RADIOIMMUNOASSAY OF [8-ARGININE]-VASOPRESSIN

1975 ◽  
Vol 80 (3) ◽  
pp. 444-452 ◽  
Author(s):  
P. Czernichow ◽  
U. Merkelbach ◽  
M. B. Vallotton
Keyword(s):  
Antidiuretic Hormone ◽  
Arginine Vasopressin ◽  
Association Constant ◽  
Specific Activity ◽  
Limit Of Detection ◽  
High Specific Activity ◽  
High Association ◽  
Theoretical Maximum

ABSTRACT A radioimmunological method for the determination of the human antidiuretic hormone, [8-arginine]-vasopressin (AVP), is described in detail. The antiserum has been raised in rabbits injected with AVP adsorbed onto charcoal particles and is used at a final dilution of 1:200 000. It contains antibodies directed specifically against the C-terminal tripeptide and possesses a high association constant of 8.2× 1012 1/mole. AVP is labelled in monoiodinated form (as proven after pronase digestion) at a high specific activity close to the theoretical maximum after purification from unlabelled hormone on Sephadex gel. The standard curves are characterized by a Bo of 55 %, a limit of detection ascertained at least at 3 pg (10 % displacement) and a 50 % displacement achieved with 35.5 pg. The index of precision λ ranges from 0.033 to 0.042. The conditions of the assay (buffer's composition and pH, timing) were systematically varied and tested. In addition the result of immunization of chicken with AVP is reported. The assay is adequate for the measurement of AVP in urine and plasma and will be described in forthcoming papers.

THE DEVELOPMENT OF A RADIOIMMUNOASSAY FOR ARGININE-VASOPRESSIN: PRODUCTION OF ANTISERA AND LABELLED HORMONE; SEPARATION TECHNIQUES; SPECIFICITY AND SENSITIVITY OF THE ASSAY IN AQUEOUS SOLUTION

1972 ◽  
Vol 52 (2) ◽  
pp. 279-288 ◽  
Author(s):  
C. R. W. EDWARDS ◽  
T. CHARD ◽  
MURIEL J. KITAU ◽  
MARY L. FORSLING ◽  
J. LANDON
Keyword(s):  
Arginine Vasopressin ◽  
Specific Activity ◽  
Limit Of Detection ◽  
High Specific Activity ◽  
Ion Exchange Resin ◽  
Cross Reactivity ◽  
Amino Acid Sequences ◽  
Simple Method ◽  
Specificity And Sensitivity ◽  
Chloramine T

SUMMARY A radioimmunoassay for vasopressin was developed using antibodies produced against conjugated and non-conjugated arginine vasopressin. Despite the fact that the vasopressin molecule has only eight amino acids, cross reactivity studies showed that these antibodies were specific for different amino acid sequences. Labelled hormone of high specific activity (350–800 μCi/μg) was produced by a modification of the chloramine-T method. Unreacted iodide was removed by the batchwise addition of an ion-exchange resin. Other techniques of purification produced no advantage over this simple method. Several methods of separating antibody-bound and free hormone were studied. All except chromatoelectrophoresis proved satisfactory. Ammonium sulphate or ethanol precipitation of bound hormone was chosen because of simplicity, speed and reproducibility. The lower limit of detection of the assay was 80 pg arginine vasopressin/ml diluent buffer. Therefore an extraction and concentration procedure is necessary for the measurement of basal circulating levels of the hormone.

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.

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.

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

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.

ULTRAFILTRATION STUDIES OF STEROID-PROTEIN BINDING

1961 ◽  
Vol 23 (2) ◽  
pp. 129-137 ◽  
Author(s):  
P. S. CHEN ◽  
I. H. MILLS ◽  
F. C. BARTTER
Keyword(s):  
Protein Binding ◽  
Specific Activity ◽  
Binding Protein ◽  
High Specific Activity ◽  
Wide Range

SUMMARY A method is described for determination of the extent of protein binding of steroids by ultrafiltration after addition of radioactively-labelled steroid tracers of high specific activity. Results obtained with specifically labelled 14C-steroids did not differ significantly from those with steroids randomly labelled with tritium. A number of steroids have been studied with this technique. The 'S' shaped curve obtained with some corticosteroids by plotting percentage ultrafilterable against steroid added to the plasma confirmed the presence of the corticosteroid-binding protein which has greater affinity for steroid than albumin. This method of plotting the ultrafiltration data afforded a method of assessing the amount of binding protein (or the number of sites). Cortisone, 17-hydroxy-11-deoxycorticosterone, cortisol, and Δ1 cortisol showed this type of curve. Binding of aldosterone, progesterone and 17-ketosteroids occurred to the same extent in plasma as in 5% albumin. The ultrafilterable fraction of these steroids in plasma was constant over a wide range of total steroid concentration. Binding of testosterone was greater with plasma than with albumin, and did not decrease upon addition of large amounts of testosterone.

