Reductive-Amination-Mediated Hapten—Carrier Conjugation

Dissolve the carrier protein (or another carrier that contains amine groups) in 0.1M sodium carbonate, 0.15 M NaCl, pH 8.5, at a concentration of 2mg/ml. 

Add peptide hapten to the carrier solution to obtain a concentration of about 2mg/ml. Alternatively, determine the molar ratio of peptide to carrier. Ratios of 20 1 to 40 1 (peptide carrier) usually result in good immunogens. 

Add fresh glutaraldehyde to the peptide/carrier solution to obtain a 1 percent final concentration. Mix well. 

Caution Use of a fume hood is recommended when working with glutaraldehyde. Avoid contact with skin and clothing. Do not breathe vapors. 

React for 2-4 hours at 4°C. Periodically mix the solution or use a gentle rocker. 

Reductive-Amination-Mediated Hapten-Carrier Conjugation 

Hapten molecules containing aldehyde residues may be cross-linked to carrier molecules by use of reductive amination (chapter 3, Section 4). At alkaline pH values, the aldehyde groups form intermediate Schiff bases with available amine groups on the carrier. Reduction of the resultant Schiff bases with sodium cyanoborohydride or sodium borohydride creates a stable conjugate held together by secondary amine bonds. 

Add the aldehyde-containing oligosaccharide to the carrier solution at a concentration sufficient to obtain at least a 20-fold molar excess of hapten to carrier. 

Adding a much greater molar excess of oligosaccharide to couple through reducing ends (i.e., up to 200-fold excess) will help to drive the conjugation reaction to completion. 

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Reductive Amination-Mediated Hapten-Carrier Conjugation... 

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