THE DEVELOPMENT OF A RADIOIMMUNOASSAY FOR OXYTOCIN: RADIO-IODINATION, ANTIBODY PRODUCTION AND SEPARATION TECHNIQUES

1970 ◽  
Vol 46 (2) ◽  
pp. 269-278 ◽  
Author(s):  
T. CHARD ◽  
M. J. KITAU ◽  
J. LANDON
Keyword(s):  
Lymph Node ◽  
Human Plasma ◽  
Rapid Method ◽  
Ammonium Sulphate ◽  
Antibody Production ◽  
Specific Activity ◽  
High Specific Activity ◽  
Separation Techniques ◽  
Ammonium Sulphate Precipitation

SUMMARY A simple and rapid method is described for labelling oxytocin with 131I at a high specific activity. This method is compared with those of previous workers. A satisfactory antiserum has been raised by direct intra-lymph node injection of oxytocin adsorbed to carbon microparticles. A number of methods for separating antibody-bound from free oxytocin are described, and reasons given for preferring a procedure using ammonium sulphate precipitation. These data form the basis for developing a radioimmunoassay intended for the determination of oxytocin in human plasma.

scholarly journals The determination of oestradiol and oestrone in the plasma of the domestic fowl by a method involving the use of labelled derivatives

1968 ◽  
Vol 106 (1) ◽  
pp. 77-86 ◽  
Author(s):  
J. E. O'Grady
Keyword(s):  
Human Plasma ◽  
Paper Chromatography ◽  
Domestic Fowl ◽  
Specific Activity ◽  
High Specific Activity ◽  
Double Labelling ◽  
Plasma Samples ◽  
Labelling Technique ◽  
Preliminary Purification

1. A method involving the use of triple-labelled derivatives has been developed for the determination of total oestrone and oestradiol in the plasma of the domestic fowl. The double-labelling technique devised by Svendsen (1960) for the determination of free oestrogens in human plasma was modified to enable the total oestrogen recovery to be determined for each sample. 2. [6,7−3H2]Oestradiol-17β is added to the plasma samples (1–10ml.), which are hydrolysed with acid and the phenolic steroids then extracted and partially purified. The extract is esterified with iodobenzene-p[35S]-sulphonyl chloride of high specific activity. After addition of standard oestrogen [131I]iodobenzene-p-sulphonates the esters are finally purified by paper chromatography. 3. The oestrogens are determined by comparing the 3H/35S and 131I/35S ratios in the purified esters with similar ratios of appropriate standards. 4. With this procedure the recoveries of oestrone and oestradiol after hydrolysis were 70–85% and 72–84% respectively, and after hydrolysis and preliminary purification 38–53% and 39–51% respectively. With this procedure up to 500ng. of oestradiol can be determined. The sensitivity of the technique for oestrone is 3·0ng. and for oestradiol 2·1ng. 5. The ranges of oestradiol and oestrone concentrations found in six plasma samples were 8·3–21·4ng./ml. and 15·2–31·6ng./ml. respectively.

PROPERTIES OF BOVINE Ac-GLOBULIN CONCENTRATES AND METHODS OF PREPARATION

1963 ◽  
Vol 41 (1) ◽  
pp. 2409-2421 ◽  
Author(s):  
Nobuo Aoki ◽  
Charles R. Harmison ◽  
Walter H. Seegers
Keyword(s):  
Human Plasma ◽  
Ammonium Sulphate ◽  
Protein Concentration ◽  
Room Temperature ◽  
Specific Activity ◽  
Barium Carbonate ◽  
Dry Weight ◽  
Physical Chemical ◽  
Bovine Plasma ◽  
Refrigerator Temperature

A procedure is described for retaining bovine plasma Ac-globulin activity as one part of the protein from plasma for every 1000 parts removed. The yields averaged 15%. The procedure involves removal of prothrombin with barium carbonate, isoelectric fractionation, fractionation with ammonium sulphate, chromatography on Amberlite IRC-50, and a second fractionation with ammonium sulphate. The procedure requires 2 days; however, the first day completes up to chromatography and the concentrate at that time is quite useful for many purposes. It is more stable than the product obtained after chromatography and the yields are higher. In absence of salts Ac-globulin is quite insoluble at pH 5.0. The final product usually contained some impurity. With the analytical ultra-centrifuge the S20in 0.1 M potassium chloride solution was found to be 4.2 at a protein concentration of 12.4 mg/ml. The specific activity was 1500 U./mg dry weight. Bovine plasma contains 120 U./ml or about 9 mg/100 ml. Assuming the same specific activity for human plasma the concentration is most likely near 1 mg/100 ml. The best stability conditions found were: 50% glycerol, pH 7.0, and 0.1 M calcium chloride. Under those conditions at room temperature all activity was retained 6 to 7 hours, at refrigerator temperature 24 hours, and at −60 °C for 1 month. In rabbits, antibodies were readily produced. Oxidizing agents destroyed the activity, while reducing agents did not, nor did they tend to stabilize. SH blocking agents destroyed the activity. The loss of activity in the presence of 0.0025 M parachloromercuribenzoate was recovered with 0.04 M cysteine. The molecule deteriorated while attempts were made to obtain physical chemical data; consequently, the molecular weight was calculated from an amino acid analysis and found to be 98,800. The reliability of this value is problematical. Human plasma was analyzed and found to contain 13 U./ml Ac-globulin. After 4 days storage, at room temperature, the prolonged prothrombin time of that plasma was completely restored with 13 units of Ac-globulin, which is equivalent to 8 μg.

