Functional opsonic activity of human serum antibodies to inner core lipopolysaccharide (galE) of serogroup B meningococci measured by flow cytometry.

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 (r(2) = 0.89). There was a correlation between phagocytic ingestion and initiation of intracellular oxidative burst (r(2) = 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 (r(2) = 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.

Enzyme linked immunosorbent assay (ELISA) for the detection of serum antibodies to the inner core lipopolysaccharide of Neisseria meningitidis group B.

We have developed a solid-phase ELISA to study the human immune response to inner core lipopolysaccharide (LPS) of Neisseria meningitidis (Nm) using structurally defined glycolipids from a genetically defined mutant (galE) of a serogroup B Nm strain. Previous studies had demonstrated that a galE (inner core) LPS epitope is conserved in approximately 70% Nm strains and was accessible to antibody in fully encapsulated wild-type Nm strains. A murine monoclonal antibody, MAb B5, raised to a galE mutant of serogroup B Nm strain, immunotype L3 (B.15.P1.7,16) was used to determine the specificity of the inner core LPS ELISA by inhibition studies using purified galE LPS and human sera. The intra-assay coefficient of variation (CV) was 5-6% and inter-assay CV was 19-22%. Using this ELISA, significant differences in the geometric mean titres (GMTs) of naturally occurring serum antibodies (specific to inner core LPS) between healthy adults (18-65 years, N=54) and healthy infants (3-4 months, N=144) of both IgG and IgM classes were found (P<0.0001). GMTs were expressed in galE arbitrary units (AU/ml) (95% confidence intervals): IgG antibodies in adults 5.7 (5. 0,6.9) and in infants 1.1 (1.0,1.3); IgM antibodies in adults 7.7 (5. 7,10.4), and in infants 0.85 (0.7,1.1). In age-matched children aged 26-113 months a difference (P=0.04) in specific IgG was found in healthy infants and infants in the acute phase of invasive Nm disease (GMT (95%CI) in AU/ml: in healthy infants 7.7 (5.3,11.0), in acute phase infants 4.2 (2.5,7.2). However, there was no difference in specific IgM (P=0.98) between these groups healthy infants 4.7 (3. 1,7.0), acute phase 4.6 (2.9, 7.4). In eleven children (5-181 months) there were differences in the GMTs of specific IgG and IgM (P=0.02, P=0.008 respectively) between paired acute and convalescent sera (GMT) (95%CI) in AU/ml: IgG acute 1.95 (0.98, 3.8), convalescent 5.2 (2.2,12.4); IgM acute 1.78 (1.05,3.0), convalescent 4.38 (2.6,7.3). We conclude that ELISA is a specific, sensitive and reproducible method for the detection of antibodies to inner core LPS of Nm and that an epitope defined by MAb B5 can be immunogenic in infants and adults. These findings are relevant to the potential candidacy of inner core LPS as a vaccine.