title: A crossreactive antipeptide monoclonal antibody with specificity for lysyl-lysine.
abstract: Synthetic peptides meeting certain guidelines have been used as immunogens to generate antibodies with predefined specificity. We have raised and characterized using established methods a monoclonal antibody against a synthetic peptide corresponding to the 18-amino acid carboxyterminal sequence (A194-211) of the platelet-derived growth factor (PDGF) A chain expressed by the U343 human glioma cell line. This antibody was generated in order to carry out structure-function studies on this region of PDGF whose biological significance is not yet clear. Anti-PDGF-A194-211 was found to be a low titre, IgM kappa molecule, with a Kd of 2.8 x 10(-7) M. When antibody reactivity was tested with parent PDGF-AAL (A chain homodimer containing a carboxyterminal extension) significant binding was observed. Surprisingly, 125I-PDGF-AAS, consisting of truncated A chains but lacking the extension was also bound. Moreover, poly-L-lysine, beta-thromboglobulin, PDGF-A194-211, and myoglobin competed dose-dependently with 125I-PDGF-AAL for antibody. 125I-bovine serum albumin was also bound. Examination of the primary sequence of proteins and peptides bound by the antibody revealed only one shared structural motif: a lysyl-lysine moiety. Selected small synthetic peptides containing this and other sequences were used as potential competitors of 125I-PDGF-A194-211 in antibody binding. Lysyl-lysyl-glycyl-glutamic acid [corrected] and lysyl-lysine competed, whereas lysyl-leucine did not. These results suggest that as few as two amino acid residues constitute a functional antigenic determinant and contrast with most previous estimates of the minimum number of residues required. Furthermore, we show that guidelines governing the design of synthetic peptides for their use as antigens to produce monoclonal antibodies of predetermined specificity may be unreliable.
authors.affiliation: Department of Hematology, University of New South Wales, Prince of Wales Hospital, Randwick, NSW, Australia.
authors.forename: L M
authors.initials: LM
authors.lastname: Khachigian
authors.forename: G
authors.initials: G
authors.lastname: Evin
authors.forename: F J
authors.initials: FJ
authors.lastname: Morgan
authors.forename: D A
authors.initials: DA
authors.lastname: Owensby
authors.forename: C N
authors.initials: CN
authors.lastname: Chesterman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001667:Binding, Competitive; D003429:Cross Reactions; D004151:Dipeptides / Q000276:immunology*; D000939:Epitopes; D007132:Immunoglobulin Isotypes / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000276:immunology; D010982:Platelet-Derived Growth Factor / Q000737:chemistry / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D004151:Dipeptides; D000939:Epitopes; D007132:Immunoglobulin Isotypes; D010455:Peptides; D010982:Platelet-Derived Growth Factor; C024575:lysyllysine