Purification and characterization of lipooligosaccharides from four strains of "Haemophilus somnus"

Abstract

Lipooligosaccharides (LOSs) from four strains of "Haemophilus somnus" were purified and their electrophoretic profile, composition, endotoxic activity, and antigenic properties were analyzed. The LOSs were most efficiently purified by enzyme digestion, hot aqueous phenol extraction, and ultracentrifugation. Each LOS could be separated into two to six distinct bands with apparent Mrs of 3280 to 4960, following electrophoresis in polyacrylamide gels. Each LOS contained dodecanoic, tetradecanoic, and 3-hydroxytetradecanoic fatty acids; a high proportion of hexose, 3-deoxy-D-manno-octulosonic acid, and phosphate; and a small amount of heptose; glucosamine was present in both the oligosaccharide and the lipid A. Each "H. somnus" LOS demonstrated endotoxic activity, as determined by gelation of Limulus ameobocyte lysate, the dermal Schwartzman reaction, and mouse lethality. Antiserum to purified "H. somnus" LOS cross-reacted with all strains of "H. somnus" tested by enzyme-linked immunosorbent assay (ELISA), but not to any Enterobacteriaceae, Pseudomonas, or Pasteurella species tested. "H. somnus" LOS was a poor immunogen, but inhibition, dot blot, and sandwich ELISA data indicated that antibodies made to LOS were predominantly, though not exclusively, to lipid A. Monoclonal antibodies directed to "H. somnus" LOS confirmed that lipid A and non-lipid A determinants were present.