title: Production and characterization of rabbit polyclonal sera against Shiga toxins Stx1 and Stx2 for detection of Shiga toxin-producing Escherichia coli.
abstract: STEC has emerged as an important group of enteric pathogens worldwide. In this study, rabbit polyclonal Stx1 and Stx2 antisera were raised and employed in the standardization of immunoassays for STEC detection. Using their respective antisera, the limit of detection of the toxin was 35.0 pg for Stx1 and 5.4 pg for Stx2. By immunoblotting, these antisera recognized both toxin subunits. Cross-reactivity was observed in the A subunit, but only Stx2 antiserum was able to neutralize the cytotoxicity of both toxins in the Vero cell assay. Six stx-harboring E. coli isolates were analyzed for their virulence traits. They belonged to different serotypes, including the O48:H7, described for the first time in Brazil. Only three strains harbored eae, and the e-hly gene and hemolytic activity was detected in five strains. Three isolates showed new stx2 variants (stx(2v-ha) and stx(2vb-hb)). The ELISA assay detected all six isolates, including one VCA-negative isolate, while the immunodot assay failed to detect one isolate, which was VCA-positive. In contrast, the colony-immunoblot assay detected only one VCA-positive isolate. Our results demonstrate that among the immunoassays developed in this study, the immunodot, and particularly the ELISA, appear as perspective for STEC detection in developing countries.
authors.affiliation: Bacteriology Laboratory, Butantan Institute, Avenida Vital Brazil, 1500, São Paulo, SP, 05503-900, Brazil.
authors.forename: Meire R B
authors.initials: MR
authors.lastname: Mendes-Ledesma
authors.forename: Letícia B
authors.initials: LB
authors.lastname: Rocha
authors.forename: Vanessa
authors.initials: V
authors.lastname: Bueris
authors.forename: Gladys
authors.initials: G
authors.lastname: Krause
authors.forename: Lothar
authors.initials: L
authors.lastname: Beutin
authors.forename: Marcia R
authors.initials: MR
authors.lastname: Franzolin
authors.forename: Luiz R
authors.initials: LR
authors.lastname: Trabulsi
authors.forename: Waldir P
authors.initials: WP
authors.lastname: Elias
authors.forename: Roxane M F
authors.initials: RM
authors.lastname: Piazza
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000032:analysis / Q000096:biosynthesis*; D002470:Cell Survival; D002522:Chlorocebus aethiops; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004927:Escherichia coli Infections / Q000382:microbiology*; D006801:Humans; D015151:Immunoblotting; D016133:Polymerase Chain Reaction; D011817:Rabbits; D022622:Shiga Toxin 1 / Q000032:analysis* / Q000276:immunology; D022641:Shiga Toxin 2 / Q000032:analysis* / Q000276:immunology; D054323:Shiga-Toxigenic Escherichia coli / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D014709:Vero Cells; D014774:Virulence
chemical_list: D000907:Antibodies, Bacterial; D004269:DNA, Bacterial; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2