PROCEDURE FOR DETERMINATION OF ALDOSTERONE IN HUMAN PERIPHERAL PLASMA BY DOUBLE-ISOTOPE DERIVATIVE ASSAY, AND ITS APPLICATION FOR MEASUREMENT OF SECRETORY RATE AND URINARY EXCRETION

1967 ◽  
Vol 45 (12) ◽  
pp. 1919-1936 ◽  
Author(s):  
Wojciech Nowaczynski ◽  
Jack Silah ◽  
Jacques Genest
Keyword(s):  
Acetic Anhydride ◽  
Urinary Excretion ◽  
Specific Activity ◽  
High Specific Activity ◽  
Plasma Aldosterone ◽  
Chromatographic Purification ◽  
Secretory Rate ◽  
Peripheral Plasma ◽  
Double Isotope

This is a report of a procedure for the determination of peripheral plasma aldosterone, in which 14C-labeled aldosterone serves as the marker, and the acetylating agent is relatively inexpensive tritium-labeled acetic anhydride of high specific activity. A new method for the chromatographic purification of aldosterone and aldosterone diacetate is presented. This procedure provides a better separation of these two compounds from the unlabeled and labeled impurities.

The use of p-Iodophenyl[125I] isothiocyanate for determination of N-terminal amino acids in nanomole quantities of protein

1975 ◽  
Vol 28 (10) ◽  
pp. 2289 ◽  
Author(s):  
CJ Burrell ◽  
PD Cooper ◽  
JM Swan
Keyword(s):  
Amino Acids ◽  
Specific Activity ◽  
High Specific Activity ◽  
Coupling Reaction ◽  
Molar Content ◽  
Terminal Residue ◽  
Terminal Amino

Identification of the N-terminal residue of 2.5-5 nmol of protein was possible by means of a modified Edman reaction and p-iodophenyl[125I] isothiocyanate of high specific activity. The second and third residues could also be identified. Yields of the terminal residue were 25-50%, allowing approximate quantitation of the molar content of protein. Use of the isotope allowed certain steps of the degradative cycle to be examined in some detail, and preparation of this relatively non- volatile labelled Edman reagent was safe, cheap and convenient. ��� The synthesis and properties of the p-iodophenylthiohydantoins of 19 amino acids, and some side products of the coupling reaction, are described.

Determination of thyrotropin in serum by time-resolved fluoroimmunoassay evaluated.

1985 ◽  
Vol 31 (10) ◽  
pp. 1706-1709 ◽  
Author(s):  
H L Kaihola ◽  
K Irjala ◽  
J Viikari ◽  
V Näntö
Keyword(s):  
Specific Activity ◽  
High Specific Activity ◽  
Reference Interval ◽  
Time Resolved ◽  
Standard Curve ◽  
Highly Sensitive ◽  
Wide Range ◽  
Sandwich Type ◽  
Serum Tsh

Abstract We evaluated a new, highly sensitive time-resolved fluoroimmunoassay for thyrotropin (TSH) in serum. This direct immunometric "sandwich"-type assay involves two monoclonal antibodies against TSH, one immobilized, the other labeled with europium. Extremely high specific activity of the label and the use of labeled antibody in large excess make the method sensitive enough to measure TSH values falling below the normal reference interval. The standard curve is nearly linear over a wide range of TSH concentrations (standard concentrations range from 0.25 to 324 milli-int. units/L). The lowest concentration detectable was 25 micro-int. units/L. The CV for the assay was less than 6% at 0.5 milli-int. unit/L or higher, 11.3% at 0.1 milli-int. unit/L. For a CV of 10% the lower limit of the working range would be around 0.1 milli-int. unit/L. The interassay CV was 6.7 to 11.8% for TSH concentrations of 0.31 to 19.6 milli-int. units/L. The 95% confidence interval for sera from 111 healthy persons was 0.6-3.8 (range 0.3-3.8) milli-int. units/L. For hyperthyroid patients and thyroid cancer patients treated with thyroxin after thyroidectomy, serum TSH values were all below the reference interval (most were less than 25 micro-int. units/L).

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