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. 2001 May;69(5):3203-13.
doi: 10.1128/IAI.69.5.3203-3213.2001.

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

J S Plested  1 , B L Ferry, P A Coull, K Makepeace, A K Lehmann, F G MacKinnon, H G Griffiths, M A Herbert, J C Richards, E R Moxon
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"V体育ios版" Functional opsonic activity of human serum antibodies to inner core lipopolysaccharide (galE) of serogroup B meningococci measured by flow cytometry

J S Plested et al. Infect Immun. 2001 May.

Abstract

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 VSports手机版. 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. .

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FIG. 1
FIG. 1
FCM profile comparing surface labeling of ethanol-fixed and live bacteria, 5 × 108 organisms per tube. (a) N. meningitidis MC58 wild type. (b) N. meningitidis MC58 galE mutant. Bacteria were opsonized with (C and D) or without (A and B) MAb B5 (approximately 0.11 μg/ml), and antibody binding was detected using anti-mouse IgG-FITC. The percentage of fluorescent organisms is indicated above gate M1 (see text).
FIG. 2
FIG. 2
(a) Phagocytosis of ethanol-fixed N. meningitidis strain MC58 by PMNms from healthy adult donor opsonized with MAb B5 in the presence of a human complement source. (b) Effect of increasing the concentration of MAb B5 on phagocytosis of N. meningitidis L3 galE LPS-coated beads by PMNms from a healthy adult donor.
FIG. 3
FIG. 3
Effect of prior incubation of MAb B5 (0.55 μg/ml) with various amounts of purified N. meningitidis (galE) LPS on phagocytosis of ethanol-fixed N. meningitidis organisms by PMNms. (A) Wild-type strain MC58. (B) MC58 galE mutant.
FIG. 4
FIG. 4
(a) Phagocytosis by PMNms of fluorescently stained ethanol-fixed N. meningitidis strain MC58 opsonized with nine paired infant sera taken early (open bars) (A) and later (hatched bars) (B) in invasive N. meningitidis disease (Table 2). Heterologous human complement was added to all sera in equal amounts. ∗, significant difference (P < 0.05, one-tailed t test) between pairs of sera. (b) Phagocytosis of fluorescently labeled ethanol-fixed N. meningitidis strain MC58 opsonized with four paired infant sera taken during acute (A) and convalescent (B) phases of N. meningitidis disease with PMNms. Sera were either nonadsorbed (hatched bars) or adsorbed (open bars) with native L3 galE LPS. Negative control serum (NC) was the human complement source. OP activity of convalescent-phase sera preadsorbed with galE LPS was significantly lower than that of the same nonadsorbed sera (P < 0.05, one-tailed t test).
FIG. 5
FIG. 5
Typical dot plot analysis of phagocytosis of N. meningitidis serogroup B strain MC58 opsonized with paired acute- or convalescent-phase sera from an infant during (A and B) or following (C and D) invasive meningococcal disease with PMNms. Sera in panels B and D were preadsorbed with purified N. meningitidis LPS (2 ng/ml) from the L3 galE mutant of MC58.
FIG. 6
FIG. 6
(a) Correlation between galE LPS serum IgG antibodies (ELISA titers) and phagocytosis of MC58 with normal adult sera by PMNms from healthy adult donors. (b) Correlation between phagocytosis with N. meningitidis MC58 and galE LPS serum IgG antibodies (ELISA titers) (L3 galE) against adult pooled serum standard. Serum samples were taken from nine children with culture-confirmed invasive meningococcal disease during acute and convalescent phases of disease (Table 2). A human complement source was used.
FIG. 7
FIG. 7
(a) Simultaneous measurement of phagocytosis (solid line) and quantitation of oxidative burst within the PMNm using DHR (oxidative burst) (dashed line) with fluorescently labeled (RR-X) N. meningitidis serogroup B (strain MC58) opsonized with different concentrations of MAb B5, human complement, and PMNms. (b) Correlation between phagocytosis of N. meningitidis strain MC58 by PMNms and quantitation of oxidative burst within PMNm with DHR (oxidative burst).
FIG. 8
FIG. 8
Correlation between phagocytosis by PMNms in OPA (A) and serum bactericidal killing by SBA (B) of reciprocal dilutions of MAb B5 against N. meningitidis MC58 galE mutant and human complement source. (C) Correlation between serum bactericidal killing and phagocytosis with dilutions of MAb B5 against N. meningitidis MC58 galE mutant.
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