title: Characterization and functional activity of murine monoclonal antibodies specific for α1,6-glucan chain of Helicobacter pylori lipopolysaccharide.
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.
  
  
  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.
authors: Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada K1A 0R6.
authors: Blair A
authors: BA
authors: Harrison
authors: Heriberto
authors: H
authors: Fernández
authors: Vandana
authors: V
authors: Chandan
authors: Myra Wilson
authors: MW
authors: Schuster
authors: Laura Otth
authors: LO
authors: Rademacher
authors: Claudio
authors: C
authors: Toledo
authors: Jianjun
authors: J
authors: Li
authors: Eleonora
authors: E
authors: Altman
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D019084:Fluorescent Antibody Technique, Indirect; D005936:Glucans / Q000276:immunology* / Q000378:metabolism; D008070:Lipopolysaccharides / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D050296:Microbial Viability / Q000187:drug effects; D008856:Microscopy, Fluorescence; D011485:Protein Binding
chemical_list: D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D005936:Glucans; D008070:Lipopolysaccharides; C504992:alpha-1-6-glucan; C442865:lipopolysaccharide, Helicobacter pylori