Immunogenicity of potassium thiocyanate extracted and electrofocused Pasteurella multocida X-73I antigens in chickens and mice

1982 ◽  
Vol 28 (11) ◽  
pp. 1219-1225 ◽  
Author(s):  
Kevin L. McKinney ◽  
Paul A. Rebers ◽  
Richard B. Rimler
Keyword(s):  
Antibody Response ◽  
Pasteurella Multocida ◽  
Potassium Thiocyanate ◽  
Particulate Fraction ◽  
Supernatant Fraction ◽  
Chicken Embryos ◽  
Isoelectric Points ◽  
Serotype 1 ◽  
Soluble Supernatant

The immunogenicity of antigenic fractions obtained by the extraction of Pasteurella multocida strain X-73 (serotype 1) with potassium thiocyanate (KSCN) was determined in chickens and mice. The initial KSCN extract was centrifuged at 105 000 × g, and the antigens were separated into a particulate fraction (40p) and a soluble supernatant fraction (40s). The ultracentrifuged fractions were further resolved by preparative electrofocusing. The 40p fraction was resolved into two subgroups having isoelectric points of 3.5–3.9 and 5.5–6.0; the 40s fraction was resolved into five subgroups ranging in isoelectric points from 4.4 to 9.0. The 40p fractions were antigenically similar and contained lipopolysaccharide (LPS) and protein. The 40s fractions were antigenically distinct from the 40p fractions and from each other; they contained proteins and polysaccharides but no LPS. The 40p antigens were strongly immunogenic in mice and chickens, whereas the 40s antigens were weakly immunogenic in chickens and not immunogenic in mice. The incorporation of Freund's complete adjuvant increased the immunogenicity of the 40s antigens in chickens. The 40p antigens induced greater frequencies of serological responses in chickens than the 40s antigens as detected by counterimmunoelectrophoresis and immunodiffusion. This suggested that the increased protection associated with the 40p antigens may have been the result of better antibody response. The toxicity of all the fractions was evaluated by determination of lethality for 10-day-old chicken embryos because of the sensitivity and reliability of the test. The 40p fraction had an LD50 = 0.38 μg, and the 40s fraction had an LD50 = 2.5 μg. Since the 40s fraction contained no detectable LPS, it is likely that two toxins are present, one which contains LPS and one which does not.

Antigenic analysis of Pasteurella multocida (serotype 1) by crossed immunoelectrophoresis: characterization of whole cell associated antigens

1980 ◽  
Vol 26 (12) ◽  
pp. 1392-1402
Author(s):  
J. L. Bhasin ◽  
L. Lapointe-Shaw
Keyword(s):  
Pasteurella Multocida ◽  
Cell Envelope ◽  
Potassium Thiocyanate ◽  
Antigenic Analysis ◽  
Whole Cell ◽  
Heat Stable ◽  
Crossed Immunoelectrophoresis ◽  
Serotype 1 ◽  
Reference Preparation ◽  
Antigen Antibody

Crossed immunoelectrophoresis and other related quantitative immunoelectrophoretic techniques have been used to elucidate the antigenic complexity of a reference preparation of capsular extract, potassium thiocyanate extract, lipopolysaccharide, heat-stable antigens, and free endotoxin from Pasteurella multocida serotype 1. The reactions of these cellular fractions, in crossed immunoelectrophoresis, with reference anti-whole cell immunoglobulins disclosed five antigens in the capsular extract, seven in the potassium thiocyanate extract, one to three in the lipopolysaccharide, three in the heat-stable antigens, and five in the free endoxin. Comparison of these reference antigen–antibody systems, in crossed immunoelectrophoresis, with intermediate gel containing either a reference anti-cell envelope or anticytoplasmic immunoglobulins not only revealed the presence of additional antigens but also gave insight into the probable cellular origins (i.e., cell surface, cell envelope, or cytoplasm) of various antigens unveiled by reference anti-whole cell immunoglobulins. Using the principle of tandem crossed immunoelectrophoresis and crossed-line immunoelectrophoresis the immunochemical relationships between the antigenic components of these reference antigen–antibody systems were established.

INCORPORATION OF IODIDE INTO ORGANIC COMPOUNDS IN THE GUINEA PIG THYROID

1963 ◽  
Vol 43 (1) ◽  
pp. 110-118 ◽  
Author(s):  
R. Ekholm ◽  
T. Zelander ◽  
P.-S. Agrell
Keyword(s):  
Guinea Pig ◽  
Protein Binding ◽  
Organic Compounds ◽  
Guinea Pigs ◽  
Microsomal Fraction ◽  
Thyroid Tissue ◽  
Particulate Fraction ◽  
Supernatant Fraction ◽  
Hydrolysis Of

