Surface Plasmon Resonance Analysis of Antipolysaccharide Antibody Specificity: Responses to Meningococcal Group C Conjugate Vaccines and Bacteria

García-Ojeda, Pablo A.; Hardy, Sharon; Kozlowski, Steven; Stein, Kathryn E.; Feavers, Ian M.

doi:10.1128/iai.72.6.3451-3460.2004

Cited by 16 publications

(10 citation statements)

References 69 publications

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“…The acceptor activity observed in these assays was similar to that expected for the addition of homologous meningococcal polysaccharides (Table 3). To demonstrate that a polysaccharide was being formed on these vesicles, the ELISA plates were coated with these reaction mixtures and assayed for the presence of ␣-2,9-polysialic acid with meningococcal group C-specific antibody (11). As is seen in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The assay mixture was then diluted to 400 l with enzyme-linked immunosorbent assay (ELISA) coating buffer (2), which was then used to coat Immulon 1B plates (100 l/well) at room temperature overnight. The plates were washed and then incubated with mouse monoclonal IgG antibody Mcl2075 (against OAc ϩ ) or Mcl2016 (against OAc Ϫ ) group C polysaccharide at 1:400 dilution (kindly donated by Marjorie Shapiro, FDA) (11). Plates were developed with goat anti-mouse IgG-alkaline phosphatase conjugate (Sigma) diluted 1:2,000.…”

Section: Methodsmentioning

confidence: 99%

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Characterization and Acceptor Preference of a Soluble Meningococcal Group C Polysialyltransferase

et al. 2011

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Vaccines against Neisseria meningitidis group C are based on its ␣-2,9-linked polysialic acid capsular polysaccharide. This polysialic acid expressed on the surface of N. meningitidis and in the absence of specific antibody serves to evade host defense mechanisms. The polysialyltransferase (PST) that forms the group C polysialic acid (NmC PST) is located in the cytoplasmic membrane. Until recently, detailed characterization of bacterial polysialyltransferases has been hampered by a lack of availability of soluble enzyme preparations. We have constructed chimeras of the group C polysialyltransferase that catalyzes the formation ␣-2,9-polysialic acid as a soluble enzyme. We used site-directed mutagenesis to determine the region of the enzyme necessary for synthesis of the ␣-2,9 linkage. A chimera of NmB and NmC PSTs containing only amino acids 1 to 107 of the NmB polysialyltransferase catalyzed the synthesis of ␣-2,8-polysialic acid. The NmC polysialyltransferase requires an exogenous acceptor for catalytic activity. While it requires a minimum of a disialylated oligosaccharide to catalyze transfer, it can form high-molecular-weight ␣-2,9-polysialic acid in a nonprocessive fashion when initiated with an ␣-2,8-polysialic acid acceptor. De novo synthesis in vivo requires an endogenous acceptor. We attempted to reconstitute de novo activity of the soluble group C polysialyltransferase with membrane components. We found that an acapsular mutant with a defect in the polysialyltransferase produces outer membrane vesicles containing an acceptor for the ␣-2,9-polysialyltransferase. This acceptor is an amphipathic molecule and can be elongated to produce polysialic acid that is reactive with group C-specific antibody.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Characterization and Acceptor Preference of a Soluble Meningococcal Group C Polysialyltransferase

et al. 2011

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“…For this purpose, SPR presents the most adequate technique, as it was shown that SPR is a suitable tool for examining receptor-ligand interactions (1), estimating antibody specificities (14), and calculating membrane-binding affinities of peptides (19). Based on these findings, we chose SPR to quantify the aMptD-receptor interactions.…”

Section: Discussionmentioning

confidence: 99%

Peptide aMptD-Mediated Capture PCR for Detection ofMycobacterium aviumsubsp.paratuberculosisin Bulk Milk Samples

