Sensitive and specific detection of enteropathogenic and enterohemorrhagic Escherichia coli using recombinant anti-intimin antibody by immunofluorescence assay

Caravelli, Andressa; Luz, Daniela; Andrade, Fernanda B.; Moraes, Cláudia Trigo Pedroso de; Maranhão, Andréa Queiroz; Piazza, Roxane Maria Fontes

doi:10.1016/j.diagmicrobio.2013.08.021

Cited by 6 publications

(5 citation statements)

References 11 publications

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“…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. Indeed, even employing the recombinant antibody such as single chain fragment variable (scFv-intimin),147, 148 merely by immunofluorescence the scFv-intimin was able to detect tEPEC, aEPEC, and EHEC isolates, showing that intimin can be a target for EPEC and EHEC diagnosis after bacterial permeabilization 148 …”

Section: Typical and Atypical Enteropathogenic E Colimentioning

confidence: 99%

Diarrheagenic Escherichia coli

Gomes

Elias

Scaletsky

et al. 2016

Brazilian Journal of Microbiology

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412

353

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Most Escherichia coli strains live harmlessly in the intestines and rarely cause disease in healthy individuals. Nonetheless, a number of pathogenic strains can cause diarrhea or extraintestinal diseases both in healthy and immunocompromised individuals. Diarrheal illnesses are a severe public health problem and a major cause of morbidity and mortality in infants and young children, especially in developing countries. E. coli strains that cause diarrhea have evolved by acquiring, through horizontal gene transfer, a particular set of characteristics that have successfully persisted in the host. According to the group of virulence determinants acquired, specific combinations were formed determining the currently known E. coli pathotypes, which are collectively known as diarrheagenic E. coli. In this review, we have gathered information on current definitions, serotypes, lineages, virulence mechanisms, epidemiology, and diagnosis of the major diarrheagenic E. coli pathotypes.

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Section: Typical and Atypical Enteropathogenic E Colimentioning

confidence: 99%

Diarrheagenic Escherichia coli

Gomes

Elias

Scaletsky

et al. 2016

Brazilian Journal of Microbiology

Self Cite

412

353

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“…But, unfortunately diagnosis of this pathogen is not included in minimally equipped laboratories, thus tools and/or methods that can be used to identify these organisms are not available [ 6 , 7 ]. Recombinant antibodies, such as single chain fragment variable (scFv), rise as an alternative tool for that approach [ 32 , 33 ]. Diverse scFvs molecules have been previously developed for antigens detection of different DEC pathotypes.…”

Section: Discussionmentioning

confidence: 99%

“…This recombinant antibody is an excellent tool for diagnosis of EPEC and also enterohemorrhagic E . coli (EHEC) by immunofluorescence [ 33 ]. EspA and intimin proteins of EHEC O157:H7 were targets for the development of scFvs that recognize both antigens in O157 and O111 EHEC tested [ 36 ].…”

Section: Discussionmentioning

confidence: 99%

“…In order to obtain active scFv-LT and scFv-ST, the refolding methodology was adapted for each molecule after inclusion bodies solubilization and purification. The stepwise dialysis was employed for both recombinant antibody, since it has been successful used for scFv refolding [ 26 , 33 ] and, the adoption of this method combined with the use of folding additives contributed to obtain active fragments. Folding additives, such as L-arginine and oxidized/reduced glutathione, are commonly used on recombinant fragments refolding, assist correct folding of molecules and avoid aggregation [ 27 , 49 , 51 ].…”

Section: Discussionmentioning

confidence: 99%

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Single Chain Variable Fragments Produced in Escherichia coli against Heat-Labile and Heat-Stable Toxins from Enterotoxigenic E. coli

et al. 2015

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BackgroundDiarrhea is a prevalent pathological condition frequently associated to the colonization of the small intestine by enterotoxigenic Escherichia coli (ETEC) strains, known to be endemic in developing countries. These strains can produce two enterotoxins associated with the manifestation of clinical symptoms that can be used to detect these pathogens. Although several detection tests have been developed, minimally equipped laboratories are still in need of simple and cost-effective methods. With the aim to contribute to the development of such diagnostic approaches, we describe here two mouse hybridoma-derived single chain fragment variable (scFv) that were produced in E. coli against enterotoxins of ETEC strains.Methods and FindingsRecombinant scFv were developed against ETEC heat-labile toxin (LT) and heat-stable toxin (ST), from previously isolated hybridoma clones. This work reports their design, construction, molecular and functional characterization against LT and ST toxins. Both antibody fragments were able to recognize the cell-interacting toxins by immunofluorescence, the purified toxins by ELISA and also LT-, ST- and LT/ST-producing ETEC strains.ConclusionThe developed recombinant scFvs against LT and ST constitute promising starting point for simple and cost-effective ETEC diagnosis.

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“…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].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2014

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BackgroundEnteropathogenic and enterohemorrhagic Escherichia coli (EPEC/EHEC) are human intestinal pathogens responsible for diarrhea in both developing and industrialized countries. In research laboratories, EPEC and EHEC are defined on the basis of their pathogenic features; nevertheless, their identification in routine laboratories is expensive and laborious. Therefore, the aim of the present work was to develop a rapid and simple assay for EPEC/EHEC detection. Accordingly, the EPEC/EHEC-secreted proteins EspA and EspB were chosen as target antigens.MethodologyFirst, we investigated the ideal conditions for EspA/EspB production/secretion by ELISA in a collection of EPEC/EHEC strains after cultivating bacterial isolates in Dulbecco’s modified Eagle’s medium (DMEM) or DMEM containing 1% tryptone or HEp-2 cells-preconditioned DMEM, employing either anti-EspA/anti-EspB polyclonal or monoclonal antibodies developed and characterized herein. Subsequently, a rapid agglutination latex test (RALT) was developed and tested with the same collection of bacterial isolates.Principal findingsEspB was defined as a biomarker and its corresponding monoclonal antibody as the tool for EPEC/EHEC diagnosis; the production of EspB was better in DMEM medium. RALT assay has the sensitivity and specificity required for high-impact diagnosis of neglected diseases in the developing world.ConclusionRALT assay described herein can be considered an alternative assay for diarrhea diagnosis in low-income countries since it achieved 97% sensitivity, 98% specificity and 97% efficiency.

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Sensitive and specific detection of enteropathogenic and enterohemorrhagic Escherichia coli using recombinant anti-intimin antibody by immunofluorescence assay

Cited by 6 publications

References 11 publications

Diarrheagenic Escherichia coli

Diarrheagenic Escherichia coli

Single Chain Variable Fragments Produced in Escherichia coli against Heat-Labile and Heat-Stable Toxins from Enterotoxigenic E. coli

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

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