Enzyme-linked immunosorbent assay for detection of antibodies toPseudomonas aeruginosa exoproteins

Granström, Marta; Wretlind, Bengt; Markman, B.; Pavlovskis, O R; Vasil, Michael L.

doi:10.1007/bf02013597

Cited by 15 publications

(9 citation statements)

References 14 publications

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“…Enzyme-linked immunosorbent assay (ELISA). Immunlon II polystyrene microplates (Dynatech Laboratories Inc., Alexandria, Va.) were coated overnight at 22°C with, per well, 100 ,ul of exotoxin A (2 ,ug/ml), elastase (2 pug/ml), alkaline protease (10 ,ug/ml), phospholipase C (10 p.g/ml), or LPS (10 jig/ml) in 0.02 M phosphate-buffered saline with 0.02% (wt/vol) sodium azide, pH 7.2, as previously described (16,36) with minor modifications in the antigen concentrations. Nunc immunoplates (Nunc, Thousand Oaks, Calif.) were coated with, per well, 100 RI of flagella (1 ,ug/ml) in 0.05 M carbonate coating buffer with 0.02% sodium azide, pH 9.6, as previously described (32).…”

Section: Methodsmentioning

confidence: 99%

Serum antibody response to Pseudomonas aeruginosa antigens during corneal infection

et al. 1991

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Previous studies in our laboratory have indicated that naturally resistant, inbred DBA/2J mice mount a greater serum antibody response to Pseudomonas aeruginosa 19660 than susceptible C57BL/6J mice. However, the specificity of the antibody produced was not known. The present study examines the specificity and kinetics of the humoral response of these mouse strains to potential virulence factors produced by the organism during both a primary and a secondary corneal infection administered 4 weeks after the primary infection. Serum antibody levels specific for lipopolysaccharide (LPS), exotoxin A, phospholipase C (PLC), alkaline protease, elastase, and flagella were measured by enzyme-linked immunosorbent assay. Little or no antibody to either alkaline protease or elastase was detected during either primary or secondary infection. Immunoglobulin G (IgG) antibodies specific to exotoxin A, PLC, and flagella were detected 2 weeks after primary infection, and a rapid response to these antigens was measured 1 week after secondary infection. During primary infection, detectable LPS-specific antibody was only IgM, while IgG appeared only after secondary infection. The kinetics of the humoral response in susceptible C57BL/6J mice-were similar to those in resistant DBA/2J mice, although the magnitude of the response varied according to the antigen tested. These results indicate that LPS, exotoxin A, PLC, and flagella are present or produced in amounts that are immunogenic during corneal infection by P. aeruginosa 19660 in the mouse strains tested.Pseudomonas aeruginosa is an opportunistic pathogen which causes severe corneal infections in humans and experimental animals (6,8,14,37,38). The infection is usually preceded by corneal trauma. Thereafter, the infection rapidly spreads and may result in permanent ocular damage. Previous experimental studies from our laboratory have indicated that DBA/2J mice can spontaneously restore corneal clarity within a few weeks after infection and are therefore classified as naturally resistant (4, 18). On the other hand, C57BL/6J mice initially exhibit the same severity of ocular infection at 24 to 72 h postinfection but are unable to restore corneal clarity within a period of 4 weeks and are classified as susceptible (4, 18). Consequently, corneal infection in these mice usually leads to corneal perforation, phthisis bulbi (shrinkage), or both. However, when the mice were reinfected in the contralateral control eye 2 months after the primary corneal infection, most of the mice restored corneal clarity within 3 to 6 days after the secondary infection (7). Recent studies suggest that genetic factors of the host regulate the corneal response of the two mouse strains and that one to two resistance and susceptibility genes may be involved (2, 5). Extending these studies further, we have recently described a correlation between the dissimilar corneal response of the two mouse strains with the ability to mount a specific, rapid, and protective humoral response to P. aeruginosa prior to the develo...

