Patent ID: 6218160
Filing Date: 2001-04-17
Classification: C07K,G01N

Abstract:
A method for the site-specific covalent conjugation of an enzyme and an antibody through carbohydrate residues, comprising:(a) contacting an enzyme with an oxidizing reagent, said reagent being present in a greater than a 1:1 molar ratio amount to the molar amount of enzyme present, to generate an aldehyde group on the enzyme;(b) contacting an antibody with an oxidizing reagent, said reagent being present in a greater than a 1:1 molar ratio amount to the molar amount of antibody present, to generate an aldehyde group on the antibody;(c) contacting the antibody-aldehyde of step (b) with a molar excess of a dihydrazide or dihydrazine containing compound to form a hydrazone bond between the aldehyde group of the antibody-aldehyde and a hydrazide or hydrazine group of the dihydrazide or dihydrazine containing compound to form an antibody-hydrazide or antibody-hydrazine intermediate;(d) removing excess unreacted dihydrazide or dihydrazine containing compound from the mixture of step (c);(e) contacting the antibody-hydrazide or antibody-hydrazine conjugate from step (d) with the enzyme-aldehyde of step (a), to form a second hydrazone bond between the aldehyde group of the enzyme-aldehyde and an unreacted hydrazide or hydrazine group of the dihydrazide or dihydrazine containing compound thereby generating an antibody-enzyme conjugate.