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#24738289   // To Up

[Immunobiological properties of Bordetella pertussis lipopolysaccharide in the acellular pertussis vaccine].

Study of Bordetella pertussis lipopolysaccharide (LPS) immunobiological properties in the acellular pertussis vaccine.
E M ZaÄ­tsev, A V Poddubikov, M V Britsina, N U Mertsalova, M N Ozeretskovskaia, I G Bazhanova, N G Plekhanova

1742 related Products with: [Immunobiological properties of Bordetella pertussis lipopolysaccharide in the acellular pertussis vaccine].

100μg100μg100μg25100μg125

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#22527330   2012/04/15 To Up

Bordetella Pertussis Toxin does not induce the release of pro-inflammatory cytokines in human whole blood.

Pertussis Toxin (PTx) is one of the most important virulence factors of Bordetella pertussis, the cause of whooping cough. Therefore, the inactivated toxin is an obligatory constituent of acellular pertussis vaccines. It is described in the literature that both native PTx and recombinant Pertussis Toxin (PTg) activate human monocytes whereas others report an inhibition of mammalian monocytes during pertussis infection. B. pertussis, as a Gram-negative bacterium, harbours naturally lipopolysaccharide (LPS, also known as endotoxin), one of the strongest stimulators of monocytes. The latter is triggered via the interaction of endotoxin with inter alia the surface receptor CD14. Consequently, it is necessary to consider a potential contamination of Pertussis Toxin preparations with LPS. First, we determined the LPS content in different preparations of PTx and PTg. All preparations examined were contaminated with LPS; therefore, possible PTx- and PTg-driven monocyte activation independently of LPS was investigated. To meet these aims, we examined monocyte response to PTx and PTg while blocking the LPS receptor CD14 with a specific monoclonal antibody (anti-CD14 mAb). In addition, all toxin preparations examined underwent an LPS depletion. Our results show that it is contaminating LPS, not Pertussis Toxin, which activates human monocytes. Blocking the CD14 receptor prevents Pertussis Toxin-mediated induction of pro-inflammatory cytokines in human monocytes. The depletion of LPS from Pertussis Toxin leads to the same effect. Additionally, the PTx toxicity after LPS depletion procedure was confirmed by animal tests. In contrast, the original Pertussis Toxin preparations not treated as mentioned above generate strong monocyte activation. The results in this publication allow the conclusion that purified Pertussis Toxin preparations do not induce the release of pro-inflammatory cytokines in human whole blood.
Christina Bache, Ingo Spreitzer, Bjoern Becker, Bettina Loeschner, Ute Rosskopf, Kay-Martin Hanschmann, Michael Schwanig, Christian K Schneider, Bernhard Lieb, Thomas Montag

1488 related Products with: Bordetella Pertussis Toxin does not induce the release of pro-inflammatory cytokines in human whole blood.

2ug5ug2ug5ug2ug96 tests50 ul16 Arrays/Slide 100 UG5ug 100ul16 Arrays/Slide

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#20943873   2010/10/13 To Up

Performance of commercial enzyme-linked immunosorbent assays for detection of antibodies to Bordetella pertussis.

Measuring antibodies to Bordetella pertussis antigens is mostly done by enzyme-linked immunosorbent assays (ELISAs). We compared the performance of ELISA kits that were commercially available in Germany. Eleven measured IgG antibodies, and nine measured IgA antibodies. An in-house ELISA with purified antigens served as a reference method. Samples included two WHO reference preparations, the former Food and Drug Administration (FDA)/Center for Biologics Evaluation and Research (CBER) reference preparations, serum samples from patients with clinically suspected pertussis, and serum samples from patients having received a combined tetanus, diphtheria, and pertussis (Tdap) vaccination. Kits using pertussis toxin (PT) as an antigen showed linearity compared to the WHO Reference preparation (r2 between 0.82 and 0.99), and these kits could quantify antibodies according to the reference preparation. ELISA kits using mixed antigens showed no linear correlation to the reference preparations. Patient results were compared to results of in-house ELISAs using a dual cutoff of either ≥100 IU/ml anti-PT IgG or ≥40 IU/ml anti-PT IgG together with ≥12 IU/ml anti-PT IgA. The sensitivities of kits measuring IgG antibodies ranged between 0.84 and 1.00. The specificities of kits using PT as an antigen were between 0.81 and 0.93. The specificities of kits using mixed antigens were between 0.51 and 0.59 and were thus not acceptable. The sensitivities of kits measuring IgA antibodies ranged between 0.53 and 0.73, and the specificities were between 0.67 and 0.94, indicating that IgA antibodies may be of limited diagnostic value. Our data suggest that ELISAs should use purified PT as an antigen and be standardized to the 1st International Reference preparation.
M Riffelmann, K Thiel, J Schmetz, C H Wirsing von Koenig

1601 related Products with: Performance of commercial enzyme-linked immunosorbent assays for detection of antibodies to Bordetella pertussis.

