Detection of attachment of enterotoxigenic Escherichia coli (ETEC) to human small intestinal cells by enzyme immunoassay.
Simple immunoassays were developed to study the binding between enterocytes of the small intestine and other cell types, and enterotoxigenic Escherichia coli (ETEC). CFA/I or CFA/II pilus protein or CFA-positive E. coli bacteria were immobilised in wells of microtitre plates and incubated with vesicles or crude mucus prepared from human brush border enterocytes. Binding of the cell preparations was detected by adding specific rabbit anti-brush border IgG followed by urease-labelled goat anti-rabbit IgG and urea substrate. The binding of purified CFA/I to human or rabbit small intestine, human oral epithelial cells or Caco-2 cells was detected with specific anti-CFA/I IgG. Both human brush border and mucus-derived preparations were able to attach to ETEC. The binding was CFA-specific and strong enough to withstand several washings. In contrast, CFA/I did not bind to small intestinal cells of non-human small intestinal origin, indicating that there may be important differences in affinity between receptors present on human small intestinal cells and cells of non-human small intestinal origin. Antibodies directed against human small intestinal and non-small intestinal cells did not cross-react with either preparation, indicating that receptors between these different cell sources are different. The EIA proved useful during the identification of a newly-recognised 15 kDa bacterial surface component of ETEC strain H10407P, which may function as a putative attachment factor. The EIAs developed in this study were easy to perform and multiple tests could be performed on small samples, including biopsy samples obtained during endoscopy.
Mynott
T L
TL
School of Agriculture, La Trobe University, Bundoora, Victoria, Australia.
Luke
R K
RK
Chandler
D S
DS
D000818:Animals; D001422:Bacterial Adhesion / Q000502:physiology*; D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism; D015153:Blotting, Western; D001963:Bromelains / Q000378:metabolism; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004847:Epithelial Cells; D004848:Epithelium / Q000502:physiology; D004926:Escherichia coli / Q000502:physiology*; D036781:Fimbriae Proteins; D006801:Humans; D007124:Immunoenzyme Techniques; D007421:Intestine, Small / Q000166:cytology / Q000382:microbiology*; D008871:Microvilli / Q000502:physiology; D009093:Mucus / Q000166:cytology / Q000502:physiology; D011817:Rabbits
D001426:Bacterial Proteins; C022924:colonization factor antigens; D036781:Fimbriae Proteins; D001963:Bromelains