Method of conjugating an activated ester to an amine-containing biological material

摘要

A coupling agent which is an activated ester such as N-maleimido-6-aminocaproyl-HNSA (mal-sac-HNSA) is formed by reacting 4-hydroxyl-3-nitrobenzene sulfonic acid sodium salt (HNSA) with a carboxylic acid moiety of a compound such as N-maleimido-6-aminocaproic acid. The coupling agent is reacted with an amino group of an amine-containing biological material such as a protein at a pH of about 5.5 to 10.0 and HNSA is released. The released HNSA is spectroscopically measured at a wavelength of from about 350 nm to about 500 nm to precisely monitor and control conjugating of the coupling agent to the biological material. The resulting product is coupled to a sulfhydryl group or other group of another material to provide cross-linking between the two. This enables joining the biological material to one another, to a support matrix, to a label, to a hapten, and to other materials. An immunotoxin for therapeutic use can be prepared by conjugating the coupling agent to an antibody such as a monoclonal antibody with tumor cell specificity and then joining the resultant conjugate to a cytotoxic molecule.