title: New flow cytometric method to quantify the inhibition of enteropathogenic Escherichia coli adhesion by anti-adhesin antibodies.
abstract: BACKGROUND
  Pathogenesis of enteropathogenic Escherichia coli (EPEC) infections can be divided in three stages. The first one is the intestinal colonization mediated by bacterial adhesins. The second and third stages are characterized by an intimate attachment of bacteria to the enterocytes. Little information is available on the specific immune response against EPEC. Here, we describe and validate a new approach to quantify the function of anti-EPEC adhesin antibodies (Abs).
  
  
  METHODS
  We developed a new method to quantify the function of anti-adhesin Abs by flow cytometry. We used pEGFP-E22 (a rabbit EPEC E22 strain expressing the GFP protein) and HeLa cells. The adhesion of E22 bacteria to HeLa cells is mediated by AF/R2, the specific E22 adhesin. We performed short-time interaction (30 min) between pEGFP-E22 and HeLa cells. After extensive washes, 10,000 HeLa cells were acquired by flow cytometry and bacterial adhesion was quantified. Different sera were used to inhibit bacterial adhesion and recombinant MPB-Afr2G (Afr2G is the main AF/R2 subunit) was also tested in this system.
  
  
  RESULTS
  We first verified that GFP expression by E22 did not modify bacterial adhesion. We then showed that this flow cytometry approach allowed easy quantification of bacterial adhesion and inhibition mediated by a specific anti-AF/R2 serum. Moreover, recombinant AF/R2 protein reversed the effect of the anti-AF/R2 serum. Finally, we validated our method using sera from E22 orally infected rabbits. We detected and quantified with this method functional specific anti-AF/R2 Abs in their sera. In addition, we correlated our results with an anti-AF/R2 enzyme-linked immunosorbent assay.
  
  
  CONCLUSIONS
  We have developed a new method to detect and quantify specific anti-EPEC adhesin Abs by flow cytometry. This method is easy to use and highly reproducible. Its development could be extended to the search of specific anti-adhesin Abs in human EPEC infections.
authors.affiliation: UMR 1225 Intéractions Hôtes-Agents Pathogènes, Institut National de la Recherche Agronomique-Ecole Nationale Vétérinaire de Toulouse, 23 chemin des Capelles, F-31076 Toulouse Cédex 3, France. s.boullier@envt.fr
authors.forename: Séverine
authors.initials: S
authors.lastname: Boullier
authors.forename: Christian
authors.initials: C
authors.lastname: Tasca
authors.forename: Alain
authors.initials: A
authors.lastname: Milon
mesh_terms: D018830:Adhesins, Escherichia coli / Q000276:immunology*; D000818:Animals; D000906:Antibodies / Q000032:analysis* / Q000097:blood / Q000494:pharmacology; D001422:Bacterial Adhesion / Q000187:drug effects / Q000276:immunology*; D004926:Escherichia coli / Q000276:immunology* / Q000472:pathogenicity; D004927:Escherichia coli Infections / Q000276:immunology / Q000503:physiopathology; D005434:Flow Cytometry / Q000379:methods*; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D007413:Intestinal Mucosa / Q000276:immunology; D008164:Luminescent Proteins; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000276:immunology; D015203:Reproducibility of Results
chemical_list: D018830:Adhesins, Escherichia coli; D000906:Antibodies; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins