Characterisation of a monoclonal antibody and its use to purify the cytotoxin of Helicobacter pylori.
The vacuolating cytotoxin (VacA) is a major virulence factor of Helicobacter pylori which is not yet well characterised and is difficult to obtain in large quantities. Here we describe the production of a monoclonal antibody that recognises the native but not the denatured form of VacA. The antibody can be efficiently used in affinity chromatography for one-step purification of large quantities of VacA from culture supernatants. Elution at acidic pH dissociates the oligomeric molecule into monomers that reanneal in a time-dependent fashion. The purified cytotoxin is able to bind, and to intoxicate HeLa cells.
Reyrat
J M
JM
IRIS, Chiron-S.p.A., Siena, Italy.
Charrel
M
M
Pagliaccia
C
C
Burroni
D
D
Lupetti
P
P
de Bernard
M
M
Ji
X
X
Norais
N
N
Papini
E
E
Dallai
R
R
Rappuoli
R
R
Telford
J L
JL
D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis / Q000302:isolation & purification; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000276:immunology* / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D006367:HeLa Cells / Q000378:metabolism; D016480:Helicobacter pylori / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D008807:Mice, Inbred BALB C; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; C074689:VacA protein, Helicobacter pylori