Detection of lipid A by monoclonal antibodies in S-form lipopolysaccharide after acidic treatment of immobilized LPS on Western blot

Abstract

Lipopolysaccharides (LPSs) were separated by SDS-PAGE, transferred onto polyvinylidene difluoride membranes, and hydrolyzed under acid conditions known to liberate lipid A from a broad variety of bacterial species. Independent of whether bis- or monophosphorylated lipid was set free, it remained bound to the membrane during subsequent immunostaining with monoclonal antibodies (MAbs) specific for the 1,4'-bis- or 4'-monophosphorylated lipid A backbone. Lipid A could be detected in the smooth (S)-form LPS from Enterobacteriaceae (Salmonella enterica, Citrobacter, Escherichia coli, Klebsiella pneumoniae, Serratia marcescens and Yersinia enterocolitica), Pseudomonas aeruginosa, Chromobacterium violaceum, Acinetobacter, Vibrio cholerae and Legionella pneumophila, thus allowing LPS-phenotype determination. The procedure can be used for the majority of Gram-negative bacteria, since LPSs containing 2,3-diamino-2,3-dideoxy-glucose or glucosamine in the lipid A disaccharide backbone could be detected. It was shown to be sensitive (up to 20 ng of hydrolyzed LPS could be detected), and could be easily performed using isolated LPS or whole-cell lysates with or without prior digestion with proteinase K. In addition to opening several other application possibilities, the procedure may be particularly useful in cases where silver-staining of the O-chain in SDSpolyacrylamide gels fails and specific antibodies against the O-antigen are not available.