Strains of enterotoxigenic Escherichia coli (ETEC) are responsible for significant rates of morbidity and mortality among children, particularly in developing countries. The majority of clinical and public health laboratories are capable of isolating and identifying Salmonella, Shigella, Campylobacter, and Escherichia coli O157:H7 from stool samples, but ETEC cannot be identified by routine methods. The method most often used to identify ETEC is polymerase chain reaction for heat-stable and heat-labile enterotoxin genes, and subsequent serotyping, but most clinical and public health laboratories do not have the capacity or resources to perform these tests. In this study, polyclonal rabbit and monoclonal mouse IgG2b antibodies against ETEC heat-labile toxin-I (LT) were characterized and the potential applicability of a capture assay was analyzed. IgG-enriched fractions from rabbit polyclonal and the IgG2b monoclonal antibodies recognized LT in a conformational shape and they were excellent tools for detection of LT-producing strains. These findings indicate that the capture immunoassay could be used as a diagnostic assay of ETEC LT-producing strains in routine diagnosis and in epidemiological studies of diarrhea in developing countries as enzyme linked immunosorbent assay techniques remain as effective and economical choice for the detection of specific pathogen antigens in cultures. Key words: detection -antibodies -heat-labile toxin -Escherichia coliDiarrhea caused approximately two million deaths per year worldwide in the last decade (WHO-UNICEF 2002), and was responsible for an estimated 16-32% (median 21%) of mortality in children aged 0-4 years in developing countries (Kosek et al. 2003). One of the major etiologic agents is enterotoxigenic Escherichia coli (ETEC), known to be endemic in essentially all developing countries. Besides ETEC strains are also usually responsible for the acute diarrhea that affects visitors to the tropics (Ericsson 2003). In Brazil, these pathogens are responsible for up to 20% of cases of infantile diarrhea, region and season dependent (Reis et al. 1982, Trabulsi et al. 1985, Gomes et al. 1991, Almeida et al. 1998, Souza et al. 2002, Fernandes-Filho et al. 2003, Franzolin et al. 2005, Barreto et al. 2006 erogeneous family of lactose-fermenting E. coli, belonging to a wide variety of O antigenic types. These strains produce enterotoxins (heat labile and/or heat stable), and colonization factors, which allow the organisms to readily colonize the small intestine and in this way cause diarrhea (Sack 1980, Wolf 1997, Nataro & Kaper 1998.Since ETEC can be recognized by the enterotoxins it produces, diagnosis must depend upon identifying either heat-labile toxins (LT) and/or heat-stable toxin (ST). One or both toxins may be expressed by ETEC. LT belongs to a structurally and functionally related AB 5 enterotoxin family, in which the A subunit has the toxic activity and B subunit is responsible for toxin binding to cell receptor.Enzyme-linked immunosorbent assay (ELISA) techniques emplo...

Enterotoxigenic Escherichia coli (ETEC) produce heat-labile (LT) and/or heat-stable enterotoxins (ST). Despite that, the mechanism of action of both toxins are well known, there is great controversy in the literature concerning the in vitro production and release of LT and, for ST, no major concerns have been discussed. Furthermore, the majority of published papers describe the use of only one or a few ETEC isolates to define the production and release of these toxins, which hinders the detection of ETEC by phenotypic approaches. Thus, the present study was undertaken to obtain a better understanding of ST and LT toxin production and release under laboratory conditions. Accordingly, a collection of 90 LT-, ST-, and ST/LT-producing ETEC isolates was used to determine a protocol for toxin production and release aimed at ETEC detection. For this, we used previously raised anti-LT antibodies and the anti-ST monoclonal and polyclonal antibodies described herein. The presence of bile salts and the use of certain antibiotics improved ETEC toxin production/release. Triton X-100, as chemical treatment, proved to be an alternative method for toxin release. Consequently, a common protocol that can increase the production and release of LT and ST toxins could facilitate and enhance the sensitivity of diagnostic tests for ETEC using the raised and described antibodies in the present work.

Intimins are outer membrane proteins expressed by enteric bacterial pathogens capable of inducing intestinal attachment-and-effacement lesion (A/E). Through immunoblotting, immunofluorescence, flow citometry and immunogold we observed that the obtained polyclonal antibody against conserved intimin region recognizes the different intimin subtypes and suggests that it can be used as a tool for EPEC and EHEC detection. Besides, immuno-dot assay seems to be a possible alternative as a capture method.

A capture enzyme-linked immunosorbent assay (ELISA), which detects LT-I toxin produced by enterotoxigenic Escherichia coli strains, has been developed. This capture assay was performed using the IgG enriched fraction of anti-LT-I antiserum and IgG2b anti-LT-I monoclonal antibody and allowed a clear distinction between E. coli LT-I -producing and non-producing strains. The estimated accuracy of the assay is 78% for sensitivity, 94% for specificity and 92% for efficiency. Thus, the capture immunoassay is a sensitive tool for detection of E. coli, which produces heat-labile enterotoxin, and is suitable for use in clinical laboratories and epidemiological surveys in developing world.

Escherichia coli producing heat-labile enterotoxin is responsible for numerous cases of diarrhea worldwide, leading to considerable morbidity and mortality. The B subunits of this toxin are responsible for the binding to the receptor, the complex ganglioside GM1 which has galactose as its terminal sugar. In this study we showed that analogs of galactose (gal) and N-acetylgalactosamine (GalNAc) interfere with the binding of heat-labile toxin to GM1. Antibodies to lectins which mimic sugar structures and neoglycoprotein were employed. These compounds were able to inhibit heat-labile toxin activity efficiently in Vero cells: 37 microg of IgG-enriched fraction from an antiserum inhibited up to 70% of this activity, and 50% of the binding of heat-labile toxin to GM1. Neoglycoprotein was more efficient than antibodies, since 2.5 microg of this ligand completely abolished the activity of heat-labile toxin on Vero cells. These data suggest that these molecules could be developed for prophylaxis and diagnosis of diarrhea caused by heat-labile toxin.