Conjugation of biomolecules using Diels-Alder cycloaddition
AssigneeThe United States of America as represented by the Department of Health and Human Services
InventorVince Pozsgay
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Abstract
A method is provided for covalently linking carbohydrates, proteins, nucleic acids, and other biomolecules under neutral conditions, using a Diels-Alder cycloaddition reaction. In an example, activated carbon-carbon double bonds were attached to free amino sites of a carrier protein, and a conjugated diene was attached to a carbohydrate hapten. Spontaneous coupling of the carbohydrate and the protein components under very mild conditions provided glycoconjugates containing up to 37 carbohydrate hapten units per carrier protein molecule. The method is also applicable to the immobilization of biomolecules on gel or solid supports. The conjugated products are useful as immunogens and as analytical and diagnostic reagents.
Claims24
A method of coupling a first biomolecule to a second biomolecule, comprising:
- (a) covalently attaching a diene moiety to the first biomolecule to form a diene component;
- (b) covalently attaching a dienophile to the second biomolecule to form a dienophile component;
- and
- (c) contacting the diene component with the dienophile component under conditions that permit a cycloaddition reaction to occur between the components, wherein one of said first and second biomolecules is a polysaccharide hapten or antigen, and the other of said first and second biomolecules is a peptide, protein, or polysaccharide carrier.
A method of coupling a polysaceharide hapten or antigen to a gel or solid support, comprising:
- (a) covalently attaching a diene moiety to a substrate selected from the group consisting of the polysaccharide hapten or antigen and the support, to form a diene component;
- (b) covalently attaching a dienophile to the substrate not selected in step (a) to form a dienophile component;
- and
- (c) contacting the diene component with the dienophile component under conditions that permit a cycloaddition reaction to occur between the components.
Dependencies are inferred from the claim text.