Stratmann

Dohmann

Heinzmann

et al. 2006

Appl Environ Microbiol

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A peptide-mediated capture PCR for the detection of Mycobacterium avium subsp. paratuberculosis in bulk milk samples was developed and characterized. Capture of the organism was performed using peptide aMptD, which had been shown to bind to the M. avium subsp. Paratuberculosis (Johne's disease) is a chronic and incurable granulomatous enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (29). The disease occurs worldwide with increasing frequency (32) and has a considerable economic impact on the livestock industry (21). Calves are mostly infected in early life, with high shedding and clinical disease commonly occurring at 2 to 5 years of age (37). Previous attempts to eradicate the disease from infected herds have frequently failed, despite a high degree of organizational and financial efforts (3,26). This is most likely due to the ubiquitous presence of the pathogen in the environments of infected herds (39) and its very great tenacity (7,20,52).An economically feasible alternative to eradication would be a control program aiming at the early identification and removal of high shedders, thereby reducing environmental contamination and infectious pressure on the herd. High shedders are more likely to secrete M. avium subsp. paratuberculosis in milk (50), and in addition, in herds with high shedders the pathogen is more likely to enter the milk by fecal contamination (8). Therefore, bulk milk might be a suitable diagnostic substrate for such an approach. Since the general infrastructure for testing of bulk milk from farms (i.e., untreated raw milk) is established (25), regular testing of this milk for the presence of M. avium subsp. paratuberculosis could allow early detection of herds with high shedders. The most convenient and amenable methods for such detection of M. avium subsp. paratuberculosis DNA in milk are enrichment via immunomagnetic separation (17,28,38) and peptide-mediated capture (47) followed by PCR, as these methods can be adapted to high-throughput testing using standard laboratory automation.In the study presented here, we followed this approach, using the phage display-derived peptide aMptD (46) for the capture of M. avium subsp. paratuberculosis in milk samples. The aMptD peptide was shown to bind to the surface-exposed MptD protein of M. avium subsp. paratuberculosis (ORF 3733C) (31), which is part of an M. avium subsp. paratuberculosis-specific pathogenicity island (46), and therefore this method, in contrast to the previously described peptide-mediated capture assay (47), is based on a defined receptor-ligand interaction. Furthermore, we thoroughly elucidated the strain and species cross-specificity of peptide aMptD for M. avium subsp. paratuberculosis by performing competitive capture assays, and we determined the kinetics and affinity of the receptor-ligand interaction by surface plasmon resonance (SPR) (27) in BIAcore biosensor experiments. Finally, in order to

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“…3 This is often observed for Gram-negative pathogenic bacteria like Neisseria meningitidis. 4 Glycans are also important signaling systems, mediators of cell-cell interactions, including self-self 5 and self-pathogen interactions 6,7 and participate in protein quality control systems. 8,9 The National Academy of Sciences recently issued a roadmap for transforming glycoscience due to glycans' central role in biology, their impact in medicine, energy generation and material sciences.…”

Section: Introductionmentioning

confidence: 99%

Direct Evidence for Hydrogen Bonding in Glycans: A Combined NMR and Molecular Dynamics Study

et al. 2013

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ABSTRACT:We introduce the abundant hydroxyl groups of glycans as NMR handles and structural probes to expand the repertoire of tools for structure-function studies on glycans in solution. To this end we present the facile detection and assignment of hydroxyl groups in a wide range of sample concentrations (0.5 to 1700 mM) and temperatures, ranging from -5 to 25 °C. We then exploit this information to directly detect hydrogen bonds, well known for their importance in molecular structural determination through NMR. Via HSQC-TOCSY, we were able to determine the directionality of these hydrogen bonds in sucrose. Furthermore, by means of Molecular Dynamics simulations in conjunction with NMR, we establish that one out of the three detected hydrogen bonds arises from inter-molecular interactions, which may shed light on glycan-glycan interactions and glycan recognition by proteins.

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Surface Plasmon Resonance Analysis of Antipolysaccharide Antibody Specificity: Responses to Meningococcal Group C Conjugate Vaccines and Bacteria

Abstract: Antibody (Ab) responses to polysaccharides (PS), such as

Cited by 16 publications

References 69 publications

Characterization and Acceptor Preference of a Soluble Meningococcal Group C Polysialyltransferase

Characterization and Acceptor Preference of a Soluble Meningococcal Group C Polysialyltransferase

Peptide aMptD-Mediated Capture PCR for Detection ofMycobacterium aviumsubsp.paratuberculosisin Bulk Milk Samples

Direct Evidence for Hydrogen Bonding in Glycans: A Combined NMR and Molecular Dynamics Study

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