DETERMINATION OF HUMAN GROWTH HORMONE (HGH) IN PLASMA BY A DOUBLE ANTIBODY RADIOIMMUNOASSAY

1966 ◽  
Vol 53 (1) ◽  
pp. 101-120 ◽  
Author(s):  
E. Cerasi ◽  
L. Della Casa ◽  
R. Luft ◽  
A. Roovete
Keyword(s):  
Present Method ◽  
Human Growth ◽  
Antibody Technique ◽  
Double Antibody ◽  
Insulin In Plasma ◽  
Assay Procedure ◽  
Absolute Measure ◽  
Assay Methods ◽  
Per Se

ABSTRACT The double antibody technique of Hales & Randle (1963) for determination of insulin in plasma was developed for the assay of HGH in plasma. The precision and reproducibility of the method as well as the recovery of HGH added to plasma were highly satisfactory. Increased HGH values were obtained in acromegaly and during insulin hypoglycaemia, while decreased values were observed during glucose infusion. Successful treatment of acromegaly was accompanied by the return of plasma HGH levels toward normal. The plasma HGH values obtained with the present method cannot be considered as reflecting the absolute amounts of HGH, since several factors in the assay procedure, mainly the incubation time and the concentration of HGH antibodies used, markedly influenced the values obtained. It is questioned whether the radioimmunological assay methods for HGH per se give an absolute measure of this hormone in plasma.

SOME FACTORS OF SIGNIFICANCE IN THE DOUBLE ANTIBODY IMMUNOASSAY OF INSULIN

1967 ◽  
Vol 54 (2) ◽  
pp. 347-361 ◽  
Author(s):  
K. Brunfeldt ◽  
K. R. Jorgensen
Keyword(s):  
Guinea Pig ◽  
Insulin Concentration ◽  
Precipitation Reaction ◽  
Antibody Technique ◽  
Complement Activity ◽  
Double Antibody ◽  
Insulin In Plasma ◽  
Antibody Method ◽  
Heparin Preparation

ABSTRACT Differences were demonstrated between 4 insulin antibody (guinea pig) precipitating antisera (rabbit), int. al. in their ability to cross react with human γ-globulin components. This cross reaction is presumably the reason why »negative« insulin values can be found at higher concentrations of certain precipitating antisera. Independently of the precipitating antiserum it was possible, in some cases, to demonstrate a difference between the insulin concentration in serum and in heparinized plasma. This difference could be eliminated by the addition of heparin, by long-lasting freezing, and by dilution of the serum. Hence, the higher values found in certain cases in serum, as compared with plasma are presumably due to the presence of complement. It is essential in the use of the double antibody technique for the determination of insulin in plasma and serum that a heparin preparation with anti-complement activity should be used and that the concentration range of the precipitating antiserum should be fixed. The double antibody method can be used with or without pre-precipitation if the above mentioned criteria are fulfilled. Pre-precipitation, however, is to be preferred, as the precipitation reaction here occurs under constant conditions.

RADIOIMMUNOASSAY OF HUMAN GROWTH HORMONE USING THE DOUBLE ANTIBODY TECHNIQUE

1968 ◽  
Vol 57 (4) ◽  
pp. 549-556 ◽  
Author(s):  
F. Melani ◽  
U. Gröschel-Stewart ◽  
J. Lawecki
Keyword(s):  
Growth Hormone ◽  
Human Growth ◽  
Biologically Active ◽  
List Type ◽  
Purification Step ◽  
Antibody Technique ◽  
Double Antibody ◽  
Starch Gel ◽  
Radio Immunoassay

ABSTRACT The method of Roos et al. (1963) for the preparation of HGH yields a highly purified biologically active hormone preparation suitable for clinical purposes and, after one additional purification step (chromatography on Sephadex G-200 according to Hunter (1965), for radioiodination. In the double antibody technique described, the labelled hormone can be used for radio-immunoassay without starch-gel or Sephadex purification. The method is simple and sensitive enough to permit the determination of HGH in serum.

