Immunomodulative properties of conjugates composed of detoxified lipopolysaccharide and capsular polysaccharide of Vibrio cholerae O135 bound to BSA-protein carrier

Biologia ◽  
2010 ◽  
Vol 65 (5) ◽  
Author(s):  
Jana Korcová ◽  
Eva Machová ◽  
Pavol Farkaš ◽  
Slavomír Bystrický
Keyword(s):  
Vibrio Cholerae ◽  
Capsular Polysaccharide ◽  
Size Exclusion ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Antibodies ◽  
Polymer Carrier ◽  
Protein Carrier ◽  
Exclusion Chromatography ◽  
Polysaccharide Antigens ◽  
Surface Polysaccharide

AbstractO135 serotype Vibrio cholerae isolated from Slovak river was used as a source of surface polysaccharide antigens. Following detoxification procedure, fractions of polysaccharides were separated by size exclusion chromatography. Two resultant fractions were the capsular polysaccharide (M w ∼ 197,000 Da) and the lipopolysaccharide fragment (M w ∼ 13,300 Da). These materials were used for preparation of four novel glycoconjugates. Two of them containing detoxified lipopolysaccharide as antigen were prepared by original chemical method using the new biocompatible polymer as carrier of antigen. Additionally, other two conjugates were prepared by direct linking of capsular and detoxified lipopolysaccharide antigens to the protein carrier using adipic acid dihydrazide spacer. The immunogenicities (induced IgM, IgG, IgA antibodies) of all conjugates were determined by enzyme-linked immunosorbent assay. Polymer containing conjugates elicited higher levels of specific anti-lipopolysaccharide IgM and IgG antibodies in comparison with other conjugates without polymer carrier. Enhanced IgM vibriocidal activity of mice antisera was also evident here.

scholarly journals Vibrio cholerae O139 Conjugate Vaccines: Synthesis and Immunogenicity of V. cholerae O139 Capsular Polysaccharide Conjugates with Recombinant Diphtheria Toxin Mutant in Mice

2000 ◽  
Vol 68 (9) ◽  
pp. 5037-5043 ◽  
Author(s):  
Zuzana Kossaczka ◽  
Joseph Shiloach ◽  
Virginia Johnson ◽  
David N. Taylor ◽  
Richard A. Finkelstein ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Diphtheria Toxin ◽  
Capsular Polysaccharide ◽  
Critical Level ◽  
Covalent Attachment ◽  
Igg Antibodies ◽  
Vibrio Cholerae O139 ◽  
Relevant Dose ◽  
Surface Polysaccharide ◽  
Outbred Mice

ABSTRACT Epidemiologic and experimental data provide evidence that a critical level of serum immunoglobulin G (IgG) antibodies to the surface polysaccharide of Vibrio cholerae O1 (lipopolysaccharide) and of Vibrio cholerae O139 (capsular polysaccharide [CPS]) is associated with immunity to the homologous pathogen. The immunogenicity of polysaccharides, especially in infants, may be enhanced by their covalent attachment to proteins (conjugates). Two synthetic schemes, involving 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) as activating agents, were adapted to prepare four conjugates of V. cholerae O139 CPS with the recombinant diphtheria toxin mutant, CRMH21G. Adipic acid dihydrazide was used as a linker. When injected subcutaneously into young outbred mice by a clinically relevant dose and schedule, these conjugates elicited serum CPS antibodies of the IgG and IgM classes with vibriocidal activity to strains of capsulatedV. cholerae O139. Treatment of these sera with 2-mercaptoethanol (2-ME) reduced, but did not eliminate, their vibriocidal activity. These results indicate that the conjugates elicited IgG with vibriocidal activity. Conjugates also elicited high levels of serum diphtheria toxin IgG. Convalescent sera from 20 cholera patients infected with V. cholerae O139 had vibriocidal titers ranging from 100 to 3,200: absorption with the CPS reduced the vibriocidal titer of all sera to ≤50. Treatment with 2-ME reduced the titers of 17 of 20 patients to ≤50. These data show that, like infection with V. cholerae O1, infection with V. cholerae O139 induces vibriocidal antibodies specific to the surface polysaccharide of this bacterium (CPS) that are mostly of IgM class. Based on these data, clinical trials with the V. cholerae O139 CPS conjugates with recombinant diphtheria toxin are planned.

