Patent ID: 8492129
Filing Date: 2013-07-23
Classification: A61K,G01N

Abstract:
1. A method of reacting a polypeptide first chemical entity and an enzyme or fluorescent material second chemical entity to form a conjugate in which the first and second chemical entities are covalently bound with respect to each other, comprising the steps of forming a conjugation reaction mixture by bringing into simultaneous contact in aqueous conditions, the first chemical entity, the second chemical entity and a thiol generator, wherein the thiol generator reacts with the polypeptide first chemical entity in a thiolation reaction resulting in formation of a free sulfhydryl group on the polypeptide first chemical entity, wherein reaction of the thiol generator with a primary amine on the polypeptide first chemical entity takes place at a pH below 8, and the free sulfhydryl group reacts with the enzyme or fluorescent second chemical entity to form the conjugate, and wherein the enzyme or fluorescent second chemical entity is modified by the attachment of at least two molecules that bind sulfhydryl groups and is therefore polyvalent with respect to its reactivity with sulfhydryl groups, and further wherein the molar ratio of the enzyme or fluorescent second chemical entity to thiol generator is 1:1 or less.