ABSTRACT Guinea pigs, kept on a iodine-sufficient diet, were injected with Na131I and the thyroids excised from 45 seconds to 5 days later. The thyroid tissue was homogenized and separated into a combined nuclear-mitochondrial-microsomal fraction and a supernatant fraction by centrifugation at 140 000 g for one hour. Protein bound 131iodine (PB131I) and free 131iodide were determined in the fractions and the PB131I was analysed for monoiodotyrosine (MIT), diiodotyrosine (DIT) and thyroxine after hydrolysis of PB131I. As early as only 20 minutes after the Na131I-injection almost 100% of the particulate fraction 131I was protein bound. In the supernatant fraction the protein binding was somewhat less rapid and PB131I values above 90% of total supernatant 131I were not found until 3 hours after the injection. In all experiments the total amount of PB131I was higher in the supernatant than in the corresponding particulate fraction. The ratio between supernatant PB131I and pellet PB131I was lower in experiments up to 3 minutes and from 2 to 5 days than in experiments of 6 minutes to 20 hours. Hydrolysis of PB131I yielded, even in the shortest experiments, both MIT and DIT. The DIT/MIT ratio was lower in the experiments up to 2 hours than in those of 3 hours and over.

Co-expression of recombinant single chain variable fragment recognizing blood antigen fused with sumo and chaperones in Escherichia coli

2018 ◽  
Vol 40 (4) ◽  
Author(s):  
Dang Thi Ngoc Ha ◽  
Le Thi Thu Hong ◽  
Truong Nam Hai
Keyword(s):  
Recombinant Antibody ◽  
Blood Type ◽  
Soluble Form ◽  
Supernatant Fraction ◽  
Blood Group Antigens ◽  
Single Chain ◽  
E Coli ◽  
Recombinant Scfv ◽  
Single Chain Variable Fragments

Single chain variable fragments (scFv) have widely been used in research, diagnosis and treatment, but the scFv is considered as difficult protein for expression in E. coli. In previous studies, we expressed a construction of recombinant single chain variable fragments again antigen specific for blood type A (antiA-scFv) individually or fused with Trx or SUMO. However, soluble fraction was low abandant and only approximately 40% when fused with Trx, the other cases were expressed in form of inclusion body. Therefore, it was difficult for purification, refolding and activity assesment. In thispaper, we demonstrated a suitable construction for soluble production of antiA-scFv fused with SUMO (SM/antiA-scFv) in presence of chaparones. Under fermentation with 0.1 mM IPTG at 20oC, the SM/antiA-scFv was entirely expressed in soluble form. Importantly, after cleavage from SUMO with SUMOprotease, antiA-scFv was still maintained in the supernatant fraction. Therefore, it can help ensure bioactivity and is useful for purification process. To the best of our knowledge, this is the first report showing soluble recombinant scFv fused with SUMO in presence of chaperone for determination of blood group antigens. Thus, this result facilitates the optimal study of soluble expression, purification and bioactivity determination of the antiA-scFv recombinant antibody. 

Polarographic Determination of Zinc in Compounded Rubber

1950 ◽  
Vol 23 (4) ◽  
pp. 975-980
Author(s):  
F. C. J. Poulton ◽  
L. Tarrant
Keyword(s):  
Zinc Oxide ◽  
Ammonium Acetate ◽  
Potassium Thiocyanate ◽  
Acetate Buffer Solution ◽  
Acid Electrolyte ◽  
Buffer Solution ◽  
Ammonium Acetate Buffer ◽  
Ammonium Acetate Buffer Solution ◽  
Gravimetric Procedure

Abstract Reasons are advanced for the unsatisfactory nature of some of the older methods for the determination of very small amounts of zinc in compounded rubber, particularly in latex mixings. The polarographic technique offers a possible solution, but most of the commoner electrolytes for the electroreduction of this metal are alkaline, and give rise to similar errors as are met in the gravimetric procedure. The development of a suitable acid electrolyte was therefore undertaken, and ways of dealing with likely interferences were examined. The electroltye finally recommended is a potassium thiocyanate-ammonium acetate buffer solution; iron, when present, is reduced to the ferrous condition by potassium iodide. The method was used to determine zinc oxide in a series of mixings of known composition ranging from 0.8 to 40 per cent. In all except the highest proportions of zinc oxide, the figures obtained agree well with the theoretical.

scholarly journals Detection of G1 proteins in Chinese hamster cells synchronized by isoleucine deprivation or mitotic selection.

1975 ◽  
Vol 66 (1) ◽  
pp. 95-101 ◽  
Author(s):  
K D Ley
Keyword(s):  
Time Course ◽  
Sodium Dodecyl ◽  
Chinese Hamster ◽  
Protein S ◽  
S Phase ◽  
Supernatant Fraction ◽  
G1 Phase ◽  
Differential Centrifugation ◽  
Chinese Hamster Cells ◽  
Soluble Supernatant

Examination of labeling patterns of proteins in Chinese hamster cells(line CHO) revealed the presence of a class of protein(s) that is synthesized during G1 phase of the cell cycle. Cells arrested in G1 by isoleucine (Ile) deprivation were prelabeded with [14-C]Ile, induced to traverse G1 by addition of unlabeled Ile, and labeled with [3-H]Ile at hourly intervals. Cells were fractionated into neclear and cytoplasmic portions, and proteins were separated by sodium dodecyl sulfate-polyacrylamide get electrophoresis. Gel profiles of proteins in the 45,000-160,000 mol wt range from the cytoplasm of cells in G1 were similar to those from cells arrested in G1 except for the presence of a mojor peak of [1-H]Ile incorporated into a protein(s) of approximately 80,000 mol wt. Peaks of net [3-H]Ile incorporation were not detected in neclear preparations. Cellular fractionation by differential centrifugation showed the peak I protein was located in the soluble supernatant fraction of the cytoplasm. Time-course studies showed that synthesis of this protein began 1-2 h after initiation of G1 traverse; the protein reached maximum levels in 4-6 h and was reduced to undetectable levels by 9 h. A cytoplasmic protein with similar electrophoretic mobility was found in G1 phase of cells synchronized by mitotic selection. This class of proteins is synthesized by cells before entry into S phase and may be involved in initiation of DNA synthesis.