scholarly journals Preparation, Immunogenicity, and Protective Efficacy, in a Murine Model, of a Conjugate Vaccine Composed of the Polysaccharide Moiety of the Lipopolysaccharide of Vibrio cholerae O139 Bound to Tetanus Toxoid

2001 ◽  
Vol 69 (5) ◽  
pp. 3488-3493 ◽  
Author(s):  
Alain Boutonnier ◽  
Sylvain Villeneuve ◽  
Farida Nato ◽  
Bruno Dassy ◽  
Jean-Michel Fournier
Keyword(s):  
Vibrio Cholerae ◽  
Tetanus Toxoid ◽  
Capsular Polysaccharide ◽  
Protective Efficacy ◽  
Immunoglobulin M ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Antibodies ◽  
Vibrio Cholerae O139 ◽  
Specific Polysaccharide ◽  
Antibody Levels

ABSTRACT The epidemic and pandemic potential of Vibrio choleraeO139 is such that a vaccine against this newly emerged serogroup ofV. cholerae is required. A conjugate made of the polysaccharide moiety (O-specific polysaccharide plus core) of the lipopolysaccharide (LPS) of V. cholerae O139 (pmLPS) was prepared by derivatization of the pmLPS with adipic acid dihydrazide and coupling to tetanus toxoid (TT) by carbodiimide-mediated condensation. The immunologic properties of the conjugate were tested using BALB/c mice injected subcutaneously three times at 2 weeks interval and then a fourth time 4 weeks later. Mice were bled 7 days after each injection and then once each month for the following 6 months. LPS and TT antibody levels were determined by enzyme-linked immunosorbent assay using immunoplates coated with either O139 LPS or TT. Both pmLPS and pmLPS-TT conjugate elicited low levels of immunoglobulin M (IgM), peaking 5 weeks after the first immunization. The conjugate elicited high levels of IgG antibodies, peaking 3 months after the first immunization and declining slowly during the following 5 months. TT alone, or as a component of conjugate, induced mostly IgG antibodies. Antibodies elicited by the conjugate recognized both capsular polysaccharide and LPS from V. cholerae O139 and were vibriocidal. They were also protective in the neonatal mouse model of cholera infection. The conjugation of the O139 pmLPS, therefore, enhanced its immunogenicity and conferred T-dependent properties to this polysaccharide.

scholarly journals Effect of Antigen Coating Conditions on Enzyme-Linked Immunosorbent Assay for Detection of Immunoglobulin G Antibody to Neisseria meningitidis Serogroup Y and W135 Capsular Polysaccharide Antigens in Serum

2003 ◽  
Vol 10 (6) ◽  
pp. 1136-1140 ◽  
Author(s):  
Peter C. Giardina ◽  
Renee E. Evans ◽  
Daniel J. Sikkema ◽  
Dace Madore ◽  
Stephen W. Hildreth
Keyword(s):  
Immunoglobulin G ◽  
Capsular Polysaccharide ◽  
Enzyme Linked Immunosorbent Assay ◽  
Meningococcal Vaccine ◽  
Igg Antibody ◽  
Human Sera ◽  
Polysaccharide Antigens ◽  
Reference Serum ◽  
Adult Volunteers ◽  
Assay Conditions

ABSTRACT Human sera collected from 28 consenting adult volunteers were used to define assay conditions for meningococcal vaccine clinical trial serology. Immunoassay parameters were optimized with these test sera and the standard reference serum, CDC1992. Coating conditions for serogroup Y and W135 polysaccharide antigens were found to influence the predicted serum immunoglobulin G (IgG) antibody concentrations. Sera that displayed IgG antibody binding profiles most unlike that of CDC1992 were influenced the most by coating conditions. Our results suggest that presentation of specific epitopes is influenced by antigen-coating concentrations for serogroup Y and W135 polysaccharides.

