title: Phage-display derived single-chain fragment variable (scFv) antibodies recognizing conformational epitopes of Escherichia coli heat-labile enterotoxin B-subunit.
abstract: Previously we have described studies on in vitro pentamer assembly of Escherichia coli (E. coli) derived heat-labile enterotoxin B subunit (EtxB) using conventional monoclonal antibodies (Amin et al., JBC 1995: 270, 20143-50 and Chung et al., JBC 2006: 281, 39465-70). To extend these studies further we have used phage-display to select single-chain Fragment variable (scFv) antibodies against different forms of the B-subunit. Two clones exhibiting strong and differential binding were chosen for detailed characterization. A comprehensive sequence analysis was performed to assign the framework and complementary-determining regions and a nonsense mutation present in one of these (scFv-B1.3.9) was corrected. Binding analysis showed that scFv-B1.3.9 bound in ELISA to both heat-denatured monomeric B-subunits (EtxB1) and also displayed cross-reactivity towards pentameric EtxB (EtxB5), although there was no reactivity towards monoganglioside (GM1) captured EtxB5. Another antibody (scFv-B5.2.14) had a different reactivity profile and, in ELISA, bound only to EtxB5 but not to EtxB1 or to EtxB5 captured via GM1. Surprisingly, in competition experiments, the assembled pentameric B-subunit inhibited binding of scFv-B5.2.14 to immobilized EtxB5 only weakly, whereas reduced, but not oxidized, monomeric EtxB1 was an efficient competitor. These results clearly demonstrate that B1.3.9 and B5.2.14 have different specificities for cryptic epitopes not accessible in the fully assembled GM1 bound pentameric form of EtxB. Taken together our results show that we were able to successfully isolate and characterize recombinant scFvs that differentially recognize diverse denatured forms or assembly intermediates of the heat-labile enterotoxin B subunit of E. coli.
authors.affiliation: Royal College of Surgeons, Department of Surgery, Connolly Hospital, Blanchardstown, D15, Ireland.
authors.forename: Wen Yuan
authors.initials: WY
authors.lastname: Chung
authors.forename: Markus
authors.initials: M
authors.lastname: Sack
authors.forename: Rachel
authors.initials: R
authors.lastname: Carter
authors.forename: Holger
authors.initials: H
authors.lastname: Spiegel
authors.forename: Rainer
authors.initials: R
authors.lastname: Fischer
authors.forename: Timothy R
authors.initials: TR
authors.lastname: Hirst
authors.forename: Neil A
authors.initials: NA
authors.lastname: Williams
authors.forename: Roger F L
authors.initials: RF
authors.lastname: James
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D000918:Antibody Specificity / Q000235:genetics / Q000276:immunology*; D001427:Bacterial Toxins / Q000032:analysis / Q000235:genetics / Q000276:immunology*; D001666:Binding Sites, Antibody / Q000235:genetics / Q000276:immunology; D003429:Cross Reactions / Q000235:genetics / Q000276:immunology; D004768:Enterotoxins / Q000032:analysis / Q000235:genetics / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D029968:Escherichia coli Proteins / Q000032:analysis / Q000235:genetics / Q000276:immunology*; D005677:G(M1) Ganglioside / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D020836:Protein Structure, Quaternary / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D017422:Sequence Analysis, DNA / Q000379:methods
chemical_list: D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D004768:Enterotoxins; D000939:Epitopes; D029968:Escherichia coli Proteins; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D005677:G(M1) Ganglioside; C027538:heat-labile enterotoxin, E coli