title: Targeting of specific domains of diphtheria toxin by site-directed antibodies.
abstract: Antibodies highly selective for two functionally distinct regions of diphtheria toxin (DTx) were prepared using synthetic peptide conjugates as immunogens. Three peptides were selected for synthesis: sequence DTx141-157 on fragment A, which contains the putative protein elongation factor (EF-2) ADP-ribosyltransferase site; DTx224-237 on fragment B, selected on the basis of forming a predicted surface loop; and DTx513-526 on fragment B, forming a part of the region containing the putative receptor binding domain. All of the anti-peptide antibodies recognized the corresponding peptide, and also reacted with the toxin, specifically with the fragment containing the sequence against which they were raised, confirming the utility of this approach in generating fragment-specific antibodies. The anti-peptide antibody with the highest binding titre both to the peptide and to the native toxin was the one prepared against the sequence with the highest surface and loop likelihood indices of the three peptides selected. The similarity of the reactivity profiles with peptide and native and denatured toxin is consistent with the prediction that the region selected occurs in a surface loop and that the structure of the peptide is similar to the conformation of this region in the native protein. The epitopes for two of the anti-peptide antibodies were mapped. The results indicated that even though the antisera were raised to peptides containing 14 amino acids (aa) they were directed predominantly against a narrow region within the peptide, consisting of only 5-6 aa residues.(ABSTRACT TRUNCATED AT 250 WORDS)
mesh_terms: D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial / Q000276:immunology*; D004167:Diphtheria Toxin / Q000276:immunology*; D004168:Diphtheria Toxoid / Q000276:immunology; D000939:Epitopes / Q000276:immunology*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000276:immunology; D010446:Peptide Fragments / Q000276:immunology; D011487:Protein Conformation; D013329:Structure-Activity Relationship
chemical_list: D000907:Antibodies, Bacterial; D004167:Diphtheria Toxin; D004168:Diphtheria Toxoid; D000939:Epitopes; D009842:Oligopeptides; D010446:Peptide Fragments