Comparison of exopolysaccharides from mucoid and nonmucoid strains of Clavibacter michiganensis subspecies sepedonicus

1993 ◽  
Vol 39 (3) ◽  
pp. 291-296 ◽  
Author(s):  
Paul J. Henningson ◽  
Neil C. Gudmestad
Keyword(s):  
Molecular Weight ◽  
High Performance ◽  
Size Exclusion ◽  
Enzyme Linked Immunosorbent Assay ◽  
Low Molecular Weight ◽  
Neutral Sugar ◽  
Clavibacter Michiganensis ◽  
Serological Reaction ◽  
Molecular Weights ◽  
Exclusion Chromatography

The exopolysaccharides produced by six strains of Clavibacter michiganensis ssp. sepedonicus were isolated and purified by liquid chromatography. Neutral sugar composition and molecular weights were determined for each polysaccharide fraction, using gas chromatography and high-performance size-exclusion chromatography. The serological reaction of each fraction was tested using enzyme-linked immunosorbent assay. Exopolysaccharide from nonmucoid strains contained only low molecular weight polysaccharides (1.5 × 103 to 1.1 × 104). Exopolysaccharide from mucoid and intermediate strains could be separated into low (4.0 × 103 to 1.1 × 104) molecular weight and high (5.0 × 105 to 1.6 × 106) molecular weight fractions. High molecular weight polysaccharides were composed almost exclusively of galactose, glucose, and fucose. The ratios of these sugars were highly variable among strains. Low molecular weight polysaccharides were primarily composed of galactose with significant and varying amounts of glucose, rhamnose, mannose, and ribose. All polysaccharide fractions except one, produced by a nonmucoid strain, reacted in the immunoassay test.Key words: exopolysaccharide, polysaccharide, Clavibacter, michiganensis, sepedonicus.

scholarly journals Enrichment of plasma extracellular vesicles for reliable quantification of their size and concentration for biomarker discovery

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Marija Holcar ◽  
Jana Ferdin ◽  
Simona Sitar ◽  
Magda Tušek-Žnidarič ◽  
Vita Dolžan ◽  
...  
Keyword(s):  
Biomarker Discovery ◽  
Plasma Source ◽  
Extracellular Vesicle ◽  
Size Exclusion ◽  
Enzyme Linked Immunosorbent Assay ◽  
Enrichment Method ◽  
Transmission Electron ◽  
Relative Standard ◽  
Exclusion Chromatography ◽  
Reliable Quantification

AbstractHuman plasma is a complex fluid, increasingly used for extracellular vesicle (EV) biomarker studies. Our aim was to find a simple EV-enrichment method for reliable quantification of EVs in plasma to be used as biomarker of disease. Plasma of ten healthy subjects was processed using sedimentation rate- (sucrose cushion ultracentrifugation—sUC) and size- (size exclusion chromatography—SEC) based methods. According to nanoparticle tracking analysis (NTA), asymmetrical flow field-flow fractionation coupled to detectors (AF4-UV-MALS), miRNA quantification, transmission electron microscopy and enzyme-linked immunosorbent assay, enrichment of EVs from plasma with sUC method lead to high purity of EVs in the samples. High nanoparticle concentrations after SEC resulted from substantial contamination with lipoproteins and other aggregates of EV-like sizes that importantly affect downstream EV quantification. Additionally, sUC EV-enrichment method linked to quantification with NTA or AF4-UV-MALS is repeatable, as the relative standard deviation of EV size measured in independently processed samples from the same plasma source was 5.4% and 2.1% when analyzed by NTA or AF4-UV-MALS, respectively. In conclusion, the sUC EV-enrichment method is compatible with reliable measurement of concentration and size of EVs from plasma and should in the future be tested on larger cohorts in relation to different diseases. This is one of the first studies using AF4-UV-MALS to quantify EVs in blood plasma, which opens new possible clinical utility for the technique.

scholarly journals The Vi Conjugate Typhoid Vaccine Is Safe, Elicits Protective Levels of IgG Anti-Vi, and Is Compatible with Routine Infant Vaccines

