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.

Flow cytometric assessment of the survival ratio of Francisella tularensis in aerobiological samples

“…A common problem with antibodies is, however, that they often show a varying degree of cross‐reaction with other species. Even when monoclonal antibodies are used, specificity can be a problem for some genera of bacteria [27]. However, the genus Francisella is antigenically coherent and the subspecies are indistinguishable by serological methods [30].…”

Variable cross-reactivity of Pseudomonas aeruginosa lipopolysaccharide-core-specific monoclonal antibodies and its possible relationship with serotype

“…Among these LPS-core-specific human mAbs, we compared in viuo protective activity with in vitro binding activity determined by different methods. Flow cytometry has recently been used for analysing bacterial cell surface antigens (Klebba et al, 1990;Evans et al, 1990;Nelson et al, 1990). Applying flow cytometry to our system (Fig.…”

“…Flow cytometry was carried out basically according to the method of Evans et al (1990). P. aerugbsa cells were cultured in heart infusion broth to late exponential-phase, washed with PBS and fixed with 1 % (v/v) formaldehyde at 25 "C for 1 h. Washed cells were resuspended in PBS to an A600 of 0.5 (approximately 1 x 108 c.f.u.…”

Recent advances of flow cytometry in fundamental and applied microbiology

“…Lipopolysaccharide structure and its role in bacterial adhesion and pathogenicity have been investigated using flow cytometry and monoclonal antibodies, sometimes combined with other techniques [67,113,132,133,147,214]. In Gram-positive bacteria [9], pathogenic fungi [88], and parasitic flagellates [1, 3, 39], monoclonal antibodies have also been employed to define phenotypic variations in epitope expressions of outer surface components in relation to the pathogenicity of the microbial cell.…”