NHS Ester-Mediated Hapten—Carrier Conjugation

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 

since the amines on the protein will be protonated and therefore be less reactive toward the sulfo-NHS esters that are formed. In addition, the hydrolysis rate of the esters is dramatically slower at acid pH. Thus, the active species may be isolated in a 

Add to the solution in step 1 a quantity of EDC and sulfo-NHS (both from Pierce) to obtain a concentration of 2 mM EDC and 5 mM sulfo-NHS. To aid in aliquoting the correct amount of these reagents, they may be quickly dissolved in 

Rgure 284 The carbodiimide EDC can be used in the presence of sulfo-NHS to create reactive sulfo-NHS ester groups on a carrier protein. Subsequent coupling with an amine-containing hapten can be done to create amide bond linkages. 

Mix and react for 15 min at room temperature to form the sulfo-NHS esters. 

Allow the reaction to continue at room temperature for 2 hours. 

Purify the hapten-carrier conjugate by gel filtration or dialysis. 

Add 2-mercaptoethanol to the reaction solution to obtain a final concentration of 20 mM. Mix and incubate for 10 min at room temperature. 

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NHS Ester-Maleimide Heterobifunctional Crosslinker-Mediated Hapten-Carrier Conjugation... 

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