Fluorescence-Activated Cell Sorter Analysis of Binding by Lipopolysaccharide-Specific Monoclonal Antibodies to Gram-Negative Bacteria

Abstract

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.