ABSTRACT
BACKGROUND: The outer core region of H. pylori lipopolysaccharide (LPS) contains α1,6-glucan previously shown to contribute to colonizing efficiency of a mouse stomach. The aim of the present study was to generate monoclonal antibodies (mAbs) specific for α1,6-glucan and characterize their binding properties and functional activity. MATERIALS AND METHODS: BALB/c mice were injected intraperitoneally with 10(8) formalin-fixed H. pylori O:3 0826::Kan cells 3× over 56 days to achieve significant titer. Anti-α1,6-glucan-producing hybridomas were screened by indirect ELISA using purified H. pylori O:3 0826::Kan LPS. One clone, 1C4F9, was selected for further characterization. The specificities of mAbs were determined by indirect and inhibition ELISA using structurally defined H. pylori LPS and synthetic oligosaccharides, and whole-cell indirect ELISA (WCE) of clinical isolates. They were further characterized by indirect immunofluorescent (IF) microscopy and their functional activity in vitro determined by serum bactericidal assays against wild-type and mutant strains of H. pylori. RESULTS: The generated anti-α1,6-glucan IgM, 1C4F9, has demonstrated an excellent specificity for the glucan chain containing 5 to 6 α1,6-linked glucose residues and showed surface accessibility by IF microscopy with H. pylori cells adherent to gastric adenocarcinoma cells monolayers. Of 38 isolates from Chile, 17 strains reacted with antiglucan mAbs in WCE (OD450 ≥ 0.2). Bactericidal activity was observed against selective wild-type and mutant H. pylori strains exhibiting OD450 values of ≥ 0.45 in WCE. CONCLUSIONS: Anti-α1,6-glucan mAbs could have potential application in typing and surveillance of H. pylori isolates as well as offer insights into structural requirements for the development of LPS-based vaccine against H. pylori infections.
Subject(s)
Antibodies, Bacterial/immunology , Antibodies, Monoclonal/immunology , Glucans/immunology , Lipopolysaccharides/immunology , Animals , Antibodies, Bacterial/isolation & purification , Antibodies, Bacterial/metabolism , Antibodies, Monoclonal/isolation & purification , Antibodies, Monoclonal/metabolism , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique, Indirect , Glucans/metabolism , Lipopolysaccharides/metabolism , Mice , Mice, Inbred BALB C , Microbial Viability/drug effects , Microscopy, Fluorescence , Protein BindingABSTRACT
Previous studies have shown that the LPS of Helicobacter pylori isolated from North American and European hosts predominantly expresses type 2 Lewis x (Le(x)) and Le(y) epitopes, whilst the LPS from Asian strains has the capacity to express type 1 Le(a) and Le(b) structures. The aim of this study was to evaluate the expression of Le antigens and the cytotoxin-associated antigen (CagA) by H. pylori isolates from Chile. A total of 38 isolates were screened. The expression of Le antigens and CagA was determined by whole-cell indirect ELISA, using commercially available monoclonal anti-Le and polyclonal anti-CagA antibodies. LPS profiles of H. pylori isolates were assessed by gel electrophoresis and Western blotting. Expression of Le(x) and/or Le(y) epitopes was confirmed in 32/38 isolates (84 %), whilst 9/38 isolates (24 %) expressed type 1 Le(b) blood group determinants, in addition to type 2 Le(x) and Le(y) structures. Six strains (16 %) were non-typeable. The majority of H. pylori strains examined were CagA-positive (83.3 %).