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Section: Methodsmentioning

confidence: 99%

Serum antibody response to Pseudomonas aeruginosa antigens during corneal infection

et al. 1991

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“…Increased antibody levels to exotoxin A or phosphatase C were observed in 15 of 22 patients with P. aeruginosa septicemia, otitis, and pneumonia. In an examination of 112 serum samples from healthy subjects of different age groups (0–3, 4–15, 16–75 years old), Granstom et al [43]showed that antibodies (IgG) to alkaline phosphatase and phospholipase C significantly increased in older children and adults. However, antibody levels to elastase and exotoxin A were not increased.…”

Section: Antigenic Properties Of Exoproductsmentioning

confidence: 99%

Pseudomonas aeruginosaantigens as potential vaccines

Stanislavsky

Lam

1997

FEMS Microbiol Rev

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Pseudomonas aeruginosa is one of the most important opportunistic bacterial pathogens in humans and animals. This organism is ubiquitous and has high intrinsic resistance to antibiotics due to the low permeability of the outer membrane and the presence of numerous multiple drug efflux pumps. Various cell-associated and secreted antigens of P. aeruginosa have been the subject of vaccine development. Among pseudomonas antigens, the mucoid substance, which is an extracellular slime consisting predominantly of alginate, was found to be heterogenous in terms of size and immunogenicity. High molecular mass alginate components (30-300 kDa) appear to contain conserved epitopes while lower molecular mass alginate components (10-30 kDa) possess conserved epitopes in addition to unique epitopes. Surface-exposed antigens including O-antigens (O-specific polysaccharide of LPS) or H-antigens (flagellar antigens) have been used for serotyping due to their highly immunogenic nature. Chemical structures of repeating units of O-specific polysaccharides have been elucidated and these data allowed the identification of 31 chemotypes of P. aeruginosa. Conserved epitopes among all serotypes of P. aeruginosa are located in the core oligosaccharide and the lipid A region of LPS and immunogens containing these epitopes induce cross-protective immunity in mice against different P. aeruginosa immunotypes. To examine the protective properties of OM proteins, a vaccine containing P. aeruginosa OM proteins of molecular masses ranging from 20 to 100 kDa has been used in pre-clinical and clinical trials. This vaccine was efficacious in animal models against P. aeruginosa challenge and induced high levels of specific antibodies in human volunteers. Plasma from human volunteers containing anti-P. aeruginosa antibodies provided passive protection and helped the recovery of 87% of patients with severe forms of P. aeruginosa infection. Vaccines prepared from P. aeruginosa ribosomes induced protective immunity in mice, but the efficacy of ribosomal vaccines in humans is not yet known. A number of recent studies indicated the potential of some P. aeruginosa antigens that deserve attention as new vaccine candidates. The outer core of LPS was implicated to be a ligand for binding of P. aeruginosa to airway and ocular epithelial cells of animals. However, heterogeneity exists in this outer core region among different serotypes. Epitopes in the inner core are highly conserved and it has been demonstrated to be surface-accessible, and not masked by O-specific polysaccharide. The use of an in vivo selection/expression technology (IVET) by a group of researchers identified a number of P. aeruginosa proteins that are expressed in vivo and essential for virulence. Two of these in vivo-expressed proteins are FptA (ferripyochelin receptor protein) and a homologue of an LPS biosynthetic enzyme. Our laboratory has identified a highly conserved protein, WbpM, and P. aeruginosa with a deficiency in this protein produces only rough LPS and became serum sensitive. Res...

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“…Antigenic properties of exoproducts P. aeruginosa produces a number of secreted virulence factors including exotoxin A, exoenzyme S, proteases I (neutral protease) and III (alkaline phosphatase), elastase, hemolysins (thermolabile phosphatase C and thermostable acid glycolipid), enter- Table 1 Probable role of extracellular products of P. aeruginosa in virulence and immunogenicity Extracellular antigen Role in virulence Antibody or protective response Reference Proteases : (i) Elastase Induces hemorrhagic lesions and necrosis, Abs in patients with chronic and/or active [18^20] tissue invasion, destruction of host defenses, P. aeruginosa infections; provide protections against e.g. complements and opsonins severe lung lesions in animal models (ii) Alkaline protease Destruction of non-speci¢c host defenses, Abs detected in burn wound patients [21] tissue invasion Phospholipase C Destruction of pulmonary surfactant Rise in Ab titer in CF patients colonized with [22,23] Generation of in£ammatory mediators P. aeruginosa, and in animal model infections Rhamnolipids…”

Section: Introductionmentioning

confidence: 99%

“…Increased antibody levels to exotoxin A or phosphatase C were observed in 15 of 22 patients with P. aeruginosa septicemia, otitis, and pneumonia. In an examination of 112 serum samples from healthy subjects of di¡erent age groups (0^3, 4^15, 16^75 years old), Granstom et al [43] showed that antibodies (IgG) to alkaline phosphatase and phospholipase C signi¢-cantly increased in older children and adults. However, antibody levels to elastase and exotoxin A were not increased.…”

