Detection of antibodies against lipopolysaccharides of Escherichia coli and Salmonella R and S strains by immunoblotting (original) (raw)
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Journal of Infectious Diseases, 1989
A panel of eight murine monoclonal antibodies (MAbs) were produced against heat-killed Escherichia coli J5 and shown to react with J5 lipopolysaccharide (LPS) by enzyme-linked immunosorbent assay (ELISA). These antibodies were then assayed by a suspension ELISA for reactivity with up to 20 heterologous smooth or rough isolates of gram-negative bacteria, which were assayed after heat or formalin treatment, or as live cells. Extracted LPS from the same bacteria were tested for reactivity with the MAbs by direct ELISA. The MAbs demonstrated broad cross-reactivity with most heat-treated bacteria. In contrast, cross-reactivity of the MAbs with live or formalin-treated bacteria was limited almost exclusively to E. coli J5, Hemophilus species, or rough mutants of Salmonella minnesota. Reactivity with extracted LPSs and lipid A varied considerably depending on the MAb. Further, when Western blotting was used as the assay only four of eight MAbs reacted with J5 LPS, and none of the MAbs reacted with LPS from smooth S. minnesota or any of its rough mutants. Adsorption of the MAbs with acid hydrolyzed, boiled, or live E. coli J5 prior to ELISA of the MAbs with J5 LPS supported evidence that none of the MAbs were specific for lipid A and that reactivity was greater with boiled than with live cells. Thus, the cross-reactivity of antibodies to E. coli J5 LPS is dependent on the physical state of the bacteria or LPS used for assay, the assay used, and the specificity of the antibody. Antiserum to the rough mutant of Escherichia coli 0111:B4 (J5) or to the deep rough mutant of Salmonella minnesota R595 can protect animals and humans from the lethal effects of endotoxic septicemia [1-9]. This area of research has been highly controversial because some investigators have not demonstrated protection with such antiserum [10-14]. Proposed protective mechanisms of the biologic
FEMS Microbiology Letters, 2001
Hybridomas secreting immunoglobulin A (IgA) monoclonal antibodies (MAbs) against Salmonella enteritidis lipopolysaccharide (LPS) were generated after mucosal immunization of BALB/c mice with heat killed bacteria. Antigen binding properties and specificity of the produced MAbs were studied in ELISA and immunoblotting with purified LPS. Two IgA MAbs agglutinated all Salmonella OD1 strains and all S. enteritidis clinical isolates. MAb 178H11 recognized O:9 antigen of subserogroup OD1 LPS. MAb 177E6/A9 reacted also with OD3 LPS antigen and agglutinated OD3 strains. These data suggest the existence of different O:9 antigen subspecificities, one presented in subgroup OD1 and the other common for OD1 and OD3. Thus the produced IgA MAbs prove to be useful reagents, which could differentiate OD1 and OD3 from OD2 strains. ß : S 0 3 7 8 -1 0 9 7 ( 0 1 ) 0 0 0 7 1 -4
FEMS Microbiology Letters, 2000
A strain of Citrobacter sedlakii showing serological cross-reaction with Escherichia coli O157 antisera was demonstrated to produce a lipopolysaccharide O-antigen having an identical structure with that of the E. coli O157 O-antigen. A strain of Citrobacter freundii showing similar cross-reaction with E. coli O157 specific monoclonal antibody was shown to produce a lipopolysaccharide O-antigen composed of a trisaccharide repeating unit having the structure [^2)-K-D Rhap-(1^3)-L-D-Rhap-(1^4)-L-D-Glcp-(1^]. This O-antigen differs from that of the E. coli O157 O-antigen and also lacks a component 2-substituted 4-amino-4,6-dideoxy-K-D-mannopyranosyl residue implicated as the common epitope in the lipopolysaccharide O-antigens of previously investigated bacterial species showing serological cross-reactivity with E. coli O157 antisera. The C. freundii O-antigen presents an interesting example of structural mimicry within a bacterial polysaccharide antigen. ß : S 0 3 7 8 -1 0 9 7 ( 0 0 ) 0 0 3 2 0 -7
Journal of Clinical Microbiology, 1991
To facilitate the identification and serotyping of Salmonella species, we established a wide variety of murine monoclonal antibodies (MAbs) that were reactive with the lipopolysaccharides (LPSs) of Salmonella serogroups B to E. An effective approach for generating LPS-reactive hybridomas was used; this required immunization of mice with LPS-coated bacteria. To screen for diagnostically useful MAbs, the MAbs were tested by enzyme-linked immunosorbent assay against a set of purified LPSs from smooth and rough Salmonella strains. At least four major groups of antibody specificities were identified: Salmonella (i) BO specific, (ii) CO specific, (iii) DO specific, and (iv) EO specific. For a more detailed epitope analysis, a panel of eight different serogroup-specific MAbs which were shown to bind the O-antigenic polysaccharide chains, yielding characteristic ladder patterns in Western blots (immunoblots) against the LPS of Salmonella serogroups B to E, were selected. The availability of...
Carbohydrate Research, 1989
Partial structures of enterobacterial lipopolysaccharides (LPS) of the Rechemotype, consisting of lipid A and 3-deoxy-D-manno-2-octulosonic acid (Kdo), as well as oligosaccharides and derivatives of Kdo were synthesized and used to characterize the epitope specificity of monoclonal antibodies against Re-mutant LPS. High-molecular-weight antigens, obtained after copolymerization of the respective ally1 glycosides with acrylamide, and the haptenic oligosaccharides were used in immunoprecipitation, immune hemolysis, and in inhibition assays. A monoclonal antibody (clone 20, IgM) recognizing a terminal Kdop group was shown to require for its binding the a-anomeric configuration and OH-4 and OH-5 groups, whereas the C-7-C-8 chain was of minor importance. Another monoclonal antibody (clone 25, IgG,), which recognizes a (2+4)-linked Kdo disaccharide, was
Lipopolysaccharide (LPS) is an important structural component of the outer cell membrane complex of gram negative microorganisms. Its causative role in gram negative bacteria-induced diseases and broad applications in different kinds of cell stimulation experiments provided a conceptual basis for studies directed at the isolation, purification, and detailed chemical characterization of LPS. The main problem with LPS purification protocols is the contamination of the end product with nucleic acids and proteins in variable proportions which could potentially interfere with downstream applications. In this study, a simple procedure for purification of LPS from Escherichia coli (E.coli) and Salmonella typhi (S.typhi) with high purity and very low contaminating nucleic acids and proteins based on the hot phenol-water extraction protocol has been introduced. The purity of extracted LPS was evaluated by silver and coomassie blue staining of SDS-PAGE gels and HPLC analysis. Limulus Amebocyt...