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.

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 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.

Purification and immunochemical quantitation of Penicillium roqueforti acid aspartyl proteinase

1997 ◽  
Vol 64 (1) ◽  
pp. 105-113 ◽  
Author(s):  
EMMANUELLE BRACQ ◽  
ANNIE LEVIEUX ◽  
DIDIER LEVIEUX
Keyword(s):  
Enzyme Activity ◽  
Sandwich Elisa ◽  
Exchange Chromatography ◽  
Site Directed Mutagenesis ◽  
Size Exclusion ◽  
Activity Measurement ◽  
Penicillium Roqueforti ◽  
Protein Ratio ◽  
Culture Filtrates ◽  
Exclusion Chromatography

Acid aspartyl proteinase of Penicillium roqueforti was purified from culture filtrates using FPLC ion-exchange chromatography on Mono Q and size exclusion chromatography on Superdex 75. The purity obtained allowed us to produce, using rabbits, a specific antiserum which was then used to develop a sandwich ELISA. The test was sensitive (detection limit, 0·25 ng/ml), reproducible (CV, 3·1–6·9%) and closely correlated with enzyme activity measurement (r=0·995, P<0·001). This ELISA should be a valuable tool for monitoring acid aspartyl proteinase level in culture filtrates and for determining the enzyme activity[ratio ]enzyme protein ratio of acid aspartyl proteinase obtained after genetic recombinations or site-directed mutagenesis.

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 Radiolabeling and Quantitative In Vivo SPECT/CT Imaging Study of Liposomes Using the Novel Iminothiolane-99mTc-Tricarbonyl Complex

2017 ◽  
Vol 2017 ◽  
pp. 1-8 ◽  
Author(s):  
Zoltán Varga ◽  
Imola Cs. Szigyártó ◽  
István Gyurkó ◽  
Rita Dóczi ◽  
Ildikó Horváth ◽  
...  
Keyword(s):  
Specific Activity ◽  
Nuclear Imaging ◽  
Imaging Study ◽  
Size Exclusion ◽  
In Vivo Studies ◽  
The Novel ◽  
Tricarbonyl Complex ◽  
Biodistribution Studies ◽  
Exclusion Chromatography

The in vivo biodistribution of liposomal formulations greatly influences the pharmacokinetics of these novel drugs; therefore the radioisotope labeling of liposomes and the use of nuclear imaging methods for in vivo studies are of great interest. In the present work, a new procedure for the surface labeling of liposomes is presented using the novel 99mTc-tricarbonyl complex. Liposomes mimicking the composition of two FDA approved liposomal drugs were used. In the first step of the labeling, thiol-groups were formed on the surface of the liposomes using Traut’s reagent, which were subsequently used to bind 99mTc-tricarbonyl complex to the liposomal surface. The labeling efficiency determined by size exclusion chromatography was 95%, and the stability of the labeled liposomes in bovine serum was found to be 94% over 2 hours. The obtained specific activity was 50 MBq per 1 μmol lipid which falls among the highest values reported for 99mTc labeling of liposomes. Quantitative in vivo SPECT/CT biodistribution studies revealed distinct differences between the labeled liposomes and the free 99mTc-tricarbonyl, which indicates the in vivo stability of the labeling. As the studied liposomes were non-PEGylated, fast clearance from the blood vessels and high uptake in the liver and spleen were observed.

scholarly journals Copolymers Formation by Photopolymerization of (Meth)acrylates Containing Dissolved Polyheteroarylenes

2009 ◽  
Vol 2009 ◽  
pp. 1-11 ◽  
Author(s):  
Dmitriy A. Sapozhnikov ◽  
Tat'yana V. Volkova ◽  
Antonina A. Sakharova ◽  
Rashid G. Gasanov ◽  
Vanda Yu. Voytekunas ◽  
...  
Keyword(s):  
Infrared Spectroscopy ◽  
Size Exclusion ◽  
Transfer Reactions ◽  
Resonance Spectroscopy ◽  
Vinyl Monomers ◽  
The Novel ◽  
Model Compounds ◽  
Spin Resonance ◽  
Exclusion Chromatography ◽  
Kinetics Of

Radical photopolymerization of (meth)acrylates in the presence of dissolved polyheteroarylenes has been investigated. The kinetics of radical polymerization of unsaturated monomers in the presence of polyheteroarylenes and model compounds has been studied by Differential Scanning Photocalorimetry and Infrared Spectroscopy. From the results of investigations into the kinetics and the polymer structures (Fourier Transform Infrared Spectroscopy, Nuclear Magnetic Resonance, Size-exclusion Chromatography, Thermogravimetric analysis), it has been established that radical photopolymerization of vinyl monomers in the presence of polyheteroarylenes leads to the formation of copolymers owing to chain transfer reactions and/or chain termination by the relevant condensation polymer. Using Electron Spin Resonance Spectroscopy the novel radicals upon the addition of model compounds for the polyheteroarylenes have been detected, and a mechanism of copolymer formation has been proposed.

