title: Monoclonal antibodies against Vero cells that protect against diphtheria toxin.
abstract: Mice were immunized with a cell line (Vero) that possesses a high number of membrane receptors for diphtheria toxin. Spleen cells from these mice were fused with SP2/0-Ag14 cells and two cell lines (1A2 and 2D2) isolated by screening for the ability of their secreted antibodies to inhibit binding of radiolabeled diphtheria toxin to Vero cells. These antibodies protected Vero cells from the inhibition of protein synthesis mediated by diphtheria toxin. The antibodies were purified, iodinated, and their binding characteristics investigated. At 4 degrees C, the association of 1A2 and 2D2 with Vero cells was saturable (KD approximately 10(-8) M) and indicated about 10(6) binding sites/cell. Diphtheria toxin did not inhibit the binding of either radiolabeled antibody. Monoclonal antibody 1A2 completely inhibited 125I-2D2 binding and vice versa. Trypsin or phospholipase C treatment of Vero cells had no effect on the ability of the monoclonal antibodies to bind to the cells. These findings suggest that: (1) the two monoclonal antibodies recognize the same or closely related epitopes and (2) the antibodies bind a domain distinct from the toxin binding site or to a subcomponent of the diphtheria toxin receptor that is present at many other cell surface sites. These antibodies offer a powerful tool to study the structure, processing and mode of action of diphtheria toxin receptors.
authors.affiliation: Department of Toxinology, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, Maryland 21701.
authors.forename: B J
authors.initials: BJ
authors.lastname: Rönnberg
authors.forename: B C
authors.initials: BC
authors.lastname: Lidgerding
authors.forename: J L
authors.initials: JL
authors.lastname: Middlebrook
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000915:Antibody Affinity; D001667:Binding, Competitive; D002460:Cell Line; D006224:Cricetinae; D004167:Diphtheria Toxin / Q000378:metabolism / Q000633:toxicity*; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D051379:Mice; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic / Q000276:immunology*; D014709:Vero Cells / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D004167:Diphtheria Toxin; C587884:Hbegf protein, mouse; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic