ELISA for toxin detection using supernatant of Stx-positive and—negative isolates with scFv fragments.

<p>The assay was performed in triplicate and the ROC curve determined the cut-off as 0.028.</p

ELISA for Stx toxin detection by Fab and scFv fragments.

<p>The assay was performed in triplicate and considered positive when * p>0.05 by Student’s t-test versus control.</p

Structural analyses of antibody fragments.

<p>A. Peptide mapping and the corresponding peptides, with the epitopes highlighted in pink. B. Structure prediction of both recombinant antibodies and Stx2, subunit A (purple) and subunit B (green), with the recognized peptides highlighted in pink as well as the antibody CDRs of recombinant antibodies. Also, the variable chains are represented, heavy (blue) and light (purple). In the Fab structure, the constant chain is shown in light blue, as well as the scFv linker.</p

Cytotoxicity neutralization assay.

<p>(A) scFv and (B) Fab. Dulbecco’s modified Eagle medium (DMEM) was used as negative control, and anti-Stx2 mAb used as positive neutralizing control. The assay was performed in triplicate, and significant neutralization was considered when * p > 0.05 by Student’s <i>t</i>-test versus the negative control. p < 0.05 with the positive control indicated that both had the same neutralizing capacity.</p

scFv and Fab purification.

<p>A. RNA extraction from Stx2 IgG-producing hybridoma. Molecular marker (lane 1); Total RNA from anti-Stx2 IgG-producing hybridoma (mAb 2E11) (lane 2). B. scFv gene cloning confirmation. Molecular marker (lane 1); PCR product (lane 2). C. scFv fragment purification. Molecular weight ladder (lane 1); scFv elutions (lanes 2 and 3). D. Phage gene cloning confirmation. Molecular marker (lane 1); Double digestion (lane 2). Phage ELISA, results of independent experiments, performed in triplicate, are expressed as the means ± SEM * p < 0.05 compared with control. E. Electrophoretic analysis of Fab fragment purification. Molecular weight ladder (lane 1); Fab fragment purified (lane 2).</p

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

<div><p>Background</p><p>Enteropathogenic and enterohemorrhagic <i>Escherichia coli</i> (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.</p><p>Methodology</p><p>First, 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.</p><p>Principal findings</p><p>EspB 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.</p><p>Conclusion</p><p>RALT 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.</p></div

Rapid agglutination latex test reactivity (%) with bacterial isolates.

<p>tEPEC (typical enteropathogenic <i>E. coli</i>); aEPEC (atypical enteropathogenic <i>E. coli</i>); EHEC (enterohemorrhagic <i>E. coli</i>); DEC/LEE⁻ (LEE-negative diarrheagenic <i>E. coli</i>); NVF <i>E. coli</i> (fecal <i>E. coli</i> negative for DEC virulence factors).</p>a<p><i>Proteus mirabilis</i>.</p><p>Rapid agglutination latex test reactivity (%) with bacterial isolates.</p

EspA and EspB production in different culture media.

<p>Atypical EPEC (aEPEC), typical EPEC (tEPEC) and EHEC isolates were cultivated in DMEM or DMEM-T or DMEM-PC. The supernatants were tested by indirect ELISA for EspA detection using anti-EspA IgG-enriched fraction (30 µg/mL) (<b>A</b>) and anti-EspA MAb (5 µg/mL) (<b>B</b>) and for EspB detection using anti-EspB IgG-enriched fraction (30 µg/mL) (<b>C</b>) and anti-EspB MAb (10 µg/mL) (<b>D</b>). The optical densities obtained for the isolates reacted with anti-EspA or anti-EspB polyclonal or monoclonal antibodies were analyzed by GraphPrism 5.01, using Student’s <i>t</i> test and two-away ANOVA. The differences were considered statistically significant when p≤0.05.</p

EspB production in different culture media.

<p>LEE-positive and LEE-negative isolates were cultivated in DMEM or DMEM-T or DMEM-PC. The supernatants were tested by indirect ELISA for EspB detection using anti-EspB IgG-enriched fraction (30 µg/mL) (<b>A</b>) and anti-EspB MAb (10 µg/mL) (<b>B</b>). The mean optical densities for LEE-negative and LEE-positive isolates were determined. The cut-off obtained by the ROC curve for anti-EspB MAb was 0.027 for DMEM and 0.0145 for DMEM-T and DMEM-PC. For anti-EspB PAb was 0.152 for DMEM, 0.135 for DMEM-T and 0.001 for DMEM-PC.</p

Typical of agglutination latex assay: negative and a semi-quantitative positive (from + to ++++) agglutination pattern with anti-EspB MAb coated beads.

<p>The test control with lysis buffer (B-PER) showed the same pattern as LEE-negative isolates.</p