LOCALIZATION OF GLUCOSE, GLUCONATE, AND GLUCOSE-6-PHOSPHATE OXIDATION SYSTEMS IN EXTRACTS OF PSEUDOMONAS FLUORESCENS

1958 ◽  
Vol 4 (1) ◽  
pp. 1-7 ◽  
Author(s):  
R. G. Eagon
Keyword(s):  
Pseudomonas Fluorescens ◽  
Particulate Fraction ◽  
Supernatant Fraction ◽  
Oxidation Of Glucose

Cell-free extracts of Pseudomonas fluorescens, strain OSU 64, derived by means of sonic oscillation and alumina grinding, were assayed to determine the localization of enzymes necessary for the oxidation of glucose, gluconate, and glucose-6-phosphate. Glucose and gluconate were oxidized to 2-ketogluconate by both the supernatant and particulate fractions of the extracts. Approximately 40% of the activity for the oxidation of glucose was demonstrated to be in the supernatant fraction and 60% in the particulate fraction. Activity for the oxidation of gluconate was found to be located with approximately 50% in each fraction. Oxidation of glucose-6-phosphate was observed only in the soluble portion.

scholarly journals SELECTIVE MERCURIMETRIC TITRATION ASSAY OF CHLORIDE CONCENTRATION IN THE WATER OF GREEN COCONUTS USING NOVEL INDICATOR SYSTEM

2017 ◽  
Vol 9 (3) ◽  
pp. 268
Author(s):  
Shivaji Rangnath Labhade
Keyword(s):  
Statistical Treatment ◽  
Chloride Concentration ◽  
Potassium Thiocyanate ◽  
Indicator System ◽  
Coconut Water ◽  
Stoichiometric Ratio ◽  
Indicator Solution ◽  
End Point ◽  
Red Color

Objective: A selective mercurimetric titration procedure is proposed for the assay of chloride concentration in the water of green coconut using mercury(II) nitrate [(Hg(NO3)2] reagent and iron(III) nitrate [Fe(NO3)3] with synthetically prepared mercury(II) thiocyanate [Hg(SCN)2] indicator system.Methods: An indicator solution was prepared by titrating Hg(NO3)2 against potassium thiocyanate (KSCN) till a red color end point using Fe(NO3)3. Then a known amount of Hg(NO3)2 was added to indicator solution and titrated against the water of green coconut till the original red color reappeared.Results: The concentration of chloride present in the volume of coconut water utilized in between these two end points was found to be reacting in the 2:1 stoichiometric ratio with the Hg(NO3)2 taken in the second step of the titration. The statistical treatment of the experimental data obtained by using standard solutions of sodium chloride (NaCl) indicates that the procedure is precise and accurate. The phosphate, sulfate, organic compounds and inorganic minerals present in the coconut water did not interfere with the measurement of chloride by this procedure. Both the cationic mineral value (was also determined by complexometric titration) and chloride concentration in the coconut water were found to be decreased with the development of the coconuts.Conclusion: The proposed procedure of determination of chloride concentration in the water of green coconut is simple, reliable and inexpensive. This procedure is excellent for determination of chloride in the acidic solution without precise adjustment of the pH for detection of the end point. Owing to the homogenous reaction condition no titration errors those are commonly encountered by co-precipitation in the argentometric assay of chloride. 

Determination of Molybdenum in Fertilizers by Atomic Absorption Spectrophotometry

1967 ◽  
Vol 50 (6) ◽  
pp. 1269-1273
Author(s):  
William L Hoover ◽  
Sonja C Dtjren
Keyword(s):  
Atomic Absorption ◽  
Isoamyl Alcohol ◽  
Potassium Thiocyanate ◽  
Atomic Absorption Spectrophotometry ◽  
Water Soluble ◽  
Complexing Agent ◽  
Alcohol Fraction ◽  
Low Levels ◽  
High Concentrations

Abstract A procedure for determining low levels of molybdenum in fertilizers by atomic absorption is proposed. With potassium thiocyanate as complexing agent, molybdenum is extracted in an isoamyl alcohol fraction to separate the fraction containing molybdenum from the water-soluble fraction containing materials that would interfere in the atomic absorption procedure. However, the procedure cannot be used with samples that have high concentrations of iron. Tests on the recovery of molybdenum in four fertilizers indicate that the procedure is reliable to levels as low as 2.5 ppm of molybdenum

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