Development and evaluation of a radioimmunoassay for Arg8-vasopressin, after extraction with Sep-Pak C18.

1985 ◽  
Vol 31 (6) ◽  
pp. 861-863 ◽  
Author(s):  
K A Ysewijn-Van Brussel ◽  
A P De Leenheer
Keyword(s):  
Human Plasma ◽  
Arginine Vasopressin ◽  
Specific Activity ◽  
Cross Reaction ◽  
High Avidity ◽  
Plasma Samples ◽  
Displacement Analysis ◽  
Double Antibody ◽  
Chloramine T ◽  
Dilution Curves

Abstract We describe a specific double-antibody radioimmunoassay for measuring arginine vasopressin (AVP) in human plasma. Antisera of high avidity were obtained from rabbits that had been injected with AVP coupled to bovine thyroglobulin. The antibody reacts with both the tripeptide tail and the pentapeptide ring of the molecule, thereby eliminating cross reaction with oxytocin. Synthetic AVP was labeled with 125I by a modification of the Chloramine-T technique. The specific activity of the labeled hormone was 29 MBq/micrograms of AVP, as estimated by self-displacement analysis. The assay involves Sep-Pak C18 extraction of acidified (pH 4) plasma. Recovery of [3H]AVP added to plasma averaged 86.6 (SD 6.1)% (n = 14). Dilution curves for plasma showed linearity of response with concentration. The overall sensitivity was 0.3 ng/L when 2-mL plasma samples were extracted. The intra-assay CV was 7.8% at 4.8 ng/L (n = 12) and the interassay CV was 12.3% (n = 16) and 6.3% (n = 14) at 2.7 and 4.1 ng/L concentrations, respectively.

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.

Simple extraction method and radioimmunoassay for somatostatin in human plasma.

1981 ◽  
Vol 27 (6) ◽  
pp. 888-891 ◽  
Author(s):  
T L Peeters ◽  
Y Depraetere ◽  
G R Vantrappen
Keyword(s):  
Human Plasma ◽  
Plasma Proteins ◽  
Extraction Method ◽  
Plasma Samples ◽  
Analytical Recovery ◽  
Simple Extraction ◽  
The Mean ◽  
Radioimmunological Determination ◽  
Dilution Curves

Abstract Interference by human plasma proteins in the radioimmunological determination of somatostatin was eliminated by subjecting plasma samples to acid--ethanol precipitation. The assay was performed on the lyophilized supernate from 300 microliters of human plasma, with reagents that are commercially available. Sensitivity was 2.6 pg, corresponding to 8.7 ng/L of plasma. The intra-assay CV was 8%; the inter-assay CV was 12% for a low (30 ng/L) and 15% for a high (100 ng/L) standard. The mean analytical recovery of exogenous somatostatin from plasma was 95%, and the standard synthetic cyclic somatostatin showed parallel dilution curves with extracted plasma samples. Neither gastrin HG-17 and HG-34, pentagastrin, secretin, glucagon, vasoactive intestinal polypeptide, substance P, nor pancreatic polypeptide interfered in the assay system. Mean immunoreactive somatostatin in 20 normal fasting subjects was 31.5 (SD 15.6) ng/L and ranged from 14 to 67 ng/L.

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.

Homologous radioimmunoassay for human parathyrin (residues 53-84).

