Identification of enteropathogenic and enterohaemorrhagic Escherichia coli strains by immunoserological detection of intimin

Abstract

Aims: To evaluate the sensitivity and specificity of polyclonal and monoclonal antibodies (Mabs) against intimin in the detection of enteropathogenic and enterohaemorrhagic Escherichia coli isolates using immunoblotting. Methods and Results: Polyclonal and Mabs against the intimin‐conserved region were raised, and their reactivities were compared in enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) isolates using immunoblotting analysis. In comparison with rat antiserum, rabbit anti‐intimin IgG‐enriched fraction had a stronger recognition pattern to a wide spectrum of intimin types in different EPEC and EHEC serotypes. On the other hand, murine monoclonal IgG2b specific to intimin, with dissociation constant of 1·3 × 10−8 mol l−1, failed in the detection of some of these isolates. Conclusion: All employed antibodies showed 100% specificity, not reacting with any of the eae‐negative isolates. The sensitivity range was according to the employed antisera, and 97% for rabbit anti‐intimin IgG‐enriched fraction, followed by 92% and 78% sensitivity with rat antisera and Mab. Significance and Impact of the Study: The rabbit anti‐intimin IgG‐enriched fraction in immunoblotting analysis is a useful tool for EPEC and EHEC diagnoses.

Diarrheagenic Escherichia coli

“…Concerning intimin detection, a rabbit polyclonal sera raised against the conserved region of intimin (Int388-667) 143 was employed in order to detect tEPEC isolates expressing α, β, γ, δ and ɛ intimin reported an application of immunoblotting with 100% specificity and 97% sensitivity in the detection of eae positive E. coli strains 144, 145, 146. These authors clearly demonstrated that polyclonal rabbit antisera is suitable for immunoblotting as a diagnostic tool, and showed that protein denaturation and linearization is a critical step for anti-intimin antibody accessibility.…”

Development of a Rapid Agglutination Latex Test for Diagnosis of Enteropathogenic and Enterohemorrhagic Escherichia coli Infection in Developing World: Defining the Biomarker, Antibody and Method

“…We analyzed in this study a collection of 71 aEPEC [17], 31 tEPEC [18], [32] and 23 EHEC [44], belonging to different serotypes characterized as LEE-positive isolates. We also included for ELISA cut-off definition and specificity of the RALT, 20 LEE-negative diarrheagenic E. coli (DEC/LEE − ), 20 fecal E. coli negative for DEC virulence factors (NVF E. coli ) isolates and 20 Enterobacteriaceae isolates ( Aeromonas hydrophila , Edwardsiella tarda , Enterobacter cloacae , Enterococcus faecalis , Klebsiella pneumoniae , Morganella morganii, Pseudomonas aeruginosa , Proteus mirabilis , Providencia spp ., Salmonella spp.…”

“…As LEE-encoded virulence factors are common to EPEC and EHEC strains, intimin has been considered the first target for diagnosis [29], mainly its conserved region (Int 388–667 ) [30], [31]. Essentially, intimin detection in EPEC and EHEC isolates is appropriate by immunofluorescence and/or immunoblotting, i.e., after bacterial permeabilization, allowing anti-intimin antibody accessibility [32]–[34].…”

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

“…No entanto, estes testes não são factíveis na realidade de países em desenvolvimento por serem economicamente inviáveis. Além disso, a detecção do gene não garante a expressão do fator de virulência correspondente (ABE et al, 2009;MENEZES et al, 2010;NARA et al, 2010;VILHENA-COSTA et al, 2006).…”

“…Vários protocolos foram testados na tentativa de remover esta proteína da membrana: lise por ureia, polimixina, EDTA, aquecimento, detergentes, porém nenhum processo foi eficiente o bastante para detecção da intimina por ELISA ou immuno dot. A explicação para este fato seria a hipótese de após rompimento celular, por ser transmembrânica, grande porção de intimina ficaria presa nas porções celulares lisadas e não é liberada para o sobrenadante; ou quando liberada, aparece em quantidades insuficientes e somente detectadas na sua forma desnaturada e linearizada por immunoblotting (MENEZES et al, 2010 -posterioremente, 0,5 mL do cultivo seria permeabilizado com 4% de Triton-X-100 por 10 min; fixado com 1% de formaldeído por 20 min e incubado com 1%…”