Biotinylation of bacterial lipopolysaccharide and its applications to electron microscopy.
We describe a procedure for lipopolysaccharide (LPS) biotinylation using N-biotinyl-L-lysine and application of the biotinylated LPS (Bi-LPS) to localization of LPS binding sites and subcellular distribution. Biotinylation of LPS was confirmed by enzyme-linked immunosorbent assay (ELISA), gel immunodiffusion, and immunodot techniques. The biological and toxicological activity of the Bi-LPS was tested by Limulus amoebocyte lysate (LAL) assays and histopathological examinations, respectively. Results showed that biotin was conjugated to LPS without disrupting the biological/toxicological activity of the molecule, which indicates that the biotin is directly linked to the polysaccharide portion of LPS. Localization of binding sites and subcellular distribution of Bi-LPS in human platelets and monocytes were studied by electron microscopy using an avidin-biotin-horseradish peroxidase (HRP) or streptavidin-gold method. Platelet surfaces were intensely stained by the reaction product of horseradish peroxidase (HPR) 5 min after incubation, and Bi-LPS was localized in small vesicles and vacuoles of platelets and in the phagocytic vacuoles of monocytes 60 min post incubation. Bi-LPS provides a reliable, stable, and sensitive tool for determination of LPS binding sites and subcellular distribution.
D000818:Animals; D001665:Binding Sites; D001792:Blood Platelets / Q000032:analysis* / Q000648:ultrastructure; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007118:Immunoassay; D005779:Immunodiffusion; D007124:Immunoenzyme Techniques; D008033:Limulus Test; D008070:Lipopolysaccharides / Q000032:analysis* / Q000378:metabolism / Q000633:toxicity; D008099:Liver / Q000187:drug effects; D008239:Lysine / Q000031:analogs & derivatives* / Q000378:metabolism; D051379:Mice; D008854:Microscopy, Electron; D009000:Monocytes / Q000032:analysis* / Q000648:ultrastructure
D008070:Lipopolysaccharides; C013411:biocytin; D008239:Lysine