Generation and refinement of peptide mimetic ligands for paratope-specificpurification of monoclonal antibodies

Paratope-specific purification of antibodies has distinct advantages over c onventional methods of antibody purification with respect to its capacity t o isolate product of high purity and immunoreactivity. The present report a ddresses the problems of identifying peptide ligands for the purification o f antibodies reactive with nonprotein antigens. Using an anti-steroid antib ody as the model, a lead sequence that bound antibody was identified from a peptide phage display library. The minimum binding unit in this sequence w as deduced using a series of truncated peptides synthesized on the heads of polyethylene pins. Replacement Net analysis of the minimum binding unit id entified peptides with increased affinity for the antibody. The affinity-ma tured peptide mimotope bound antibody in solution. By molecular modeling th e peptide was superimposable onto estrone-3-glucuronide localized in the cr ystal structure of the antibody binding pocket. In order to resolve problem s of presentation posed by the reversal of orientation of the peptide on th e affinity matrix compared with the pins, the mimotope peptide was synthesi zed in reverse sequence using D-amino acids. The resulting affinity matrix was effective for the purification of antibody. Eluted product demonstrated molecular homogeneity and high immunoreactivity. It is concluded that the combination of biological and chemical library techniques described provide s a method for the generation and affinity maturation of mimotopes for anti bodies against nonprotein antigens. (C) 2001 Academic Press.