Fluorescence-activated cell sorter analysis of binding by lipopolysaccharide-specific monoclonal antibodies to gram-negative bacteria.
Sixteen murine monoclonal antibodies (MAbs) reactive with the O-side chain, core oligosaccharide, or lipid A of Escherichia coli O111:B4 and Salmonella minnesota lipopolysaccharide (LPS) were evaluated for binding activity against wild-type and rough mutant strains using a fluorescence-activated cell sorter (FACS) and fluorescein-conjugated antiimmunoglobulin probe. O-side-chain-reactive MAbs produced immunofluorescence against homologous, smooth strains up to 500-fold higher than controls. Many core- and lipid A-reactive MAbs exhibited limited reactivity with smooth bacteria. Some core- and lipid A-associated epitopes, however, were better recognized by MAbs on intact bacteria than on isolated LPS. FACS analysis of binding by the core-reactive MAb, J8-4C10, to E. coli O26:B6 smooth bacteria revealed staining and non-staining bacterial phenotypes that were sorted and stably expressed in subculture. FACS analysis thus documented differences in the whole-cell reactivity of MAbs specific for various LPS subcomponents, differences in MAb reactivity with isolated and cell-associated LPS, and spontaneous changes in the phenotypic expression of certain LPS-associated epitopes on intact bacteria.
Evans
M E
ME
Department of Medicine, Uniformed Services University of the Health Sciences, F. Edward Hébert School of Medicine, Bethesda, MD 20814-4799.
Pollack
M
M
Hardegen
N J
NJ
Koles
N L
NL
Guelde
G
G
Chia
J K
JK
D000907:Antibodies, Bacterial / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002469:Cell Separation; D005434:Flow Cytometry; D006090:Gram-Negative Bacteria / Q000276:immunology*; D008070:Lipopolysaccharides / Q000276:immunology*; D011863:Radioimmunoassay
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D008070:Lipopolysaccharides