title: Development and characterization of recombinant antibody fragments that recognize and neutralize in vitro Stx2 toxin from Shiga toxin-producing Escherichia coli.
abstract: 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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors: Laboratório de Bacteriologia, Instituto Butantan, São Paulo, Brazil.
authors: Daniela
authors: D
authors: Luz
authors: Banting and Best Department of Medical Research, Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Canada.
authors: Gang
authors: G
authors: Chen
authors: Laboratório de Imunologia, Universidade de Brasília, Brasília, Brazil.
authors: Andrea Q
authors: AQ
authors: Maranhão
authors: Laboratório de Bacteriologia, Instituto Butantan, São Paulo, Brazil.
authors: Leticia B
authors: LB
authors: Rocha
authors: Banting and Best Department of Medical Research, Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Canada.
authors: Sachdev
authors: S
authors: Sidhu
authors: Laboratório de Bacteriologia, Instituto Butantan, São Paulo, Brazil.
authors: Roxane M F
authors: RM
authors: Piazza
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism*; D051379:Mice; D019151:Peptide Library; D012680:Sensitivity and Specificity; D022641:Shiga Toxin 2 / Q000037:antagonists & inhibitors*; D054323:Shiga-Toxigenic Escherichia coli / Q000276:immunology / Q000378:metabolism*; D057127:Single-Chain Antibodies / Q000235:genetics / Q000378:metabolism*
chemical_list: D057134:Antibodies, Neutralizing; D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library; D022641:Shiga Toxin 2; D057127:Single-Chain Antibodies