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Infection and Immunity, May 2001, p. 3203-3213, Vol. 69, No. 5
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.5.3203-3213.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Functional Opsonic Activity of Human Serum Antibodies to Inner Core Lipopolysaccharide (galE) of Serogroup B Meningococci Measured by Flow Cytometry

Joyce S. Plested,^1,2,* Berne L. Ferry,² Philip A. Coull,^1,2 Katherine Makepeace,¹ Anne K. Lehmann,³ Fiona G. MacKinnon,¹ Helen G. Griffiths,² Mark A. Herbert,¹ James C. Richards,⁴ and E. Richard Moxon¹

Molecular Infectious Disease Group, Oxford University Department of Paediatrics, John Radcliffe Hospital, Oxford OX3 9DU,¹ and Department of Clinical Immunology, Churchill Hospital, Headington, Oxford OX3 7LJ,² United Kingdom; Institute of Medicine, University of Bergen, Bergen, Norway³; and Institute of Biological Sciences, National Research Council, Ottawa K1A OR6, Canada⁴

Received 27 October 2000/Returned for modification 8 January 2001/Accepted 7 February 2001

A recently described flow cytometric opsonophagocytic assay (OPA) was adapted to quantify the functional activity of serum antibodies specifically directed against serogroup B inner core lipopolysaccharide (LPS) of Neisseria meningitidis. The percentage of human peripheral polymorphonuclear leukocytes and monocytes (PMNms) ingesting fluorescently labeled, ethanol-fixed N. meningitidis organisms (phagocytic activity) in the presence of human sera was measured to reflect the serum opsonic activity against the bacterium. The contribution to opsonophagocytic activity of antibodies to inner core LPS was estimated by comparing the opsonic activities of adult and infant sera before and after adsorbing anti-LPS antibodies from the sera using purified LPS extracted from an LPS mutant (galE) of N. meningitidis strain MC58 (B:15:P1.7,16:L3). The specificity of the assay was further investigated using monoclonal antibody (MAb) B5, which binds to an inner core LPS epitope of N. meningitidis. A dose-dependent decrease in phagocytic activity was observed when MAb B5 was incubated with LPS from an inner core LPS (galE) mutant. Similarly, the number of PMNms ingesting fluorescently labeled polystyrene beads coated with inner core (galE) LPS decreased in a dose-dependent fashion when MAb B5 was incubated with various concentrations of the homologous inner core LPS. Strong correlations were found between the concentration of serum antibodies to inner core LPS (galE) versus the phagocytic activity using healthy adult sera (r² = 0.89). There was a correlation between phagocytic ingestion and initiation of intracellular oxidative burst (r² = 0.99) using polystyrene beads coated with inner core LPS and opsonized with the same sera using the oxidative burst indicator system dihydrorhodamine123/rhodamine 123. OPA results were also found to correlate closely with the results of the serum bactericidal assay using MAb B5 against the N. meningitidis MC58 galE mutant in the presence of human complement (r² = 0.994, P = 0.003, two-tailed test). These studies demonstrate that functional antibodies are produced in humans against meningococcal inner core LPS and that the OPA is a useful approach to study the opsonic activity of antibodies to inner core LPS in health and disease.

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^* Corresponding author. Mailing address: Department of Clinical Immunology, Churchill Hospital, Oxford OX3 7LJ, United Kingdom. Phone: (01865)-226002. Fax: (01865)-225990. E-mail: Joyce.Plested{at}paediatrics.ox.ac.uk.

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Infection and Immunity, May 2001, p. 3203-3213, Vol. 69, No. 5
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.5.3203-3213.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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