Glutaraldehyde-Mediated Hapten—Carrier Conjugation

The following procedure utilizes the one-step glutaraldehyde method. A two-step method may be used to limit somewhat polymerization of the conjugate (Chapter 10, Section 1.2). Varying the pH and the amount of glutaraldehyde added to the reaction can control the yield and molecular weight of the conjugates formed. 

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

Add peptide hapten to the carrier solution to obtain a concentration of about 2 

Add fresh glutaraldehyde to the peptide/carrier solution to obtain a 1% 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 h at 4°C. Periodically mix the solution or use a gentle rocker. The conjugate may be stabilized by addition of a reductant such as sodium borohydride or sodium cyanoborohydride. Usually sodium cyanoborohydride is recommended for specific reduction of Schiff bases, but since the conjugate has already formed at this point, the use of sodium borohydride will both reduce the associated Schiff bases and eliminate any remaining aldehyde groups. Add sodium borohydride to a final concentration of 10 mg/ml. Continue to react for 1 h at 4°C. 

The reaction of glutaraldehyde with protein carriers and peptide haptens involves mainly lysine e-amine and N-terminal oc-amine groups. The conjugates formed are usually of high-molecular weight and may cause precipitation products. In addition, the orientation of the 

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