Development of immunoreagents for diagnostics of CagA-positive Helicobacter pylori infections.
BACKGROUND
Helicobacter pylori strains expressing cytotoxic CagA protein are more likely to provoke severe gastric mucosal pathology and cause adenocarcinoma development than that lacking CagA. Determination of the CagA-status of a pathogen, therefore, is regarded as informative approach in H. pylori infection diagnostics and disease risk prediction.


METHODS
Molecular cloning, recombinant protein expression in Escherichia coli, affinity chromatography, electrophoresis and commonly used techniques of hybridoma production and screening were used as well as different immunosorbent assays and Western blot procedures.


RESULTS
Four overlapping N-terminally His(6)-tagged recombinant fragments of CagA that covered the entire CagA sequence were produced and purified. An ELISA for specific anti-CagA serum antibodies detection was developed and evaluated. Utilizing recombinant fragments, the first set of monoclonal antibodies against CagA-antigen was produced and characterized. Three antibodies recognized distinct linear epitopes inside conserved regions of the cytotoxin whereas the epitope of the forth antibody was mapped in the variable area of CagA. The monoclonal antibodies allowed discriminating CagA-positive and CagA-negative H. pylori strains by means of Western blot and immunosorbent assays.


CONCLUSIONS
The use of recombinant protein technology allowed obtaining pure CagA antigen, thus providing new perspectives for development of immunodiagnostic reagents. The set of monoclonal antibodies is a valuable tool for determination of CagA-status of H. pylori infection and for the investigation of cytotoxin molecule as well.
Klimovich
Alexander V
AV
Cytology and Histology Department, St.-Petersburg State University, Russia. aklimovich@gmail.com <aklimovich@gmail.com>
Samoylovich
Marina P
MP
Gryazeva
Irina V
IV
Terekhina
Lidiya A
LA
Suvorov
Alexander N
AN
Klimovich
Vladimir B
VB
D000818:Animals; D000907:Antibodies, Bacterial / Q000302:isolation & purification; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D000942:Antigens, Bacterial / Q000032:analysis*; D001426:Bacterial Proteins / Q000032:analysis*; D001431:Bacteriological Techniques / Q000379:methods*; D015153:Blotting, Western / Q000379:methods; D016481:Helicobacter Infections / Q000382:microbiology*; D016480:Helicobacter pylori / Q000737:chemistry* / Q000302:isolation & purification / Q000472:pathogenicity; D006801:Humans; D007163:Immunosorbent Techniques; D051379:Mice; D008807:Mice, Inbred BALB C; D037521:Virulence Factors / Q000032:analysis*
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D037521:Virulence Factors; C080866:cagA protein, Helicobacter pylori