Capture immunoassay for LT detection produced by enterotoxigenic Escherichia coli in bacterial isolates

Abstract

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.

Production, characterization, and application of antibodies against heat-labile type-I toxin for detection of enterotoxigenic Escherichia coli

“…Thus, the sensitivity and specificity of the capture immunoassay standardized by Menezes et al (2003) was compared with GM1 ELISA. Despite the fact that GM1 is commercially available, the specificity of the assay is antibody dependent.…”

“…Sensitivity and specificity of capture assay in detecting LT-I -The sensitivity and specificity of the capture assay was determined as described by Menezes et al (2003) in the standardization of the capture ELISA immunoassay. Here we tested different concentrations of purified LT (from 100 µg to 0.7 ng) or supernatants of bacterial isolates.…”

“…After electrophoresis, the separated proteins were transferred to nitrocellulose membrane (Hybond C-Extra, Amersham Life Science) at 100 V for 18 h at 4 o C. The membrane was blocked with 3% BSA for 2 h and reacted with IgG-enriched fraction of polyclonal anti-LT antibody (5 µg/ml) and monoclonal LT-antibodies (18 µg/ml IgG1 or IgG2b) for 18 h at 4 o C. The membrane was then washed and incubated for 2 h with horseradish peroxidase-conjugated anti-mouse IgG diluted 1:10,000 or anti-rabbit IgG diluted 1/10,000. After washing, the substrate 3,3-Diaminobenzidine tetrahydrochloride plus hydrogen peroxide was added and the reaction was stopped by distilled water addition.Sensitivity and specificity of capture assay in detecting LT-I -The sensitivity and specificity of the capture assay was determined as described by Menezes et al (2003) in the standardization of the capture ELISA immunoassay. Here we tested different concentrations of purified LT (from 100 µg to 0.7 ng) or supernatants of bacterial isolates.…”

Caracterização genotípica de fatores de virulência de Escherichia Coli enterotoxigênica isoladas de água para consumo humano

“…As doenças veiculadas pela água são principalmente causadas por patógenos entéricos, sendo Escherichia coli de grande importância clínica, por se tratar de uma bactéria termotolerante de origem exclusivamente fecal (CABRAL, 2010). Dados epidemiológicos mostram que cerca de 700.000 mil pessoas de países em desenvolvimento morrem todos os anos por decorrência da diarreia por ETEC (MENEZES et al, 2003). E. coli associadas à infecção intestinal, tanto em crianças como em adultos, são conhecidas como E. coli diarreiogênicas (DEC) e estão agrupadas em oito patotipos: E. coli enteropatogênica (EPEC), E. coli enterotoxigênica (ETEC), E. coli enteroinvasora (EIEC), E. coli produtora de toxina Shiga (STEC), E. coli enteroagregativa (EAEC), E. coli que adere difusamente a células epiteliais (DAEC), E. coli aderente invasora (AIEC) e E. coli enteroagregativa produtora de toxina Shiga (STEAEC) (CLEMENTS et al, 2012).…”

Anticorpo recombinante anti-intimina: uma ferramenta para o diagnóstico rápido de Escherichia coli enteropatogênica e de Escherichia coli enterohemorrágica.

“…Diante disso, em nosso laboratório foram produzidos anticorpos específicos para as toxinas LT e ST de ETEC (MENEZES, 2002;MENEZES et al, 2003MENEZES et al, , 2006, para a toxina Pet de EAEC (VILHENA-COSTA et al,…”