Mediated Hapten—Carrier Conjugation

One particularly novel carrier was reported to consist of 50-70 nm colloidal gold particles of the type often used in cytochemical labeling techniques for microscopy (Pow and Crook, 1993) (Chapter 24). Adsorption of peptide antigens onto gold and subsequent injection of the complex into rabbits in an adjuvant mixture resulted in rapid production of antibody of extremely high titer. The resultant antibodies could be used in immunocytochemistry at dilutions from l-in-250,000 down to l-in-1,000,000, which is orders-of-magnitude beyond the dilutions typically used with lower-titer antibodies. 

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. 

Conjugation of Tyrosyl-Lysine to BSA Using Various Amounts of EDC 

Comparison of Tyrosyl-Lysine Conjugation to BSA and KLH Using Various Concentrations of EDC 

Peptide Hapten Containing Amines and Cartx xylates 

Amide Bond Formation Through Amines or Carboxylates on Hapten to Amines or Carboxylates on Carrier 

Rgure 277 To study the conjugation of peptides to carriers using different levels of EDC, tyrosyl-lysine was conjugated to BSA and separated after the reaction by chromatography on a Sephadex G-25 column. As the EDC level was incteased in the reaction, more peptide reacted and the peptide peak (the second peak) was depleted. The absorbance of the carrier peak (the first one) increases as more peptide is conjugated. 

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NHS Ester-Maleimide Heterobifunctional Crosslinker-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... 

Active-Hydrogen-Mediated Hapten-Carrier Conjugation 447... 

Reductive-Amination-Mediated Hapten-Carrier Conjugation 455... 

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