Synthetic peptides as antigens: Pitfalls of conjugation methods

Peptide-carrier conjugates were prepared using 9 different synthetic peptides, 3 carrier proteins and 4 coupling reagents. Residues of the carrier protein that were modified by different coupling reagents (e.g., glutaraldehyde, carbodiimides, bis-diazotized benzidine) were found to elicit specific antibodies that reacted with unrelated carrier proteins treated with the same coupling agent. To demonstrate the presence of peptide antibodies in an antiserum raised against a peptide-carrier conjugate, it was necessary to use as antigen the peptide coupled to another carrier by means of a different coupling agent. Some of the commonly used conjugation methods were found to lead to conjugates of insufficient stability and sometimes also altered the antigenic properties of the peptide moiety. These difficulties can be overcome by additional control experiments designed to test the quality and the peptide-carrier conjugates.