Generation and refinement of peptide mimetic ligands for paratope-specific purification of monoclonal antibodies.
Paratope-specific purification of antibodies has distinct advantages over conventional methods of antibody purification with respect to its capacity to isolate product of high purity and immunoreactivity. The present report addresses the problems of identifying peptide ligands for the purification of antibodies reactive with nonprotein antigens. Using an anti-steroid antibody as the model, a lead sequence that bound antibody was identified from a peptide phage display library. The minimum binding unit in this sequence was deduced using a series of truncated peptides synthesized on the heads of polyethylene pins. Replacement Net analysis of the minimum binding unit identified peptides with increased affinity for the antibody. The affinity-matured peptide mimotope bound antibody in solution. By molecular modeling the peptide was superimposable onto estrone-3-glucuronide localized in the crystal structure of the antibody binding pocket. In order to resolve problems of presentation posed by the reversal of orientation of the peptide on the affinity matrix compared with the pins, the mimotope peptide was synthesized 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 provides a method for the generation and affinity maturation of mimotopes for antibodies against nonprotein antigens.
Murray
A
A
Cancer Research Laboratories, University of Nottingham, Nottingham, NG7 2RD, United Kingdom.
Smith
R G
RG
Brady
K
K
Williams
S
S
Badley
R A
RA
Price
M R
MR
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification*; D001666:Binding Sites, Antibody / Q000276:immunology; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis* / Q000737:chemistry; D004970:Estrone / Q000031:analogs & derivatives* / Q000276:immunology; D006801:Humans; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D018716:Molecular Mimicry; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D008024:Ligands; D019151:Peptide Library; D010455:Peptides; C015856:estrone-3-glucuronide; D004970:Estrone