to the amount of homocysteine in said sample.
A method of determining the amount of homocysteine in a sample, wherein at least a portion of said homocysteine is in the free disulfide form, comprising the steps of:
- (a) preparing an admixture comprising:
- (1) said sample,
- (2) a releasing agent to release said homocysteine from the disulfide form,
- (3) an alkylating reagent having a haloketone or alpha haloaldehyde functional group, the carbonyl of said haloketone or alpha haloaldehyde function group derivatized with a protected functional group capable of being activated to chemically modify the sulfhydryl groups of homocysteine to form modified homocysteine, and
- (4) an antibody capable of specifically binding to said modified homocysteine to form an immunocomplex, and
- (5) an activating reagent capable of deprotecting said protected functional group of said alkylating reagent;
- and
- (b) examining said medium for the amount of said immunocomplex, the amount thereof being related to the amount of homocysteine in said sample.
Dependencies are inferred from the claim text.