Phage-display derived single-chain fragment variable (scFv) antibodies recognizing conformational epitopes of Escherichia coli heat-labile enterotoxin B-subunit.
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.
Chung
Wen Yuan
WY
Royal College of Surgeons, Department of Surgery, Connolly Hospital, Blanchardstown, D15, Ireland.
Sack
Markus
M
Carter
Rachel
R
Spiegel
Holger
H
Fischer
Rainer
R
Hirst
Timothy R
TR
Williams
Neil A
NA
James
Roger F L
RF
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
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