Section: Extracellular Antigenmentioning

confidence: 99%

“…complements and opsonins severe lung lesions in animal models (ii) Alkaline protease Destruction of non-speci¢c host defenses, Abs detected in burn wound patients [21] tissue invasion Phospholipase C Destruction of pulmonary surfactant Rise in Ab titer in CF patients colonized with [22,23] Generation of in£ammatory mediators P. aeruginosa, and in animal model infections Rhamnolipids Detergent-like properties, cilostatic, enhances Not determined [24,25] phospholipase C activities Exotoxin A ADP-ribosylation of elongation factor II, Abs in patients with chronic infection or active [19,26] inhibit protein synthesis exacerbation of P. aeruginosa infections otoxin, collagenase, lecithinase, lipase [2,17]. The probable role of these secreted pseudomonas antigens is summarized in Table 1.…”

Section: Extracellular Antigenmentioning

confidence: 99%

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Pseudomonas aeruginosa antigens as potential vaccines

Stanislavsky

1997

FEMS Microbiology Reviews

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Pseudomonas aeruginosa is one of the most important opportunistic bacterial pathogens in humans and animals. This organism is ubiquitous and has high intrinsic resistance to antibiotics due to the low permeability of the outer membrane and the presence of numerous multiple drug efflux pumps. Various cell-associated and secreted antigens of P. aeruginosa have been the subject of vaccine development. Among pseudomonas antigens, the mucoid substance, which is an extracellular slime consisting predominantly of alginate, was found to be heterogeneous in terms of size and immunogenicity. High molecular mass alginate components (30^300 kDa) appear to contain conserved epitopes while lower molecular mass alginate components (10^30 kDa) possess conserved epitopes in addition to unique epitopes. Surface-exposed antigens including Oantigens (O-specific polysaccharide of LPS) or H-antigens (flagellar antigens) have been used for serotyping due to their highly immunogenic nature. Chemical structures of repeating units of O-specific polysaccharides have been elucidated and these data allowed the identification of 31 chemotypes of P. aeruginosa. Conserved epitopes among all serotypes of P. aeruginosa are located in the core oligosaccharide and the lipid A region of LPS and immunogens containing these epitopes induce crossprotective immunity in mice against different P. aeruginosa immunotypes. To examine the protective properties of OM proteins, a vaccine containing P. aeruginosa OM proteins of molecular masses ranging from 20 to 100 kDa has been used in pre-clinical and clinical trials. This vaccine was efficacious in animal models against P. aeruginosa challenge and induced high levels of specific antibodies in human volunteers. Plasma from human volunteers containing anti-P. aeruginosa antibodies provided passive protection and helped the recovery of 87% of patients with severe forms of P. aeruginosa infection. Vaccines prepared from P. aeruginosa ribosomes induced protective immunity in mice, but the efficacy of ribosomal vaccines in humans is not yet known. A number of recent studies indicated the potential of some P. aeruginosa antigens that deserve attention as new vaccine candidates. The outer core of LPS was implicated to be a ligand for binding of P. aeruginosa to airway and ocular epithelial cells of animals. However, heterogeneity exists in this outer core region among different serotypes. Epitopes in the inner core are highly conserved and it has been demonstrated to be surface-accessible, and not masked by O-specific polysaccharide. The use of an in vivo selection/expression technology (IVET) by a group of researchers identified a number of P. aeruginosa proteins that are expressed in vivo and essential for virulence. Two of these in vivo-expressed proteins are FptA (ferripyochelin receptor protein) and a homologue of an LPS biosynthetic enzyme. Our laboratory has identified a highly conserved protein, WbpM, and P. aeruginosa with a deficiency in this protein produces only rough LPS and became serum sensit...

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Enzyme-linked immunosorbent assay for detection of antibodies toPseudomonas aeruginosa exoproteins

Cited by 15 publications

References 14 publications

Serum antibody response to Pseudomonas aeruginosa antigens during corneal infection

Serum antibody response to Pseudomonas aeruginosa antigens during corneal infection

Pseudomonas aeruginosaantigens as potential vaccines

Pseudomonas aeruginosa antigens as potential vaccines

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