Application of Bead-ELISA method to detect Helicobacter pylori VacA.
Helicobacter pylori is an etiological agent of gastritis, gastric ulcer and gastric cancer. In order to clarify the significance of vacuolating cytotoxin (VacA) for the pathogenesis of Helicobacter pylori infection, we established and applied the sandwich bead enzyme-linked immunosorbent assay (Bead-ELISA) for quantitative determination of VacA in the culture mediums of H. pylori and other species of Helicobacter. The minimum concentration of VacA in culture medium detected by Bead-ELISA was 25 pg VacA/ml and its sensitivity was found to be quite high compared to vacuolation assay and Western blot analysis, e.g. the minimum concentrations of VacA in culture medium required for detection by vacuolation assay and Western blotting were 11 ng/ml and 38 ng/ml, respectively. All the H. pylori strains used were found to produce VacA in the culture medium by Bead ELISA, even though some strains were negative by Western blot and vacuolation assay. The results obtained by Bead-ELISA was consistent with those by PCR amplification of a 785 bp vacA fragments. A toxin immunologically similar to VacA produced by other strains of Helicobacter such as H. muridarum (ATCC49282), H. mustelae (F10) and H. felis (ATCC49179) could not be detected by Bead-ELISA as well as Western Blot.
Nagata
H
H
Institute of Tropical Medicine, Nagasaki University, Nagasaki, 8528523, Japan.
Wada
A
A
Kurazono
H
H
Yahiro
K
K
Shirasaka
D
D
Ikemura
T
T
Aoyama
N
N
Wang
A P
AP
Makiyama
K
K
Kohno
S
S
Hirayama
T
T
D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis / Q000737:chemistry; D001426:Bacterial Proteins / Q000276:immunology / Q000302:isolation & purification*; D015153:Blotting, Western; D002478:Cells, Cultured; D017931:DNA Primers / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D016481:Helicobacter Infections / Q000175:diagnosis* / Q000382:microbiology; D016480:Helicobacter pylori / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D008863:Microspheres; D016133:Polymerase Chain Reaction; D011817:Rabbits; D012680:Sensitivity and Specificity; D014617:Vacuoles / Q000737:chemistry
D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D017931:DNA Primers; C074689:VacA protein, Helicobacter pylori