Polyclonal anti-intimin antibody: immunological characterization and its use in EPEC and EHEC diagnosis

Koga, Paula Célia Mariko; Menezes, Caroline Anunciação; Lima, Flávia Afonso; Nara, Júlia Mitico; Magalhães, Caroline Arantes; Cianciarullo, Aurora Marques; Ferreira-Júnior, Jorge Mario da Costa; Trabulsi, Luíz Rachid; Mendes-Ledesma, Meire R. B.; Piazza, Roxane Maria Fontes

doi:10.1590/s1517-83822003000500002

Cited by 13 publications

(16 citation statements)

References 4 publications

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“…The sections were deparaffinized and incubated with 0.3% H 2 O 2 in PBS for 20 min at room temperature to inactivate endogenous peroxidase. Samples were blocked with PBS containing 2% bovine serum albumin (BSA) for 30 min at room temperature and incubated for 2 h in the same buffer containing a 1:5,000 dilution of polyclonal rabbit anti-intimin IgG-enriched fraction (41,46). Sections were washed three times with PBS and incubated for 1 h at room temperature with biotin-labeled goat anti-rabbit IgG (Sigma) diluted in PBS-2% BSA.…”

Section: Methodsmentioning

confidence: 99%

Immunization of Mice with Lactobacillus casei Expressing a Beta-Intimin Fragment Reduces Intestinal Colonization by Citrobacter rodentium

Ferreira

Silva

Piazza

et al. 2011

Clin Vaccine Immunol

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Gastrointestinal infections are among the leading causes of childhood mortality around the world and are responsible for approximately 2 million deaths every year (12). Enteropathogenic Escherichia coli (EPEC) is one of the main causes of childhood diarrhea in developing countries and has been considered a worldwide pathogen (7,18,31,59). EPEC has been classified into typical or atypical according to the expression or lack of expression, respectively, of the bundle-forming pilus, encoded by the EAF plasmid (1, 50). Epidemiological studies suggest that the prevalence of atypical EPEC has increased in acute or persistent diarrhea cases in both children and adults (23,29,52,62). EPEC colonizes the intestinal mucosa, causing the characteristic attaching and effacing (A/E) lesions. The formation of the A/E lesion involves a type III secretion system (T3SS), encoded by the LEE pathogenicity island, which delivers a range of effector proteins into host cells. Using this strategy, the pathogen triggers actin polymerization, producing a pedestal-shaped structure to which the bacteria strongly attach through the interaction of the adhesin intimin with the translocated receptor Tir (27,38). Intimin is a 94-kDa protein inserted into the bacterial outer membranes from typical and atypical EPEC, enterohemorrhagic E. coli (EHEC), and the mouse pathogen Citrobacter rodentium. This adhesin is encoded by the eae gene and is essential for virulence (28,48). The N-terminal region is conserved among intimins, whereas the C-terminal portion is highly variable and has been used to classify intimin into 29 types that can be related to tropism for different intestinal tissues (26,35,49). Among them, the ␤ subtype is one of the intimins most frequently expressed by EPEC isolates (6,56,60). EPEC and EHEC infections can be prevented by breastfeeding (17,57), and antibodies against intimin are usually observed in the milk of women living in areas of endemicity (58,70). In addition, these antibodies are able to inhibit the adhesion of EPEC to epithelial cells in vitro (15,19). Several vaccination strategies using intimin have reduced colonization or mortality in animal models of infection with rabbit enteropathogenic E. coli (REPEC), EHEC, or C. rodentium (4,30,36,39), suggesting that intimin is a good candidate for the formulation of vaccines against these infections. A minimal intimin fragment able to bind to the receptor Tir is composed of the last 190 amino acids, but high-affinity binding was observed only with a larger fragment comprising the last 280 amino acid residues (Int 280 ) (8). In fact, subcutaneous immunization of mice with the Int 280 fragment from ␣-intimin reduced intestinal colonization by a C. rodentium strain expressing an intimin from the same subtype (30). However, analysis of the immunodominant regions inside Int 280 have shown that antibodies against this fragment were directed to 2 regions in the N terminus of Int 280 , more precisely, a

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Section: Methodsmentioning

confidence: 99%

Immunization of Mice with Lactobacillus casei Expressing a Beta-Intimin Fragment Reduces Intestinal Colonization by Citrobacter rodentium

Ferreira

Silva

Piazza

et al. 2011

Clin Vaccine Immunol

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“…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.…”

Section: Typical and Atypical Enteropathogenic E Colimentioning

confidence: 99%

Diarrheagenic Escherichia coli

Gomes

Elias

Scaletsky

et al. 2016

Brazilian Journal of Microbiology

384

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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“…In a previous study, the reactivity of IgG‐enriched fraction of rabbit anti‐intimin serum with tEPEC belonging to serotypes that express α, β, γ, δ and ε intimin by immunoblotting had been observed. This reactivity was confirmed using immuno‐dot assay, immunofluorescence, flow cytometry and immunogold labelling (Koga et al. 2003).…”

Section: Discussionmentioning

confidence: 78%

“…Amongst the methods for the detection of virulence factor expression, the immunoassays can be considered as first alternative, and for these purposes polyclonal and/or Mabs are raised (Koga et al. 2003; Menezes et al.…”

Section: Discussionmentioning

confidence: 99%

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

Menezes

Rocha

Koga

et al. 2010

Journal of Applied Microbiology

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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.

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Polyclonal anti-intimin antibody: immunological characterization and its use in EPEC and EHEC diagnosis

Cited by 13 publications

References 4 publications

Immunization of Mice with Lactobacillus casei Expressing a Beta-Intimin Fragment Reduces Intestinal Colonization by Citrobacter rodentium

Immunization of Mice with Lactobacillus casei Expressing a Beta-Intimin Fragment Reduces Intestinal Colonization by Citrobacter rodentium

Diarrheagenic Escherichia coli

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

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