EVALUATION OF THE DOUBLE ANTIBODY RADIOIMMUNOASSAY OF INSULIN AND THE DETERMINATION OF INSULIN IN PLASMA AND URINE IN NORMAL SUBJECTS

1969 ◽  
Vol 60 (2) ◽  
pp. 327-351 ◽  
Author(s):  
Kai R. Jørgensen
Keyword(s):  
Extreme Values ◽  
Degradation Products ◽  
Insulin Response ◽  
Normal Subjects ◽  
Precipitation Reaction ◽  
Fasting Insulin ◽  
Double Antibody ◽  
Insulin In Plasma ◽  
The Subject

ABSTRACT The use of the double antibody method for radio-immunological determination of insulin in plasma was evaluated on the basis of dilution and recovery experiments, as well as by the investigation of the reproducibility and accuracy of the method. Given the optimum conditions for the precipitation reaction, the method appears from the present investigations to be well suited for plasma insulin determinations. With the technique used for the separation of free and antibody bound insulin, the results of the insulin determination were found to be independent of the radioactive degradation products present in the tracer insulin. It was not possible to demonstrate any increased degradation of the tracer insulin by incubation in plasma or urine as compared with incubation in 0.5 % human albumin buffer. The absolute insulin values were found to be independent of the length of the incubation period. No difference was found between the fasting insulin concentrations in a group of new-born infants of non-diabetic mothers and a group of adult normal subjects. Similarly, the fasting insulin values were found to be independent of the sex of the subject investigated. After oral administration of glucose a considerable variation was found in the insulin response in a group of normal subjects. This variation, within the weight limits used, was found to be independent of the sex, age and weight of the subject investigated. A corresponding condition was found to be valid for the insulin excretion in the urine of normal subjects. It was concluded that the large variation in the insulin response in a group of normal subjects did not allow conclusions about the clinical significance of the extreme values, but that the variation alone must be taken as an expression for a pronounced biological variation.

Effects of anti-prolactin autoantibodies on serum prolactin measurements

1994 ◽  
Vol 130 (5) ◽  
pp. 434-437 ◽  
Author(s):  
Naoki Hattori ◽  
Katsuji Ikekubo ◽  
Takashi Ishihara ◽  
Kunisaburo Moridera ◽  
Megumu Hino ◽  
...  
Keyword(s):  
Direct Measurement ◽  
The Other ◽  
Serum Prolactin ◽  
Immunoradiometric Assay ◽  
Routine Method ◽  
Antibody Technique ◽  
Double Antibody ◽  
Assay Procedure ◽  
Routine Assay ◽  
Kobe City

Hattori N, Ikekubo K, Ishihara T, Moridera K, Hino M, Kurahachi H. Effects of anti-prolactin autoantibodies on serum prolactin measurements. Eur J Endocrinol 1994:130:434–7. ISSN 0804–4643 The influence of anti-PRL autoantibodies on PRL measurements determined by immunoassays was investigated in 10 patients with anti-PRL autoantibodies. Four different immunoassay systems (two double-antibody radioimmunoassays (RIAs), a single-antibody RIA and an immunoradiometric assay (IRMA)) were examined. Total and free PRL were extracted from sera by precipitating γ-globulin with polyethylene glycol with and without acidification, respectively. PRL values determined by direct measurement were compared with total PRL values. The proportion of free to total PRL levels determined by each immunoassay in sera with anti-PRL autoantibodies was significantly lower than in control sera. Values obtained by direct measurement of PRL (a routine assay procedure) in control sera were similar to total PRL values, whereas in sera with anti-PRL autoantibodies the values varied from one immunoassay to the other. In sera with anti-PRL autoantibodies, double-antibody RIA I, RIA 2 and single-antibody RIA 3 yielded values lower for PRL than for total PRL (52 ±15% in RIA 1, 40±8.8% in RIA 2.40±14% in RIA 3), while PRL levels determined by IRMA were not significantly different (112 ±14%). Immunoglobulin G purified from serum with anti-PRL autoantibodies dosedependently decreased the recovery of PRL assayed by the double-antibody technique, while it did not affect that by IRMA. These data suggest that the presence of anti-PRL autoantibodies gives variable results depending on the immunoassays used. It is noteworthy that anti-PRL autoantibodies do not cause falsely high PRL values, as in the case of antithyroid hormone autoantibodies, rather they cause an underestimate, when a double-antibody RIA, a routine method for PRL measurement, is used. Naoki Hattori, Department of Endocrinology. Kobe City General Hospital, 4-6 Minatojima Nakamachi, Chuoku, Kobe 650, Japan

Optimization of Radioimmunoassay for Human Growth Hormone by the Charcoal-Dextran Technique

1970 ◽  
Vol 16 (10) ◽  
pp. 845-848 ◽  
Author(s):  
Joseph C Meek ◽  
Mary M Stoskopf ◽  
Robert E Bolinger
Keyword(s):  
Molecular Weight ◽  
Growth Hormone ◽  
Guinea Pig ◽  
Human Growth Hormone ◽  
Serum Proteins ◽  
Human Growth ◽  
Molecular Fragments ◽  
Antibody Technique ◽  
Double Antibody

Abstract The effect of serum on the radioimmunoassay for human growth hormone was tested by use of the double-antibody technique and the charcoal-coated dextran technique. With the double-antibody technique, serum effects a falsely high reading for unknowns, while with the charcoal-dextran technique it causes falsely low values to be obtained. Part of the anomaly with the charcoal-dextran technique is the result of adsorption of small molecular fragments of radiolabeled hormone onto serum proteins of higher molecular weight. These fragments can be eliminated by frequent repurification of the labeled hormone. Another part is not related to purity of the label but is corrected by subtracting values for serum blanks that contain no antibody. The use of antiserum to human growth hormone prepared in the goat is more sensitive than that prepared in the guinea pig.