Eine Methode zur qualitativen und quantitativen Bestimmung von drei Juvenilhormonen von Insekten

1974 ◽  
Vol 29 (3-4) ◽  
pp. 161-168 ◽  
Author(s):  
K. H. Trautmann ◽  
A. Schuler ◽  
M. Suchý ◽  
H.-K. Wipf
Keyword(s):  
Quantitative Determination ◽  
Specific Activity ◽  
High Specific Activity ◽  
Ether Extract ◽  
Qualitative And Quantitative ◽  
Work Up ◽  
First Time ◽  
Very High ◽  
Tenebrio Molitor Larvae

Abstract A method is presented permitting the qualitative and quantitative determination of all three presently known hormones (JH1-3). The determination is based on the method of radioactive isotope dilution, whereby a very small known amount of tritium-labelled JH-1 is added to the ether extract of the particular species. The addition of radioactive JH-1 permits the isolation of all three hormones, because of their similar behaviour during the chosen work up. The quantitative determination was carried out by gas chromatography and the identification was confirmed with the help of retention-times and GC-MS combination. The method was checked by using an extract of Hyalophora cecropia. For the first time methyl 10,11-epoxy-3,7,11-trimethyl-2-trans-6-trans-dodecadienoate (JH-3) could also be identified as the juvenile hormone of Melo­lontha melolontha. In Vanessa io larvae, Tenebrio molitor larvae and adults and in Musca domestica larvae none of the three known hormones could be detected. The preparation of JH-1 labelled with tritium in the methyl group of the ester was accomplished with very high specific activity (4.34 Ci/mmol) of the tritiated acid with diazomethane.

scholarly journals Design and Synthesis of 99mTcN-Labeled Dextran-Mannose Derivatives for Sentinel Lymph Node Detection

2018 ◽  
Vol 11 (3) ◽  
pp. 70 ◽  
Author(s):  
Alessandra Boschi ◽  
Micòl Pasquali ◽  
Claudio Trapella ◽  
Alessandro Massi ◽  
Petra Martini ◽  
...  
Keyword(s):  
Lymph Node ◽  
Specific Activity ◽  
High Specific Activity ◽  
Specific Class ◽  
Design And Synthesis ◽  
Specific Receptors ◽  
Mannose Derivatives ◽  
Mannose Receptors ◽  
Multifunctional Ligands ◽  
Pendant Arms

Background: New approaches based on the receptor-targeted molecular interaction have been recently developed with the aim to investigate specific probes for sentinel lymph nodes. In particular, the mannose receptors expressed by lymph node macrophages became an attractive target and different multifunctional mannose derivate ligands for the labeling with 99mTc have been developed. In this study, we report the synthesis of a specific class of dextran-based, macromolecular, multifunctional ligands specially designed for labeling with the highly stable [99mTc≡N]2+ core. Methods: The ligands have been obtained by appending to a macromolecular dextran scaffold pendant arms bearing a chelating moiety for the metallic group and a mannosyl residue for allowing the interaction of the resulting macromolecular 99mTc conjugate with specific receptors on the external membrane of macrophages. Two different chelating systems have been selected, S-methyl dithiocarbazate [H2N‒NH‒C(=S)SCH3=HDTCZ] and a sequence of two cysteine residues, that in combination with a monophosphine coligand, are able to bind the [99mTc≡N]2+ core. Conclusions: High-specific-activity labeling has been obtained by simple mixing and heating of the [99mTc≡N]2+ group with the new mannose-dextran derivatives.

