Phage-display derived single-chain fragment variable (scFv) antibodies recognizing conformational epitopes of Escherichia coli heat-labile enterotoxin B-subunit

Abstract

Abbreviations: E.coli, Escherichia coli; scFv, single-chain Fv antibody fragment; ELISA, enzyme-linked immunosorbent assay; G M1 , monoganglioside-G M1 (Gal -3GalNac 1-(neu5Ac 2-3)-4Gal 1-4Glc 1-cer); Etx, heatlabile enterotoxin; EtxB 5 , heat-labile enterotoxin B pentamer; EtxB 1 , heat-labile enterotoxin B subunit monomer; Ctx, cholera toxin; mAb, monoclonal antibody; SDS-PAGE, sodium dodecylsulphate polyacrylamide gel electrophoresis; CDR, complementary determining region; V H , heavy chain of an antibody; V L , light chain of an antibody; DTT, dithiotreitol; PBS, phosphate buffer saline; MBP, myelin basic protein.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

Single Chain Variable Fragments Produced in Escherichia coli against Heat-Labile and Heat-Stable Toxins from Enterotoxigenic E. coli

“…Diverse scFvs molecules have been previously developed for antigens detection of different DEC pathotypes. In terms of ETEC, some authors described scFv against F5 and K99 colonization factors [ 34 ], as well as the LT toxin [ 35 ].…”

Cloning approach and functional analysis of anti-intimin single-chain variable fragment (scFv)

“…According to these authors, it would provide an effective, less expensive and animal-friendly alternative as a prophylactic agent against colibacillosis. Also for ETEC, but concerning anti-LT monoclonal antibodies, Chung et al [30] selected scFvs that specifically reacted with intermediate forms of the B subunit of LT toxin, with the potential to be used to study pentamer assembly in vivo. Thus, the generation of scFvs with differential binding specificities opens up key areas for the development of new therapeutics that can block toxin formation in situ and thus be used for the treatment of diarrheal diseases caused by E. coli and Vibrio cholerae [30].…”

Recombinant antibodies for specific detection of clostridial [Fe-Fe] hydrogenases

“…With the help of phage display, screening conditions can be adjusted according to specific needs. More specifically, phage displayed libraries have been used in protein engineering12, protein-protein interaction studies13, epitope mapping14, specific screening and quantification15, recognizing function-specific16 and diverse protein conformations1718 of aerobic enzymes. However, screening of antibodies specific towards O 2 labile targets has been rarely conducted.…”