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 Structure of Human Cyclophilin A in Complex with the Novel Immunosuppressant Sanglifehrin A at 1.6 Å Resolution

2005 ◽  
Vol 280 (23) ◽  
pp. 21965-21971 ◽  
Author(s):  
Joerg Kallen ◽  
Richard Sedrani ◽  
Gerhard Zenke ◽  
Juergen Wagner
Keyword(s):  
Crystal Structure ◽  
Three Dimensional ◽  
Cyclophilin A ◽  
The Other ◽  
Size Exclusion ◽  
Dimensional Structure ◽  
The Novel ◽  
Immunosuppressive Activity ◽  
Exclusion Chromatography ◽  
Sanglifehrin A

Sanglifehrin A (SFA) is a novel immunosuppressant isolated from Streptomyces sp. that binds strongly to the human immunophilin cyclophilin A (CypA). SFA exerts its immunosuppressive activity through a mode of action different from that of all other known immunophilin-binding substances, namely cyclosporine A (CsA), FK506, and rapamycin. We have determined the crystal structure of human CypA in complex with SFA at 1.6 Å resolution. The high resolution of the structure revealed the absolute configuration at all 17 chiral centers of SFA as well as the details of the CypA/SFA interactions. In particular, it was shown that the 22-membered macrocycle of SFA is deeply embedded in the same binding site as CsA and forms six direct hydrogen bonds with CypA. The effector domain of SFA, on the other hand, has a chemical and three-dimensional structure very different from CsA, already strongly suggesting different immunosuppressive mechanisms. Furthermore, two CypA·SFA complexes form a dimer in the crystal as well as in solution as shown by light scattering and size exclusion chromatography experiments. This observation raises the possibility that the dimer of CypA·SFA complexes is the molecular species mediating the immunosuppressive effect.

Immunodiagnosis of paramphistomosis using monoclonal antibody-based sandwich ELISA for detection ofParamphistomum gracilecirculating 16 kDa antigen

Parasitology ◽  
2017 ◽  
Vol 144 (7) ◽  
pp. 899-903 ◽  
Author(s):  
PANAT ANURACPREEDA ◽  
KULLANID TEPSUPORNKUL ◽  
RUNGLAWAN CHAWENGKIRTTIKUL
Keyword(s):  
Monoclonal Antibody ◽  
Detection Method ◽  
Sandwich Elisa ◽  
Enzyme Linked Immunosorbent Assay ◽  
Microtitre Plate ◽  
Serum Samples ◽  
Fecal Samples ◽  
Lower Detection Limit ◽  
Lower Detection ◽  
Sensitivity Specificity

SUMMARYIn this study, we have produced a monoclonal antibody (MoAb) against 16 kDa antigen ofParamphistomum gracile(16 kDaAgPg), and developed an accurate sandwich enzyme-linked immunosorbent assay (sandwich ELISA) for the detection of circulating 16 kDaAg in the serum and fecal samples from cattle naturally infected withP. gracile. MoAb 1D10 was immobilized on a microtitre plate, and the antigen in the samples was captured and detected with biotinylated rabbit anti-16 kDaAgPg antibody. The lower detection limit of sandwich ELISA was 3·5 pg mL−1, and no cross-reaction with other parasite antigens was evaluated. The reliability of the assay was examined using the serum and fecal samples from cattle naturally infected withP. gracile, Fasciola gigantica, Moniezia benedeni, Trichurissp.,Strongyloidessp., strongylids and non-infected animals. The sandwich ELISA showed the sensitivity, specificity and accuracy at 98·33, 100 and 99·55% (serum samples), and 96·67, 100 and 99·09% (fecal samples). Therefore, this detection method is a rapid and excellent potential assay for the accurate diagnosis of paramphistomosis.

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