Comparison of four bifunctional reagents for coupling peptides to proteins and the effect of the three moieties on the immunogenicity of the conjugates

Abstract

Peptide-carrier conjugates are widely used to raise antipeptide antibodies. In a model system using angiotensin and tetanus toxoid as the peptide and the carrier protein respectively, four cross-linking reagents were employed to study their effect on the immunogenicity of the conjugates. Optimization of the conjugation method for these heterobifunctional reagents, all succinimidyl active esters, resulted in well-defined conjugates of predictable composition. ELISA assays were performed to compare the antigenicity and the immunogenicity of the conjugates. The antipeptide antibody titres were of the order of 2 × 10 4−2 × 10 5. The anti-carrier antibody titres were high, in spite of the modification of the protein. Three of the four coupling reagents used for conjugation were of the ‘maleimide’ type: succinimidyl 6-( N-maleimido)- n-hexanoate (MHS), succinimidyl 4-( N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) and succinimidyl m-maleimidobenzoate (MBS). One coupling reagent contained an activated disulphide: succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The constrained linkers originating from SMCC and MBS induced very high linker-specific antibody levels. The more flexible non-aromatic linkers originating from MHS and SPDP showed almost no reactivity. For this reason and since the thioether linkage is more stable than the disulphide bond, we recommend MHS as the crosslinking reagent of choice.