Effect of different hapten-carrier conjugation ratios and molecular orientations on antibody affinity against a peptide antigen

Hapten-carrier conjugate immunization is an important tool in the generation of hapten-specific antibodies for analytical purposes and in the uncovering of basic vaccine-immunological mechanisms. The affinity of antibodies is known to play an important role for the resulting sensitivity of antibody-based assay systems and in deciding whether a vaccine-induced antibody response will be protective. With ovalbumin as a carrier protein and a peptide (7.2NY) representing a 19 amino acid sequence from the E. coli-derived Verotoxin 2e as a model hapten we investigated whether it was possible to influence the affinity and titre of antibodies raised against the hapten using different conjugation ratios and orientations. The peptide was coupled to ovalbumin in four conjugation ratios and two molecular orientations — terminal and central — and the conjugates were verified by mass spectrometry. Mice were immunised ten times at two-weeks intervals with low doses of the eight conjugates. Blood samples collected between each immunisation were analysed by ELISA for specific antibody titres and relative affinities. With both types of conjugations, the anti-peptide antibody titres increased in response to increasing conjugation ratios, but central conjugation resulted in markedly higher titres than terminal conjugation. The overall anti-peptide antibody affinities reached approximately similar levels with both orientations, whereas a reversed proportionality was observed between conjugation ratio and antibody affinity for terminal conjugation. Thus, it appears that the molar ratio of a peptide and its carrier may affect the resulting antibody affinities, and that a conjugation ratio between a terminally conjugated peptide and its carrier approaching one will result in relatively high antibody affinities. Furthermore, the molecular orientation of the coupled peptide has a major effect on the anti-peptide antibody titres induced.