1982 ◽  
Vol 28 (8) ◽  
pp. 1749-1753 ◽  
Author(s):  
W Hitzler ◽  
H Schmidt-Gayk ◽  
P Spiropoulos ◽  
F Raue ◽  
M Hüfner
Keyword(s):  
Primary Hyperparathyroidism ◽  
Guinea Pigs ◽  
Histidine Residue ◽  
Normal Range ◽  
Double Antibody ◽  
Heterologous System ◽  
Chloramine T ◽  
Carboxyl Terminal ◽  
Free Serum ◽  
Dilution Curves

Abstract We describe a sequential saturation double-antibody radioimmunoassay for carboxyl-terminal fragments of human parathyrin (hPTH) in serum. Standards are prepared with synthetic hPTH (residues 53-84) in hPTH-free serum. Antisera are obtained by immunizing guinea pigs with partly purified hPTH extracted from adenomatous glands. Tracer is prepared by labeling hPTH (53-84), presumably at the histidine residue, with 125I by the Chloramine T method at pH 8.6. Dilution curves for hPTH extracted from adenomas are superimposable on dilution curves for the synthetic 53-84 fragment. Dilution of sera from hyperparathyroid patients showed linearity of response with concentration in the present assay, but non-linearity in the heterologous radioimmunoassay. In contrast to the heterologous system, which discriminated 28 of 32 patients with primary hyperparathyroidism from 32 normals (normal range: undetectable to 54 pmol/L, omitting the highest and lowest values from controls), the present assay separated these groups without overlap.

A direct radioimmunoassay for tonin

1978 ◽  
Vol 56 (8) ◽  
pp. 769-773 ◽  
Author(s):  
J. Gutkowska ◽  
R. Boucher ◽  
S. Demassieux ◽  
R. Garcia ◽  
J. Genest
Keyword(s):  
Specific Activity ◽  
High Specific Activity ◽  
Submaxillary Gland ◽  
Optimal Conditions ◽  
Excellent Correlation ◽  
Submaxillary Glands ◽  
Double Antibody ◽  
Antibody Method ◽  
Chloramine T

A sensitive radioimmunoassay for the measurement of tonin is described. It is based on the use of antibodies produced in rabbits against highly purified tonin obtained from rat submaxillary gland. A radioiodinated enzyme with high specific activity was obtained by the chloramine-T method. Optimal conditions for radioimmunoprecipitation were established and the double-antibody method was used to separate bound and free tonin. The radioimmunoassay may be used to measure tonin in amounts as low as 150 pg. This radioimmunoassay was applied to the measurement of tonin in rat saliva and in homogenates of submaxillary glands. Excellent correlation was found between tonin activity measured fiuorometrically and tonin concentration obtained by the radioimmunoassay.

FRACTIONATION AND ANALYSIS OF MONKEY PITUITARY GLANDS FOR POSTERIOR LOBE HORMONES

1962 ◽  
Vol 40 (2) ◽  
pp. 283-289 ◽  
Author(s):  
Darrell N. Ward ◽  
Earl F. Walborg ◽  
Harry S. Lipscomb ◽  
Roger Guillemin
Keyword(s):  
Amino Acid ◽  
Current Distribution ◽  
Arginine Vasopressin ◽  
Specific Activity ◽  
Small Scale ◽  
Posterior Lobe ◽  
Pharmacological Activities ◽  
Pituitary Glands ◽  
Counter Current ◽  
Counter Current Distribution

ABSTRACT Fractionation of monkey pituitary glands gave an oxytocin fraction in low yield which showed a counter-current distribution coefficient equivalent to that obtained with oxytocin from other species. Fractionation and chromatography of monkey vasopressin on carboxymethyl cellulose gave arginine-vasopressin of 60% purity, based on amino acid analysis and specific activity. Counter-current distribution on a small scale gave arginine-vasopressin of 89% purity. Reports by others that monkey pituitary glands contain arginine-vasopressin, based on pharmacological activities, are substantiated by the chemical data presented here.

scholarly journals Radioimmunoassay of plasma ouabain in healthy and pregnant individuals

2000 ◽  
Vol 165 (3) ◽  
pp. 669-677 ◽  
Author(s):  
O Vakkuri ◽  
SS Arnason ◽  
A Pouta ◽  
O Vuolteenaho ◽  
J Leppaluoto
Keyword(s):  
Pregnant Women ◽  
Human Plasma ◽  
High Performance ◽  
Chemical Nature ◽  
Solid Phase ◽  
Plasma Concentrations ◽  
First Trimester ◽  
Plasma Samples ◽  
Third Trimester ◽  
Endogenous Substances