BIOASSAY OF GROWTH HORMONE AND PROLACTIN PREPARATIONS BY DETERMINATION OF LONGITUDINAL BONE GROWTH WITH TETRACYCLINE

1974 ◽  
Vol 76 (1) ◽  
pp. 35-52 ◽  
Author(s):  
K.-G. Thorngren ◽  
L. I. Hansson
Keyword(s):  
Growth Hormone ◽  
Present Method ◽  
Bone Growth ◽  
Biological Effects ◽  
Human Growth ◽  
Lower Growth ◽  
Longitudinal Bone Growth ◽  
Hypophysectomized Rats ◽  
Growth Promoting

ABSTRACT The growth stimulating effect of different growth hormone and prolactin preparations on the longitudinal bone growth in thyroxine-treated hypophysectomized rats was determined by the tetracycline method. The effect of the hormone preparations was compared with that of the 1st International Standard for growth hormone. The potency calculation showed that the tested human growth hormone preparations have a higher potency than the bovine, ovine and porcine growth hormone preparations. Also potency differences were found between hormones from the same species but prepared by different methods. The prolactin preparations have a considerably lower growth promoting activity than the growth hormone preparations. The bioassay method used in the present investigation has a favourable mean precision (λ = 0.172) and sensitivity compared with the earlier bioassay methods. The present method increases the possibility of determining the biological effects of various growth promoting substances.

Two-Stage Procedure for the Quantitative Determination of Autoprothrombin III Concentration and Some Applications

1967 ◽  
Vol 18 (01/02) ◽  
pp. 198-210 ◽  
Author(s):  
Ronald S Reno ◽  
Walter H Seegers
Keyword(s):  
Prothrombin Time ◽  
Factor Vii ◽  
Two Stage ◽  
Pronounced Increase ◽  
One Stage ◽  
Assay Procedure ◽  
Bovine Plasma ◽  
The One ◽  
Autoprothrombin C

SummaryA two-stage assay procedure was developed for the determination of the autoprothrombin C titre which can be developed from prothrombin or autoprothrombin III containing solutions. The proenzyme is activated by Russell’s viper venom and the autoprothrombin C activity that appears is measured by its ability to shorten the partial thromboplastin time of bovine plasma.Using the assay, the autoprothrombin C titre was determined in the plasma of several species, as well as the percentage of it remaining in the serum from blood clotted in glass test tubes. Much autoprothrombin III remains in human serum. With sufficient thromboplastin it was completely utilized. Plasma from selected patients with coagulation disorders was assayed and only Stuart plasma was abnormal. In so-called factor VII, IX, and P.T.A. deficiency the autoprothrombin C titre and thrombin titre that could be developed was normal. In one case (prethrombin irregularity) practically no thrombin titre developed but the amount of autoprothrombin C which generated was in the normal range.Dogs were treated with Dicumarol and the autoprothrombin C titre that could be developed from their plasmas decreased until only traces could be detected. This coincided with a lowering of the thrombin titre that could be developed and a prolongation of the one-stage prothrombin time. While the Dicumarol was acting, the dogs were given an infusion of purified bovine prothrombin and the levels of autoprothrombin C, thrombin and one-stage prothrombin time were followed for several hours. The tests became normal immediately after the infusion and then went back to preinfusion levels over a period of 24 hrs.In other dogs the effect of Dicumarol was reversed by giving vitamin K1 intravenously. The effect of the vitamin was noticed as early as 20 min after administration.In response to vitamin K the most pronounced increase was with that portion of the prothrombin molecule which yields thrombin. The proportion of that protein with respect to the precursor of autoprothrombin C increased during the first hour and then started to go down and after 3 hrs was equal to the proportion normally found in plasma.

