Analysis of Helicobacter pylori binding site on HEp-2 cells and three cell lines from human gastric carcinoma.
Helicobacter pylori (H. pylori) is a pathogen responsible for chronic gastritis and peptic ulcer diseases. It colonises the gastric mucus layer and adheres to the gastric epithelial cell surface. As this adherence is the first step of infection, it is important to study the adherence mechanism. The aim of this study was to analyse the specific binding assay of H. pylori to HEp-2 cells and three gastric phenotype cell lines, AGS, MKN-45 and AZ-521. H. pylori NCTC 11637 grown on agar plates was harvested and used in experiments. H. pylori was inoculated to pre-cultured cell monolayers. Adhered bacteria were labelled with an anti-H. pylori antibody and an FITC-conjugated secondary antibody and quantified by using a fluorescent plate reader. Microbial adherence to HEp-2 cells increased with incubation time and incubated concentration of H. pylori. No further increase was obtained with four or more hours of incubation or with a concentration of 4 x 10(7) bacteria/well or more. Scatchard analysis revealed a linear plot and the Bmax value was 88.3. Similar adherence patterns were obtained when AGS, AZ-521 and MKN-45 cells were used for adherence assays, but they had a lower binding affinity than HEp-2 cells and AZ-521. MKN-45 cells had less receptors than HEp-2 and AGS cells. In conclusion, H. pylori adhered to the cell surface could be quantified by this assay method. H. pylori adhesion to cell surfaces has a single population of binding site and one type of binding site on HEp-2, AGS, AZ-521 and MKN-45 cells.
Nishihara
K
K
Pharmacology Research Lab., Tokushima Research Center, Taiho Pharmaceutical Co. Ltd., Japan.
Nozawa
Y
Y
Nomura
S
S
Kitazato
K
K
Miyake
H
H
D018829:Adhesins, Bacterial / Q000378:metabolism; D001422:Bacterial Adhesion; D001665:Binding Sites; D002455:Cell Division; D005453:Fluorescence; D016481:Helicobacter Infections / Q000503:physiopathology; D016480:Helicobacter pylori / Q000166:cytology / Q000472:pathogenicity / Q000502:physiology*; D006801:Humans; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000378:metabolism; D013274:Stomach Neoplasms / Q000382:microbiology* / Q000473:pathology; D013997:Time Factors; D014407:Tumor Cells, Cultured
D018829:Adhesins, Bacterial; D011956:Receptors, Cell Surface