title: Design of ligands for the purification of anti-MUC1 antibodies by peptide epitope affinity chromatography.
abstract: The fine specificity of epitope recognition of the anti-MUC1 mucin monoclonal antibody, C595 has been studied using solid-phase replacement net (RNET) analysis. Two peptides (RAAP and RPPP) showed increased reactivity with C595 antibody compared with the native epitope (RPAP). These were synthesized as integral motifs within MUC1 immunodominant peptides and analyzed by fluorescence quenching (FQ) and circular dichroism (CD). They were also tested as ligands for the purification of C595 antibody using epitope affinity chromatography. Affinity matrices were compared with respect to capacity, affinity, and quality of the purified product. In FQ tests the native epitope peptide (APDTRPAPG) and the alanine substituted peptide had similar association constants when reacting with C595 antibody, whereas the proline substituted peptide (APDTRPPPG) had a higher association constant. This order of affinity for C595 was confirmed in chromatography experiments in which antibody was eluted from the former two peptide matrices at approximately the same point on the NaSCN elution gradient, whereas antibody was desorbed from APDTRPPPG at a higher NaSCN concentration. Circular dichroism analysis showed that the thermodynamically preferred conformation of these peptides in aqueous solution is the P-II extended helix, the conformation preferred for an extended bound form of the peptide held by interactions with the peptide amides. The stronger binding peptide (APDTRPPPG) has the higher population of the P-II helix in solution. In conclusion, RNET analysis is useful in the rational design of peptide ligands so that the performance of affinity matrices may be regulated.
authors.affiliation: Cancer Research Laboratories, School of Pharmaceutical Sciences, University of Nottingham, United Kingdom. pazam@pani.pharmacy.nottingham.ac.uk
authors.forename: A
authors.initials: A
authors.lastname: Murray
authors.forename: D I
authors.initials: DI
authors.lastname: Spencer
authors.forename: S
authors.initials: S
authors.lastname: Missailidis
authors.forename: G
authors.initials: G
authors.lastname: Denton
authors.forename: M R
authors.initials: MR
authors.lastname: Price
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002846:Chromatography, Affinity / Q000379:methods; D002942:Circular Dichroism; D018984:Epitopes, T-Lymphocyte / Q000276:immunology*; D008024:Ligands; D051379:Mice; D018396:Mucin-1; D009842:Oligopeptides / Q000276:immunology*; D010446:Peptide Fragments; D010455:Peptides / Q000138:chemical synthesis* / Q000276:immunology; D011487:Protein Conformation
chemical_list: D000911:Antibodies, Monoclonal; D018984:Epitopes, T-Lymphocyte; D008024:Ligands; C116055:MUC1 tandem repeat peptide; D018396:Mucin-1; D009842:Oligopeptides; D010446:Peptide Fragments; D010455:Peptides