Determination of Plasminogen in Human Plasma by the Lysis Time Method

1966 ◽  
Vol 16 (01/02) ◽  
pp. 001-017 ◽  
Author(s):  
W Berg ◽  
K Korsan-Bengtsen ◽  
J Ygge
Keyword(s):  
Human Plasma ◽  
Present Method ◽  
Blood Donors ◽  
Fibrinogen Concentration ◽  
Plasmin Activity ◽  
Time System ◽  
Lysis Time ◽  
High Dilutions ◽  
Single Determination

SummaryA one-stage lysis time system containing fibrinogen, streptokinase, thrombin, and a known, small amount of plasminogen was used to determine plasminogen in plasma.The known amount of plasminogen was added to the system in order to keep the lysis times relatively short when a highly diluted plasma was tested. High dilutions of plasma were used to reduce the influence of the plasma inhibitors.The calculation of the plasminogen concentration was made on the basis of the correlation: “plasminogen = fibrinogen/lysis time” which was valid in the system. The method allowed determination of plasminogen in plasma with varying fibrinogen concentrations, as the fibrinogen concentration in plasma was considered in the calculation.The presence of “spontaneous” plasmin activity in the plasma did not influence the plasminogen determination. Estimated by this method, the plasminogen content in plasma from 32 blood donors aged 25-45 years was 13.1 ±2.4 casein u/ml. The error of a single determination was 0.3 casein u/ml. The plasminogen content in plasma, determined with the present method, is about 3-4 times higher than the content found when a caseinolytic method is used.

Human Prothrombin Activation: Determination of Plasma and Serum Levels of Prothrombin Fragment 3 by Radioimmunoassay

1979 ◽  
Author(s):  
Daniel Walz ◽  
Thomas Brown
Keyword(s):  
Blood Clotting ◽  
Factor Xa ◽  
Heart Association ◽  
Serum Levels ◽  
Cross Reactivity ◽  
Assay Procedure ◽  
A Chain ◽  
Prothrombin Activation ◽  
Theoretical Amount

Human prothrombin activation is unique in that, in addition to the release of fragment 1.2 (FI.2) from the NH-terminus of prothrombin by factor Xa during the generation of thrombin, an additional 13 residue polypeptide, fragment 3 (F3), is autocatalytically removed from the amino-terminus of the thrombin A chain. We have developed a rapid radioimmunoassay for human F3 which incorporates short incubation times and the use of a preprecipitated second antibody; the assay can be performed in three hours. Specificity studies in buffer systems show prothrombin and prethrombin 1 cross-reacting at a level of 0.001; purified thrombin does not cross-react. In the presence of 5% BSA, prothrombin displays considerably less cross-reactivity. No immunoreactive material to F3 antibodies could be detected in 400 μL of plasma. Serum, obtained from whole blood clotting, contained measurable quantities of F3 (40-100 ng/mL). This amount in serum represents only 5-10% of the theoretical amount available should all of the fragment be hydrolytically cleaved during the conversion of prothrombin to thrombin. This assay procedure is currently being utilized to monitor the activation of purified human prothrombin in the absence and presence of selected plasma inhibitors. (Supported in part by NIH 05384-17 and the Michigan Heart Association).

Determination of Factor XIII Activity and of Factor XIII Inhibitors Using an Ammonium-Sensitive Electrode

1983 ◽  
Vol 50 (02) ◽  
pp. 563-566 ◽  
Author(s):  
P Hellstern ◽  
K Schilz ◽  
G von Blohn ◽  
E Wenzel
Keyword(s):  
Factor Xiii ◽  
Normal Subjects ◽  
The Other ◽  
Activity Measurement ◽  
Factor Xiii Deficiency ◽  
Assay Procedure ◽  
Stabilization Factor ◽  
Release Method ◽  
Sensitive Electrode

SummaryAn assay for rapid factor XIII activity measurement has been developed based on the determination of the ammonium released during fibrin stabilization. Factor XIII was activated by thrombin and calcium. Ammonium was measured by an ammonium-sensitive electrode. It was demonstrated that the assay procedure yields accurate and precise results and that factor XIII-catalyzed fibrin stabilization can be measured kinetically. The amount of ammonium released during the first 90 min of fibrin stabilization was found to be 7.8 ± 0.5 moles per mole fibrinogen, which is in agreement with the findings of other authors. In 15 normal subjects and in 15 patients suffering from diseases with suspected factor XIII deficiency there was a satisfactory correlation between the results obtained by the “ammonium-release-method”, Bohn’s method, and the immunological assay (r1 = 0.65; r2= 0.70; p<0.01). In 3 of 5 patients with paraproteinemias the values of factor XIII activity determined by the ammonium-release method were markedly lower than those estimated by the other methods. It could be shown that inhibitor mechanisms were responsible for these discrepancies.

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