0.2 mg1 mL1 mL100 μg100 1 mg200 ug100 assays

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#11580218   // To Up

International collaborative study: evaluation of proposed International Reference Reagent of pertussis antiserum (mouse) 97/642.

A freeze dried preparation of mouse serum in vials coded 97/642 containing antibodies to five pertussis antigens [pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN), fimbriae type 2 and 3 (Fim 2 and 3)] has been assessed for its suitability as an international reference reagent in an international collaborative study by thirteen laboratories in nine countries. This serum has been compared with U.S. Standard Pertussis Antiserum (mouse) Lot No. 1 (US Lot 1), which has been in use since 1995, for antibodies for each antigen. Calibration of the proposed International Reference Reagent of Pertussis Antiserum (pIRR) in terms of US Lot 1 gives results which are broadly consistent between laboratories for antibodies to each antigen, although the between-laboratory differences are larger than those seen for comparison of identical sera. Calibration of two positive control sera in terms of the pIRR gave similar between laboratory variability of estimates to that obtained when the same sera were calibrated in terms of US Lot 1. Overall continuity of estimates is maintained if units are assigned to the pIRR based on its calibration in terms of US Lot 1 in this study. Data presently available indicate that the pIRR is sufficiently stable to serve as a reference reagent. It was therefore recommended, with the agreement of all participants, that the preparation in vials coded 97/642 be established as the First International Reference Reagent for Pertussis Antiserum, mouse, with assigned unitages 16 units of anti-PT per vial, 143 units of anti-FHA per vial and 30 units of anti-PRN per vial based on its calibration in terms of US Lot 1. These unitages are also consistent with calibration of 97/642 in terms of the Japanese preparations JNIH-11 for anti-FHA and of JNIH-12 for anti-PT. Purified antigens for Fim 2 and Fim 3 are not readily available and an arbitrary value of 32 units per vial is suggested for anti-Fim 2 and 3 mixture. These recommendations were agreed by the Expert Committee on Biological Standardization of the World Health Organization.
R Gaines Das, D Xing, P Rigsby, P Newland, M Corbel

2856 related Products with: International collaborative study: evaluation of proposed International Reference Reagent of pertussis antiserum (mouse) 97/642.

1ml1ml 125 ml 1ml1ml1mlmouse ear (50 ml)10ml1ml 15 ml 10ml500 mouse tails (100 ml)

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#10225286   // To Up

Adjuvant and protective properties of native and recombinant Bordetella pertussis adenylate cyclase toxin preparations in mice.

Bordetella pertussis produces a cell-invasive adenylate cyclase toxin which is synthesised from the cyaA gene as an inactive protoxin that is post-translationally activated by the product of the cyaC gene. Purified active and inactive CyaA proteins were prepared from B. pertussis or from recombinant Escherichia coli expressing both cyaA and cyaC genes or the cyaA gene alone. respectively. In addition, a hybrid toxin (Hyb2) in which an internal region of CyaA had been replaced with the analogous region from the leukotoxin (LktA) of Pasteurella haemolytica, and which had low cell-invasive activity, was also prepared from E. coli expressing the cyaC gene. The CyaA preparations showed no evidence of toxicity in a mouse weight-gain test. Active toxin preparations were protective in mice against intranasal challenge with wild-type B. pertussis, as evidenced by lung:body weight ratios and bacterial numbers in the lungs, which were comparable to those in mice given whole-cell DPT vaccine. Hyb2 was not as protective as active CyaA and inactive CyaA preparations were not protective. Active CyaA, when co-administered with ovalbumin (OA), had a marked adjuvant effect on the anti-OA IgG antibody response which was not as apparent with inactive CyaA preparations. Similarly, active CyaA stimulated a greater anti-CyaA response than the inactive form.
K Hormozi, R Parton, J Coote

1277 related Products with: Adjuvant and protective properties of native and recombinant Bordetella pertussis adenylate cyclase toxin preparations in mice.

