Staff View: Development and characterization of recombinant antibody Fragments that recognize and neutralize in vitro Stx2 toxin from shiga toxin-producing escherichia coli

Development and characterization of recombinant antibody Fragments that recognize and neutralize in vitro Stx2 toxin from shiga toxin-producing escherichia coli

Background: Stx toxin is a member of the AB5 family of bacterial toxins: the active A subunit has N-glycosidase activity against 28S rRNA, resulting in inhibition of protein synthesis in eukaryotic cells, and the pentamer ligand B subunits (StxB) bind to globotria(tetra)osylceramide receptors (Gb3/G...

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Main Authors:	Luz, Daniela, Chen, Gang, Maranhão, Andrea Queiroz, Rocha, Leticia Barboza, Sidhu, Sachdev, Piazza, Roxane Maria Fontes
Format:	Artigo
Language:	Inglês
Published:	Plos One 2017
Subjects:	Anticorpos Diagnóstico Toxinas
Online Access:	http://repositorio.unb.br/handle/10482/22422 http://dx.doi.org/10.1371/journal.pone.0120481
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spelling	ir-10482-224222021-05-19T13:36:11Z Development and characterization of recombinant antibody Fragments that recognize and neutralize in vitro Stx2 toxin from shiga toxin-producing escherichia coli Luz, Daniela Chen, Gang Maranhão, Andrea Queiroz Rocha, Leticia Barboza Sidhu, Sachdev Piazza, Roxane Maria Fontes Anticorpos Diagnóstico Toxinas Background: Stx toxin is a member of the AB5 family of bacterial toxins: the active A subunit has N-glycosidase activity against 28S rRNA, resulting in inhibition of protein synthesis in eukaryotic cells, and the pentamer ligand B subunits (StxB) bind to globotria(tetra)osylceramide receptors (Gb3/Gb4) on the cell membrane. Shiga toxin-producing Escherichia coli strains (STEC) may produce Stx1 and/or Stx2 and variants. Strains carrying Stx2 are considered more virulent and related to the majority of outbreaks, besides being usually associated with hemolytic uremic syndrome in humans. The development of tools for the detection and/or neutralization of these toxins is a turning point for early diagnosis and therapeutics. Antibodies are an excellent paradigm for the design of high-affinity, protein-based binding reagents used for these purposes. Methods: and Findings In this work, we developed two recombinant antibodies; scFv fragments from mouse hybridomas and Fab fragments by phage display technology using a human synthetic antibody library. Both fragments showed high binding affinity to Stx2, and they were able to bind specifically to the GKIEFSKYNEDDTF region of the Stx2 B subunit and to neutralize in vitro the cytotoxicity of the toxin up to 80%. Furthermore, the scFv fragments showed 79% sensitivity and 100% specificity in detecting STEC strains by ELISA. Conclusion: In this work, we developed and characterized two recombinant antibodies against Stx2, as promising tools to be used in diagnosis or therapeutic approaches against STEC, and for the first time, we showed a human monovalent molecule, produced in bacteria, able to neutralize the cytotoxicity of Stx2 in vitro. 2017-02-06T10:44:47Z 2017-02-06T10:44:47Z 2015-03 Artigo LUZ, Daniela et al. Development and characterization of recombinant antibody fragments that recognize and neutralize in vitro Stx2 toxin from Shiga toxin-producing Escherichia coli. Plos One, v. 10, n.3, 2015. Diponível em: <http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0120481>. Acesso em: 12 dez. 2016. doi: http://dx.doi.org/10.1371/journal.pone.0120481. http://repositorio.unb.br/handle/10482/22422 http://dx.doi.org/10.1371/journal.pone.0120481 Inglês Acesso Aberto Copyright: © 2015 Luz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Fonte: http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0120481. Acesso em: 12 dez. 2016. application/pdf Plos One
institution	REPOSITORIO UNB
collection	REPOSITORIO UNB
language	Inglês
topic	Anticorpos Diagnóstico Toxinas
spellingShingle	Anticorpos Diagnóstico Toxinas Luz, Daniela Chen, Gang Maranhão, Andrea Queiroz Rocha, Leticia Barboza Sidhu, Sachdev Piazza, Roxane Maria Fontes Development and characterization of recombinant antibody Fragments that recognize and neutralize in vitro Stx2 toxin from shiga toxin-producing escherichia coli
description	Background: Stx toxin is a member of the AB5 family of bacterial toxins: the active A subunit has N-glycosidase activity against 28S rRNA, resulting in inhibition of protein synthesis in eukaryotic cells, and the pentamer ligand B subunits (StxB) bind to globotria(tetra)osylceramide receptors (Gb3/Gb4) on the cell membrane. Shiga toxin-producing Escherichia coli strains (STEC) may produce Stx1 and/or Stx2 and variants. Strains carrying Stx2 are considered more virulent and related to the majority of outbreaks, besides being usually associated with hemolytic uremic syndrome in humans. The development of tools for the detection and/or neutralization of these toxins is a turning point for early diagnosis and therapeutics. Antibodies are an excellent paradigm for the design of high-affinity, protein-based binding reagents used for these purposes. Methods: and Findings In this work, we developed two recombinant antibodies; scFv fragments from mouse hybridomas and Fab fragments by phage display technology using a human synthetic antibody library. Both fragments showed high binding affinity to Stx2, and they were able to bind specifically to the GKIEFSKYNEDDTF region of the Stx2 B subunit and to neutralize in vitro the cytotoxicity of the toxin up to 80%. Furthermore, the scFv fragments showed 79% sensitivity and 100% specificity in detecting STEC strains by ELISA. Conclusion: In this work, we developed and characterized two recombinant antibodies against Stx2, as promising tools to be used in diagnosis or therapeutic approaches against STEC, and for the first time, we showed a human monovalent molecule, produced in bacteria, able to neutralize the cytotoxicity of Stx2 in vitro.
format	Artigo
author	Luz, Daniela Chen, Gang Maranhão, Andrea Queiroz Rocha, Leticia Barboza Sidhu, Sachdev Piazza, Roxane Maria Fontes
author_sort	Luz, Daniela
title	Development and characterization of recombinant antibody Fragments that recognize and neutralize in vitro Stx2 toxin from shiga toxin-producing escherichia coli
title_short	Development and characterization of recombinant antibody Fragments that recognize and neutralize in vitro Stx2 toxin from shiga toxin-producing escherichia coli
title_full	Development and characterization of recombinant antibody Fragments that recognize and neutralize in vitro Stx2 toxin from shiga toxin-producing escherichia coli
title_fullStr	Development and characterization of recombinant antibody Fragments that recognize and neutralize in vitro Stx2 toxin from shiga toxin-producing escherichia coli
title_full_unstemmed	Development and characterization of recombinant antibody Fragments that recognize and neutralize in vitro Stx2 toxin from shiga toxin-producing escherichia coli
title_sort	development and characterization of recombinant antibody fragments that recognize and neutralize in vitro stx2 toxin from shiga toxin-producing escherichia coli
publisher	Plos One
publishDate	2017
url	http://repositorio.unb.br/handle/10482/22422 http://dx.doi.org/10.1371/journal.pone.0120481
_version_	1710449485673398272
score	13.657419

Development and characterization of recombinant antibody Fragments that recognize and neutralize in vitro Stx2 toxin from shiga toxin-producing escherichia coli

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