Hapten—carrier conjugation carbodiimide-mediated

Another method of NHS ester mediated hapten—carrier conjugation is to create reactive sulfo-NHS esters directly on the carboxylates of the carrier protein using the EDC/sulfo-NHS reaction described in Chapter 3, Section 1.2. A carbodiimide reaction in the presence of sulfo-NHS activates the carboxylate groups on the carrier protein to form amine-reactive sulfo-NHS esters. The activation reaction is done at pH... 

The reactions involved in an EDC-mediated conjugation are discussed in Chapter 3, Section 1.1 (Note EDC is l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride MW 191.7 and is sometimes referred to as EDAC). The carbodiimide first reacts with available carboxylic groups on either the carrier or hapten to form a highly reactive o-acylisourea intermediate. The activated carboxylic group then can react with a primary amine to form an amide bond, with release of the EDC mediator as a soluble isourea derivative. The reaction is quite efficient with no more than 2 hours required for it to go to completion and form a conjugated immunogen. 

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