2011 ◽  
Vol 18 (5) ◽  
pp. 730-735 ◽  
Author(s):  
Vu Dinh Thiem ◽  
Feng-Ying C. Lin ◽  
Do Gia Canh ◽  
Nguyen Hong Son ◽  
Dang Duc Anh ◽  
...  
Keyword(s):  
Adverse Reactions ◽  
Capsular Polysaccharide ◽  
Geometric Mean ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Antibodies ◽  
Type B ◽  
Protective Level ◽  
Content Type ◽  
Expanded Program On Immunization ◽  
Routine Infant

ABSTRACTTyphoid fever remains a serious problem in developing countries. Current vaccines are licensed for individuals who are 5 years old or older. A conjugate of the capsular polysaccharide (CP) ofSalmonella entericaserovar Typhi (Vi) bound to recombinant exoprotein A ofPseudomonas aeruginosa(Vi-rEPA) enhanced Vi immunogenicity and protected 2- to 5-year-olds in Vietnam. In this study, Vi-rEPA was evaluated for use in infants. A total of 301 full-term Vietnamese infants received Expanded Program on Immunization (EPI) vaccines alone or with Vi-rEPA orHaemophilus influenzaetype b-tetanus toxoid conjugate (Hib-TT) at 2, 4, and 6 months and Vi-rEPA or Hib-TT alone at 12 months. Infants were visited 6, 24, and 48 h after each injection to monitor adverse reactions. Maternal, cord, and infant sera were assayed for IgG anti-Vi and for IgG antibodies to Hib CP and the diphtheria, tetanus, and pertussis toxins at 7, 12, and 13 months. No vaccine-related serious adverse reactions occurred. In the Vi-rEPA group, the IgG anti-Vi geometric mean (GM) increased from the cord level of 0.66 to 17.4 enzyme-linked immunosorbent assay units (EU) at 7 months, declined to 4.76 EU at 12 months, and increased to 50.1 EU 1 month after the 4th dose (95% of infants had levels of ≥3.5 EU, the estimated protective level). Controls had no increase of the IgG anti-Vi GM. Infants with cord anti-Vi levels of <3.5 EU responded with significantly higher IgG anti-Vi levels than those with levels of ≥3.5 EU. Anti-diphtheria, -tetanus, and -pertussis toxin levels were similar in all groups. Vi-rEPA was safe, induced protective anti-Vi levels, and was compatible with EPI vaccines, and it can be used in infants. High cord IgG anti-Vi levels partially suppressed infant responses to Vi-rEPA.

scholarly journals Dissociation Mechanics and Stability of Type a Botulinum Neurotoxin Complex by Means of Biophysical Evaluation

2022 ◽  
Author(s):  
Shavron Hada ◽  
Jae Chul Lee ◽  
Eun Chae Lee ◽  
Sunkyong Ji ◽  
Jeong Sun Nam ◽  
...  
Keyword(s):  
Light Scattering ◽  
Botulinum Neurotoxin ◽  
Type A ◽  
Size Exclusion ◽  
Enzyme Linked Immunosorbent Assay ◽  
Biophysical Characterization ◽  
Exclusion Chromatography ◽  
Associated Proteins ◽  
Dissociated State

Abstract Biophysical characterization of type A botulinum neurotoxin (BoNT/A) complex along with its thermodynamic stability was assessed through a combination of various methods. BoNT/A exists as large complexes in association with neurotoxin associated proteins (NAPs). To evaluate its biophysical behavior, size-exclusion chromatography (SEC), multi-angled light scattering (MALS), enzyme linked immunosorbent assay (ELISA), and dynamic light scattering (DLS) were utilized. Initially, a single peak (peak 1) of SEC was observed at pH 6.0, and an additional peak (peak 2) appeared at pH 7.4 with a decrement of peak 1. Through MALS and ELISA, the peak 2 was determined to be BoNT/A dissociated from its complex. The dissociation was accelerated by time and temperature. At 37°C, dissociated BoNT/A self-associated at pH 7.4 in the presence of polysorbate 20. On the other hand, the dissociation was partly reversible when titrated back to pH 6.0. Overall, BoNT/A was more stable when associated with NAPs at pH 6.0 compared to its dissociated state at pH 7.4. The conventional analytical methods could be utilized to relatively quantify its amount in different formulations.