Improvements in the determination of the turnover rate of plasma thyroxine in avian species: application to the Japanese quail

1987 ◽  
Vol 114 (2) ◽  
pp. 191-198 ◽  
Author(s):  
E. J. Cookson ◽  
J. Glover
Keyword(s):  
Japanese Quail ◽  
Half Life ◽  
Turnover Rate ◽  
Specific Activity ◽  
Simple Procedure ◽  
High Specific Activity ◽  
Future Application ◽  
Sodium Thiocyanate ◽  
Plasma Samples

ABSTRACT The disappearance of the thyroid hormone thyroxine (T4) from plasma in fully grown male Japanese quail can be described as a first order process with a rate constant of 0·178 ± 0·013/h (mean±s.e.m., n = 8), which represents a half-life of 3·90 h. A small amount of [125I]T4 in relation to total circulating T4 was injected i.v. into Japanese quail and plasma samples were taken at appropriate time-intervals for the determination of residual plasma radioactivity. The rate of disappearance of [125I]T4 was subsequently equated to the turnover rate of the endogenous hormone. Previous methods were modified to overcome problems arising from possible disturbance of plasma T4 metabolism, recirculation of radiolabelled iodide, and to purify the [125I]T4 from the plasma samples. By using labelled T4 of very high specific activity, the amount of [125I]T4 administered was kept much smaller than has been used in previous studies on Japanese quail, thus limiting any interference with plasma T4 dynamics. To minimize any disturbance of plasma T4 metabolism, only four blood samples were taken, at three-hourly intervals after the injection of [125I]T4. The rapid turnover of T4 produced a large amount of labelled inorganic iodide, the re-entry of which into the plasma T4 pool was inhibited by s.c. administration of sodium thiocyanate 1 h before injection of[125I]T4. Assay of the true [125I]T4 turnover was significantly improved over that used in previous studies by purifying the [125I]T4 from the plasma samples chromatographically. The samples were applied to small Sephadex G-25 columns with sodium hydroxide (0·1 mol/l) as the eluant. This simple procedure clearly separated the [125I]T4 from the other radioiodinated plasma components such as free iodide, non-hormonal iodinated proteins and triiodothyronine (T3), thus enabling a more accurate assessment of the residual labelled T4 concentration in the plasma and hence the T4 half-life. The future application of this method to the study of plasma T4 turnover under various experimental conditions is discussed and the possible involvement of T4 turnover studies in the assessment of T4 to T3 conversion is considered. J. Endocr. (1987) 114, 191–198

scholarly journals Bromelain Activity of Waste Parts of Two Pineapple Varieties

2018 ◽  
Vol 2 ◽  
pp. 21-28 ◽  
Author(s):  
Edmund Ofosu Benefo ◽  
Isaac Williams Ofosu
Keyword(s):  
Enzyme Activity ◽  
Ammonium Sulphate ◽  
Crude Extract ◽  
Specific Activity ◽  
High Demand ◽  
Digestion Method ◽  
Waste Products ◽  
Food Industries ◽  
Ethanol Precipitation ◽  
Ammonium Sulphate Precipitation

Bromelain, a protease found in pineapples, is of high demand in the pharmaceutical, cosmetic and food industries. Along the pineapple processing chain, waste products such as peels, crowns, stems and cores result. These parts are usually discarded, though they contain significant amounts of the enzyme bromelain. This study sought to determine the bromelain activity of the crowns and peels of two pineapple varieties grown in Ghana;MD2andSugarloaf. The crude extract was obtained by homogenising the peels and crowns in a cold phosphate buffer and centrifuging at 3000 rpm for 15 min. Ethanol and ammonium sulphate precipitation were carried out on the extract between 30% – 80% precipitation levels. The enzyme activity was determined using the casein digestion method. Results showed that bromelain was precipitated mainly in the 30% – 60% precipitation range.Sugarloafcrowns yielded the highest enzyme activity of 20.82 U/ml and a specific activity of 194.58 U/mg at the 40% ammonium sulphate precipitation level. This was followed by theSugarloafpeels with an enzyme activity of 19.98 U/ml at 50% ethanol precipitation level. Ethanol precipitation resulted in fractions with lower bromelain activity. Enzyme activity was higher in theSugarloafvariety and also in the crowns of both varieties. The two pineapple varieties have significant levels of bromelain activity and could be exploited for commercialisation.

