However, this technique is expensive, labor-intensive, and time consuming and so, not often established for routine diagnosis. Cytotoxicity assays are the most sensitive methods for detecting active Stxs ( Paton and Paton, 1998) and have been used as “gold standard” for evaluation of immunological tests.
Numerous assays for the diagnosis of STEC have been developed including microbiological, immunological, and genetical methods ( Bettelheim and Beutin, 2003). The expression of Stx is characteristic of STEC strains and so, exploitable targets for laboratory diagnosis of these pathogens. Stxs consist of a single A subunit, with catalytic activity, linked to a ring of five B subunits, responsible for specific cell binding of the toxin ( O'Brien and Holmes, 1987). Stx2, which is 56% homologous to Stx1 at the amino acid sequence level, is clinically the most important Stx type, because it is associated with severe outcomes of human infections including HUS ( Friedrich et al., 2002 Brooks et al., 2005). Shiga toxins (Stxs) are thought to be the major virulence factor of STEC strains ( Tarr et al., 2005) and comprise a family composed of Stx1, Stx2, and their variants, which can be found in STEC strains isolated from either humans or animals ( Ito et al., 1990). coli (STEC) infection is important to achieve an appropriate and early supportive treatment in the course of infection to decrease renal damage and improve overall patient outcome ( Ake et al., 2005). Prompt and accurate diagnosis of Shiga toxin producing E. Also, it is the leading cause of acute renal failure in pediatric age and the second for chronic renal failure ( Exeni, 2001). The incidence of HUS in Argentina is one of the highest in the world, with approximately 500 new cases being observed each year in under-5-year-old children ( Rivas et al., 2010). coli.Įnterohemorrhagic Escherichia coli (EHEC) causes a spectrum of human diseases ranging from mild non-bloody diarrhea through hemorrhagic colitis to the extraintestinal manifestation hemolytic-uremic syndrome (HUS) ( Griffin and Tauxe, 1991). Our results show that anti-Stx2B IgY sandwich ELISA could be used in routine diagnosis as a rapid, specific and economic method for detection of Shiga toxin-producing E. Twenty three strains negative for stx genes, including different bacteria species, showed no activity in Vero cell assay and produced negative results in ELISA, except for two strains. All Stx variants were detected by the sandwich ELISA, with a detection limit of 115 ng/ml Stx2. Supernatants from 42 STEC strains of different serotypes and stx variants, including stx 2EDL933, stx 2vha, stx 2vhb, stx 2g, stx 1EDL933, and stx 1d were tested. Several parameters were tested in order to optimize the sandwich ELISA assay, including concentration of antibodies, type and concentration of blocking agent, and incubation temperatures. The anti-Stx2B IgY was harvested from eggs laid by hens immunized with a recombinant protein fragment.
Chicken IgY was used as capture antibody and a HRP-conjugated rabbit IgG as the detection antibody. In this study, a sandwich ELISA for determination of Shiga toxin (Stx) was developed using anti-Stx2B subunit antibodies and its performance was compared with that of the Vero cell assay and a commercial immunoassay kit. Regardless of serotype, Shiga toxins (Stx1 and/or Stx2) are uniformly expressed by all EHEC, and so exploitable targets for laboratory diagnosis of these pathogens. coli (STEC) is associated with a spectrum of diseases that includes diarrhea, hemorrhagic colitis and a life-threatening hemolytic-uremic syndrome (HUS). Vélez Sarsfield 563 (C1282AFF), Buenos Aires, ArgentinaĮnterohemorrhagic Escherichia coli (EHEC), a subset of Shiga toxin producing E. 5Servicio Sueros y Antígenos-Instituto Nacional de Producción de Biológicos ANLIS “Dr.Veterinarias, Universidad Nacional del Centro Pcia. Sanidad Animal y Medicina Preventiva, Fac. 3IncuINTA, Instituto de Virología, Centro Nacional de Investigaciones Agropecuarias, Instituto Nacional de Tecnología Agropecuaria, Calle Las Cabañas y Los Reseros s/n, Casilla de Correo 25 (1712), Castelar, Buenos Aires, Argentina.1Instituto de Patobiología, Centro Nacional de Investigaciones Agropecuarias, Instituto Nacional de Tecnología Agropecuaria, Calle Las Cabañas y Los Reseros s/n, Casilla de Correo 25 (1712), Castelar, Buenos Aires, Argentina.
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