Ouabain was recently isolated from human plasma, bovine hypothalamus and bovine adrenal in attempts to identify endogenous substances inhibiting the cell membrane sodium pump. A number of radioimmunoassays have been developed in order to study the clinical significance of ouabain. The results have been controversial with regard to the presence and chemical nature of plasma ouabain-like immunoreactivity. We have now measured ouabain in healthy and pregnant individuals using solid-phase extraction of plasma samples followed by a new radioimmunoassay with the extraordinary sensitivity of at least 2 fmol/tube (5 pmol/l). Plasma extracts, a previously isolated human plasma ouabain-like compound and bovine hypothalamic inhibitory factor displaced the tracer in parallel and eluted identically with ouabain in high-performance liquid chromatography. Plasma ouabain immunoreactivity was found to be much lower than reported previously: 12.6+/-1.3 pmol/l in healthy men (mean+/-s.e., n=20) and 9.4+/-0.7 pmol/l in women (n=14). In pregnant women (n=28) plasma ouabain concentration was 16.3+/-4.0 pmol/l during the first trimester, 18.8+/-4.3 pmol/l during the second trimester and 24.3+/-4.0 pmol/l during the third trimester (all P<0.01 compared with non-pregnant women). Plasma ouabain 3-5 days after the delivery was 13.6+/-1.1 pmol/l (n=10, P<0.05-0.01 compared with second and third trimesters). The pregnancy-related changes in the plasma concentrations of ouabain resembled those of cortisol. Therefore cortisol was measured from the same plasma samples and a significant positive correlation was found (r=0.512, P=0.006). The similar profiles of plasma ouabain and cortisol during pregnancy and their rapid decreases postpartum are consistent with the adrenal cortical origin of ouabain and also show that the secretions of these hormones are possibly under the control of same factors.

scholarly journals Protocol to analyze circulating small non-coding RNAs by high-throughput RNA sequencing from human plasma samples

2021 ◽  
Vol 2 (3) ◽  
pp. 100606
Author(s):  
Giuseppina E. Grieco ◽  
Guido Sebastiani ◽  
Daniela Fignani ◽  
Noemi Brusco ◽  
Laura Nigi ◽  
...  
Keyword(s):  
Human Plasma ◽  
Rna Sequencing ◽  
High Throughput ◽  
Plasma Samples ◽  
Human Plasma Samples ◽  
Non Coding Rnas

scholarly journals The immunological characterization of several human ribonucleases by using primary binding tests

1977 ◽  
Vol 163 (3) ◽  
pp. 419-426 ◽  
Author(s):  
E A Neuwelt ◽  
M Schmukler ◽  
M S Niziak ◽  
P B Jewett ◽  
C C Levy
Keyword(s):  
Human Plasma ◽  
Cross Reaction ◽  
The Other ◽  
Human Pancreas ◽  
Human Tissues ◽  
Antigenic Difference ◽  
Immunological Characterization ◽  
Similarities And Differences

RNAases (ribonucleases), purified from four human tissues, as well as bovine pancreatic RNAase (RNAase A), were studied by immunodiffusion methods and by two different primary binding tests. The enzymes fell into two groups immunologically, those purified from plasma and pancreas in one and those from spleen and liver in the other. No antigenic cross-reaction between the two groups was detected by any of the immunoassays used. There was a slight antigenic cross-reaction between the human and bovine pancreatic RNAases. The liver and spleen RNAases were immunologically identical by all criteria used, whereas a small but consistent antigenic difference between the human plasma and human pancreas enzymes was detected. The significance of this difference between the human plasma and pancreas RNAases is discussed in relation to similarities and differences in their properties.

Sign in / Sign up

Export Citation Format

Share Document