RESUMO
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.
Assuntos
Toxinas Bacterianas/metabolismo , Bioensaio/métodos , Escherichia coli Enterotoxigênica/metabolismo , Enterotoxinas/metabolismo , Proteínas de Escherichia coli/metabolismo , Animais , Antibacterianos/farmacologia , Toxinas Bacterianas/imunologia , Ácidos e Sais Biliares/farmacologia , Ciprofloxacina/farmacologia , Escherichia coli Enterotoxigênica/efeitos dos fármacos , Escherichia coli Enterotoxigênica/isolamento & purificação , Enterotoxinas/imunologia , Ensaio de Imunoadsorção Enzimática , Proteínas de Escherichia coli/imunologia , Feminino , Imunoglobulina G/imunologia , Lincomicina/farmacologia , Masculino , Camundongos Endogâmicos BALB C , CoelhosRESUMO
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.
Assuntos
Humanos , Animais , Criança , Camundongos , Coelhos , Anticorpos Monoclonais/biossíntese , Toxinas Bacterianas/imunologia , Enterotoxinas/biossíntese , Escherichia coli/imunologia , Imunoglobulina G/biossíntese , Anticorpos Monoclonais , Enterotoxinas/genética , Enterotoxinas/imunologia , Escherichia coli/genética , Técnicas Imunoenzimáticas , Imunoglobulina G , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , SorotipagemRESUMO
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.
Assuntos
Anticorpos Monoclonais/biossíntese , Toxinas Bacterianas/imunologia , Enterotoxinas/biossíntese , Escherichia coli/imunologia , Imunoglobulina G/biossíntese , Animais , Criança , Enterotoxinas/genética , Enterotoxinas/imunologia , Escherichia coli/genética , Proteínas de Escherichia coli , Humanos , Técnicas Imunoenzimáticas , Camundongos , Reação em Cadeia da Polimerase , Coelhos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , SorotipagemRESUMO
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.