0.1mg100μg100ul100 100μg1 mg2 100.00 ul1050

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#9092559   // To Up

Insulin-induced activation of NADPH-dependent H2O2 generation in human adipocyte plasma membranes is mediated by Galphai2.

Human fat cells possess a multireceptor-linked H2O2-generating system that is activated by insulin. Previous studies revealed that manganese was the sole cofactor required for a hormonal regulation of NADPH-dependent H2O2 generation in vitro. In this report it is shown that the synergistic activation of NADPH-dependent H2O2 generation by Mn2+ and insulin was blocked by GDPbetaS (guanosine 5'-O-(2-thiodiphosphate)), pertussis toxin and COOH-terminal anti-Galphai1-2 or the corresponding peptide. Consistently, manganese could be replaced by micromolar concentrations of GTPgammaS (guanosine 5'-O-(3-thiotriphosphate)), which increased NADPH-dependent H2O2 generation by 20-40%. Insulin shifted the dose response curve for GTPgammaS to the left (>10-fold) and increased the maximal response. In the presence of 10 microM GTPgammaS, the hormone was active at picomolar concentrations, indicating that insulin acted via its cognate receptor. The insulin receptor and Gi were co-adsorbed on anti-Galphai and anti-insulin receptor beta-subunit (anti-IRbeta) affinity columns. Partially purified insulin receptor preparations contained Galphas, Galphai2, and Gbetagamma (but no Galphai1 or Galphai3). The functional nature of the insulin receptor-Gi2 complex was made evident by insulin's ability to modulate labeling of Gi by bacterial toxins. Insulin action was mimicked by activated Galphai, but not by Galphao or Gbetagamma, indicating that insulin's signal was transduced via Galphai2. Thus, NADPH oxidase is the first example of an effector system that is coupled to the insulin receptor via a heterotrimeric G protein.
H I Krieger-Brauer, P K Medda, H Kather

2848 related Products with: Insulin-induced activation of NADPH-dependent H2O2 generation in human adipocyte plasma membranes is mediated by Galphai2.

100 ul50 ul4 Membranes/Box96T100ug5ug100 μg4 Membranes/Box100ug Lyophilized 100ul4 Membranes/Box100ug Lyophilized

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#9141706   // To Up

Antigenic polymorphism of the lipopolysaccharides from human and animal isolates of Bordetella bronchiseptica.

Six monoclonal antibodies (mAbs) against lipopolysaccharides (LPS) from Bordetella pertussis (P1P3, 60.5), B. parapertussis (PP2, PP6, PPB) and B. bronchiseptica (BRg1) were used to examine the presence of antigenic determinants of LPS on B. bronchiseptica cells. Forty-eight clinical isolates of this Gram-negative bacterium (4 canine, 3 equine, 6 porcine, 4 rabbit and 31 human) were examined. Significant cross-reactivities with the heterologous anti-pertussis and anti-parapertussis mAbs were observed. The isolates also exhibited marked antigenic polymorphism. The 48 isolates could be classified in six immunogroups. Purified LPS preparations extracted from some isolates were analysed by ELISA, thin-layer chromatography, and tricine-SDS-PAGE. The results show that four main types of antigenic polymorphism of B. bronchiseptica LPSs exist: (a) heterogeneity of the core, (b) presence or absence of O-chains, (c) differences in the hinge region between O-chain and core, and (d) differences in interactions of LPS with other cell-surface constituents. Smooth-type LPS molecules, detectable with mAb PP6, were more frequently observed in animal isolates (94%) than in human isolates (52%). Reverse frequencies were found with mAb 60.5 (48% of human isolates, 18% of animal isolates), which is unable to react with long-chain LPSs. This observation could be due to the general absence of some lectin-like receptor, specific to the O-chain, on human bronchoalveolar tissues.
Karine Le Blay, Pascale Gueirard, Nicole Guiso, Richard Chaby

2704 related Products with: Antigenic polymorphism of the lipopolysaccharides from human and animal isolates of Bordetella bronchiseptica.

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#1550816   // To Up

Site-specific antibodies directed against G protein beta and gamma subunits: effects on alpha and beta gamma subunit interaction.