PROPERTIES OF BOVINE Ac-GLOBULIN CONCENTRATES AND METHODS OF PREPARATION

1963 ◽  
Vol 41 (12) ◽  
pp. 2409-2421 ◽  
Author(s):  
Nobuo Aoki ◽  
Charles R. Harmison ◽  
Walter H. Seegers
Keyword(s):  
Human Plasma ◽  
Ammonium Sulphate ◽  
Protein Concentration ◽  
Room Temperature ◽  
Specific Activity ◽  
Barium Carbonate ◽  
Dry Weight ◽  
Physical Chemical ◽  
Bovine Plasma ◽  
Refrigerator Temperature

A procedure is described for retaining bovine plasma Ac-globulin activity as one part of the protein from plasma for every 1000 parts removed. The yields averaged 15%. The procedure involves removal of prothrombin with barium carbonate, isoelectric fractionation, fractionation with ammonium sulphate, chromatography on Amberlite IRC-50, and a second fractionation with ammonium sulphate. The procedure requires 2 days; however, the first day completes up to chromatography and the concentrate at that time is quite useful for many purposes. It is more stable than the product obtained after chromatography and the yields are higher. In absence of salts Ac-globulin is quite insoluble at pH 5.0. The final product usually contained some impurity. With the analytical ultra-centrifuge the S20in 0.1 M potassium chloride solution was found to be 4.2 at a protein concentration of 12.4 mg/ml. The specific activity was 1500 U./mg dry weight. Bovine plasma contains 120 U./ml or about 9 mg/100 ml. Assuming the same specific activity for human plasma the concentration is most likely near 1 mg/100 ml. The best stability conditions found were: 50% glycerol, pH 7.0, and 0.1 M calcium chloride. Under those conditions at room temperature all activity was retained 6 to 7 hours, at refrigerator temperature 24 hours, and at −60 °C for 1 month. In rabbits, antibodies were readily produced. Oxidizing agents destroyed the activity, while reducing agents did not, nor did they tend to stabilize. SH blocking agents destroyed the activity. The loss of activity in the presence of 0.0025 M parachloromercuribenzoate was recovered with 0.04 M cysteine. The molecule deteriorated while attempts were made to obtain physical chemical data; consequently, the molecular weight was calculated from an amino acid analysis and found to be 98,800. The reliability of this value is problematical. Human plasma was analyzed and found to contain 13 U./ml Ac-globulin. After 4 days storage, at room temperature, the prolonged prothrombin time of that plasma was completely restored with 13 units of Ac-globulin, which is equivalent to 8 μg.

STUDIES ON THE PURIFICATION OF BOVINE PLASMINOGEN (PROFIBRINOLYSIN)

1960 ◽  
Vol 38 (1) ◽  
pp. 1029-1034 ◽  
Author(s):  
Arthur Dalby ◽  
Edmond R. Cole ◽  
Edwin T. Mertz
Keyword(s):  
Calcium Phosphate ◽  
Ammonium Sulphate ◽  
Bovine Serum ◽  
Specific Activity ◽  
Gel Chromatography ◽  
Isoelectric Precipitation ◽  
Ammonium Sulphate Precipitation ◽  
Ph Range

A crude bovine plasminogen product obtained from bovine serum by 30% ammonium sulphate precipitation has been purified 20-fold by means of isoelectric precipitation and calcium phosphate gel chromatography. A threefold purification was achieved by isoelectric precipitation. Plasminogen was precipitated in greatest yield and highest specific activity at 40-fold dilution in the pH range of 5.25–5.50. The conditions under which plasminogen is eluted from calcium phosphate gel columns have been investigated. Plasminogen fractions possessing specific activity seven times that of the isoelectric-precipitated materials have been obtained by elution with phosphate buffers over the range of 0.05 to 0.1 M, pH 6.8.

scholarly journals Interleukin-6 Promotes Anti-OspA Borreliacidal Antibody Production In Vitro

2006 ◽  
Vol 13 (1) ◽  
pp. 19-25 ◽  
Author(s):  
Erik L. Munson ◽  
Dean T. Nardelli ◽  
K. H. Kevin Luk ◽  
Monica C. Remington ◽  
Steven M. Callister ◽  
...  
Keyword(s):  
Lymph Node ◽  
B Lymphocytes ◽  
Antibody Production ◽  
Interleukin 6 ◽  
In Vitro Assay ◽  
Vitro Assay ◽  
Lymph Node Cells ◽  
In Vitro Treatment

ABSTRACT Determination of the immunological mediators responsible for promoting the production of borreliacidal antibody may facilitate the development of an improved borreliosis vaccine for human and veterinary use. Previously, we developed an in vitro assay to determine if borreliacidal antibody production could be augmented by treatment with different cytokines. In this study, in vitro treatment of lymph node cells producing borreliacidal antibody with recombinant interleukin-6 (rIL-6) resulted in a fourfold enhancement of anti-OspA borreliacidal antibody. Moreover, rIL-6 enhanced Western immunoblot titers and increased the number of B lymphocytes. In contrast, treatment of anti-OspA borreliacidal antibody-producing cells with anti-IL-6 resulted in a fourfold reduction in borreliacidal activity. Treatment with anti-IL-6 also inhibited enhanced borreliacidal antibody production induced by anti-gamma interferon. These data suggest that IL-6 plays a significant role in the production of anti-OspA borreliacidal antibodies.

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