title: A comparison of immunoblotting, flow cytometry and ELISA to monitor the binding of anti-lipopolysaccharide monoclonal antibodies.
abstract: This study was designed to assess the use of flow cytometry to observe the binding, under physiological conditions, of anti-lipopolysaccharide (LPS) monoclonal antibodies (mAbs) to whole bacteria, and to compare this with the more conventional whole cell ELISA and immunoblotting techniques. The bacteria consisted of two clinical isolates of E. coli 018:K1 and 06:K5 and two isogenic mutants of the 018 parent: a non-capsulate (018:K-) and a rough mutant (018rf). Two cross-reactive anti-core mAbs and one 018 0-antigen-specific mAb were used. ELISA and flow cytometry showed that capsule and O-polysaccharide influenced the binding of mAbs to the bacteria, whilst the latter technique demonstrated that sub-populations existed. Immunoblotting showed the two anti-core mAbs to be different, one bound only to core which was not substituted with O-antigen, whilst the other bound both to substituted and unsubstituted core. This comparison for monitoring the binding of anti-LPS mAbs demonstrates the potential use of flow cytometry in bacterial cell surface research, and complements results obtained by ELISA and immunoblotting.
authors.affiliation: Bacteriology Department, University of Edinburgh Medical School, U.K.
authors.forename: D
authors.initials: D
authors.lastname: Nelson
authors.forename: W
authors.initials: W
authors.lastname: Neill
authors.forename: I R
authors.initials: IR
authors.lastname: Poxton
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology*; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D008070:Lipopolysaccharides / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C
chemical_list: D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D008070:Lipopolysaccharides