scholarly journals Degradation of Wheat Germ Agglutinin during Sourdough Fermentation

Foods ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 340
Author(s):  
Luis E. Rojas Tovar ◽  
Michael G. Gänzle
Keyword(s):  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Reductase Activity ◽  
Size Exclusion ◽  
Enzyme Linked Immunosorbent Assay ◽  
Gluten Free ◽  
Exchange Reactions ◽  
Exclusion Chromatography ◽  
Thiol Exchange ◽  
Fungal Protease

Non Celiac Wheat Sensitivity (NCWS) is an intolerance to wheat products and individuals with NCWS often adhere to a gluten free diet. However, gluten free diets are often associated with a reduced sensory and nutritional quality. Wheat Germ Agglutinin (WGA) is one of the wheat components linked to NCWS. This study explored the fate of WGA during sourdough fermentation. To assess the role of thiol-exchange reactions and proteolysis, sourdoughs were fermented with Fructilactobacillus sanfranciscensis DSM20451, F. sanfranciscensis DSM20451ΔgshR, which lacks glutathione reductase activity, or Latilactobacillus sakei TMW1.22, with or without addition of fungal protease. The conversion of WGA was determined by size exclusion chromatography of fluorescence-labeled WGA, and by enzyme-linked immunosorbent assay (ELISA). Commercial whole wheat flour contained 6.6 ± 0.7 μg WGA/g. After fermentation with L. sakei TMW1.22 and F. sanfranciscensis DSM20451, the WGA content was reduced (p < 0.05) to 2.7 ± 0.4 and 4.3 ± 0.3 μg WGA/g, respectively, while the WGA content remained unchanged in chemically acidified controls or in doughs fermented with F. sanfranciscensis DSM20451ΔgshR. Protease addition did not affect the WGA content. In conclusion, the fate of WGA during sourdough fermentation relates to thiol-exchange reactions but not to proteolytic degradation.

scholarly journals Dynamics of glucagon secretion in mice and rats revealed using a validated sandwich ELISA for small sample volumes

2016 ◽  
Vol 311 (2) ◽  
pp. E302-E309 ◽  
Author(s):  
Nicolai J. Wewer Albrechtsen ◽  
Rune E. Kuhre ◽  
Johanne A. Windeløv ◽  
Anne Ørgaard ◽  
Carolyn F. Deacon ◽  
...  
Keyword(s):  
Sandwich Elisa ◽  
Glucagon Secretion ◽  
Small Sample ◽  
Size Exclusion ◽  
Enzyme Linked Immunosorbent Assay ◽  
The Novel ◽  
Intravenous Glucose ◽  
Exclusion Chromatography ◽  
Sensitivity Specificity ◽  
Rats And Mice

Glucagon is a metabolically important hormone, but many aspects of its physiology remain obscure, because glucagon secretion is difficult to measure in mice and rats due to methodological inadequacies. Here, we introduce and validate a low-volume, enzyme-linked immunosorbent glucagon assay according to current analytical guidelines, including tests of sensitivity, specificity, and accuracy, and compare it, using the Bland-Altman algorithm and size-exclusion chromatography, with three other widely cited assays. After demonstrating adequate performance of the assay, we measured glucagon secretion in response to intravenous glucose and arginine in anesthetized mice (isoflurane) and rats (Hypnorm/midazolam). Glucose caused a long-lasting suppression to very low values (1–2 pmol/l) within 2 min in both species. Arginine stimulated secretion 8- to 10-fold in both species, peaking at 1–2 min and returning to basal levels at 6 min (mice) and 12 min (rats). d-Mannitol (osmotic control) was without effect. Ketamine/xylazine anesthesia in mice strongly attenuated ( P < 0.01) α-cell responses. Chromatography of pooled plasma samples confirmed the accuracy of the assay. In conclusion, dynamic analysis of glucagon secretion in rats and mice with the novel accurate sandwich enzyme-linked immunosorbent assay revealed extremely rapid and short-lived responses to arginine and rapid and profound suppression by glucose.

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