Journal:

Article Title: Enterotoxigenic Escherichia coli TibA Glycoprotein Adheres to Human Intestine Epithelial Cells

doi: 10.1128/IAI.69.1.52-57.2001

Figure Lengend Snippet: Binding of purified and biotinylated TibA to HCT8 monolayers. Bound TibA was detected by peroxidase-coupled streptavidin. Color development in each well was measured at 405 nm. Symbols: ♦, saturation curve performed with 0.0, 1.0, 2.4, 4.8, 9.6, 19.2, and 48.0 pmol of TibA per well; ▴, competition curve performed with 4.8 pmol of labeled TibA plus 0.0, 1.0, 4.8, 9.6, or 48.0 pmol of unlabeled TibA. Every experiment was performed at least three times. The extent of biotin labeling varied from day to day, resulting in significant differences in color development. Therefore, both curves shown here represent the average of two experiments performed in duplicate.

Article Snippet: The human ileocecal epithelial cell line HCT8 (ATCC CCL 244) was maintained in RPMI 1640 medium containing 10% fetal bovine serum, 1 mM l -glutamine, and 1 mM sodium pyruvate (Life Technologies).

Techniques: Binding Assay, Purification, Labeling

Journal:

Article Title: Enterotoxigenic Escherichia coli TibA Glycoprotein Adheres to Human Intestine Epithelial Cells

doi: 10.1128/IAI.69.1.52-57.2001

Figure Lengend Snippet: Inhibition of TibA-mediated invasion by anti-TibA antiserum. Invasion assays were performed in the absence of antibodies (▪) or in the presence of preabsorbed and affinity-purified IgG from rabbit preimmune serum or polyclonal anti-TibA antiserum. (A) Invasion of HCT8 cells relative to E. coli DH5α(pET109), representing 100% (actual invasion of this strain in the absence of antibody was 1.30% ± 0.05%). Statistically significant effects of antibody treatment on the invasion efficiency of DH5α(pET109) are indicated by ∗ (P < 0.01) or ∗∗ (P < 0.005) as determined by analysis of variance of three experiments, each performed in triplicate. (B) Invasion of HCT8 cells relative to ETEC strain H10407, representing 100% (actual invasion of this strain in the absence of antibody was 0.24% ± 0.02%). TIB3 is a tib locus deletion mutant of H10407. Data are shown as averages for three replicates. Statistically significant effects of antibody treatment on the invasion efficiency of H10407 are indicated by ∗ (P < 0.01) as determined by analysis of variance of three experiments, each performed in triplicate.

Article Snippet: The human ileocecal epithelial cell line HCT8 (ATCC CCL 244) was maintained in RPMI 1640 medium containing 10% fetal bovine serum, 1 mM l -glutamine, and 1 mM sodium pyruvate (Life Technologies).

Techniques: Inhibition, Affinity Purification, Mutagenesis