Little is known about the specific domains of G protein beta and gamma subunits which interact with each other and with the alpha subunit. We used site-specific anti-peptide antibodies directed against beta and gamma subunits to investigate domains on beta and gamma subunits involved in alpha subunit interaction. Antibodies included four against the transducin (Gt) beta subunit (residues 1-10 = MS, 127-136 = KT, 256-265 = RA, and 330-340 = SW) and two against the gamma subunit (residues 2-12 = PV and 58-68 = PE). All antisera, when affinity-purified on peptide columns, yielded antibodies capable of recognizing the denatured cognate subunit on immunoblots, but only RA, SW, PV, and PE recognized native beta gamma t subunits. Affinity purification of MS and KT antisera on columns of immobilized native Gt yielded antibodies capable of recognizing native beta gamma t subunits. The functional effects of each antibody preparation on alpha t-beta gamma t interaction were assessed by assaying the ability of the preparations to immunoprecipitate beta gamma t subunits in the presence of excess alpha subunits and by testing the inhibition of beta gamma t-dependent ADP-ribosylation of alpha t-subunits catalyzed by pertussis toxin. On the basis of the results, we conclude that the domains on beta gamma t which may be directly involved in alpha t-beta gamma t interaction include the extreme amino terminus, residues 127-136 and 256-265 of beta t, and the carboxyl terminus of gamma t.(ABSTRACT TRUNCATED AT 250 WORDS)
T Murakami, W F Simonds, A M Spiegel

1633 related Products with: Site-specific antibodies directed against G protein beta and gamma subunits: effects on alpha and beta gamma subunit interaction.

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#1771750   // To Up

Characterization of the immunogenicity of formaldehyde detoxified Pasteurella multocida toxin.

The immunogenicity of the Pasteurella multocida toxin (PMT) was studied in murine model systems. Mice were vaccinated with either formaldehyde treated pure PMT (pure toxoid) or formaldehyde treated crude extract of toxigenic P. multocida (crude toxoid). The corresponding mean anti-PMT titres, sero-conversion rates and survival rates after challenge with affinity purified PMT were compared. When assessed both by anti-PMT titres and seroconversion and challenge, pure toxoid was a more potent immunogen than crude toxoid. This greater immunogenic potency was unaffected by the addition of killed cell preparations of Bordetella bronchiseptica, non-toxigenic P. multocida and B. pertussis. Increasing anti-PMT titres and seroconversion rates were induced by increasing doses of formaldehyde treated PMT (fPMT) in the pure toxoid vaccines, but not in the vaccines containing crude toxoid. However, improved survival rates were observed for both types of vaccine, when the fPMT content was raised. Immunization of pregnant mice with vaccines containing fPMT induced protection of the offspring against challenge with PMT; the protection of the offspring corresponded to that of the mother.
A Bording, N T Foged

1307 related Products with: Characterization of the immunogenicity of formaldehyde detoxified Pasteurella multocida toxin.

5 G500 Units1 mL100 ug100 ul 100 G96 wells11 mg2x96 well plate 100ul1 mg

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#1894374   // To Up

Purification and characterization of the heat-labile toxin of Bordetella pertussis.

A procedure is described for purification of pertussis heat-labile toxin (PEHLT) from cells of Bordetella pertussis. The purification procedure, performed in the cold and in the presence of protease inhibitors, gives 1,350-fold purification with yields of about 60%. The toxin was shown to be a single-chain polypeptide of 140 kDa, pI 6.02. It was completely inactivated by heating at 56 degrees C for 60 min. Rabbit antiserum prepared against PEHLT neutralized the toxin and gave a single precipitin line on immunodiffusion. In immunodiffusion assays, this anti-PEHLT serum did not react with pertussis toxin, filamentous hemagglutinin, or preparations of pertussis adenylate cyclase. Purified PEHLT elicited dermonecrosis and atrophy of the spleen. PEHLT is extraordinarily active; 0.4 X 10(-12) g caused necrotic lesions in newborn mice, and with 18- to 20-g mice the 50% lethal dose was about 11 X 10(-9) g.
Y L Zhang, R D Sekura

1538 related Products with: Purification and characterization of the heat-labile toxin of Bordetella pertussis.

0.2 mg 5 G100μg100μg25100μg0.2 mg1 mg0.2 mg1 mg100μg25ml

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