Structure-function analyses of diphtheria toxin by use of monoclonal antibodies.
A large panel of hybridomas, secreting monoclonal antibodies (MAbs) specific for diphtheria toxin (DT) and prepared by immunization with either intact DT or its A or B fragment (DTA or DTB), have been isolated and characterized. The 213 MAbs were initially screened for reactivity to DT by enzyme-linked immunosorbent assay analyses and then were classified for their reactivity with DT, DTB, or DTA by solid-phase Western blot (immunoblot) analyses; 129 DTB-specific, 51 DTA-specific, and 33 non-fragment-assignable MAbs were obtained. Of the DTB MAbs, 118 recognize epitopes between residues 194 and 453, 10 recognize epitopes between residues 454 and 481, and 1 recognizes an epitope present in denatured toxin but not present in native DT located within the carboxyl-terminal receptor-binding region of DT (residues 482 to 535). Those MAbs that were the most protective in a cytotoxicity assay recognized native toxin in solution and inhibited binding of radiolabeled toxin to Vero cells to the greatest extent. A number of MAbs were able to detect epitopes that became more or less accessible when the toxin was preincubated at acidic (endosomal-mimicking) pH, suggesting that the epitopes they recognize may be important in the low-pH-induced insertion and/or translocation of DT across the endosomal membrane.
Rolf
J M
JM
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas 75235.
Eidels
L
L
D000911:Antibodies, Monoclonal / Q000276:immunology*; D004167:Diphtheria Toxin / Q000737:chemistry / Q000276:immunology* / Q000633:toxicity; D000939:Epitopes; D066257:Heparin-binding EGF-like Growth Factor; D006863:Hydrogen-Ion Concentration; D036341:Intercellular Signaling Peptides and Proteins; D011233:Precipitin Tests; D011487:Protein Conformation; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic / Q000378:metabolism; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D004167:Diphtheria Toxin; D000939:Epitopes; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic