K.J. Catt

Solid-Phase Radioimmunoassay in Antibody-Coated Tubes

The adsorption of antibody to polymeric surfaces has been used to develop a new method of solid-phase radioimmunoassay. Incubation is performed in antibody-coated, disposable tubes that are finally washed-out with water and counted for quantitation of the bound tracer. The method is simple, rapid, inexpensive, and suitable for automation.

ANGIOTENSIN II BLOOD-LEVELS IN HUMAN HYPERTENSION

Abstract Studies on normal subjects have shown Summary that blood-angiotensin-it levels are not markedly altered by postural change and sodium restriction, while more prominent increases are caused by acute administration of diuretics and oral contraceptive therapy. In benign and uncomplicated essential hypertension, blood-angiotensin-II levels are usually normal. However, increased levels are commonly present in patients with severe essential hypertension, renal hypertension, and malignant hypertension, with a significant correlation between diastolic pressure and blood-angiotensin-II concentration in all three forms of hypertension. The association with high blood-angiotensin-II is particularly frequent in malignant hypertension and renal hypertension, being observed in 90% of patients with these conditions. It is proposed that high levels of circulating angiotensin II may contribute to the development of renal hypertension in man, and that the ability of the diseased kidney to compensate for the pressor effects of high angiotensin levels by adjusting sodium and fluid balance may be an additional determining factor in the development of hypertension associated with renal disease.

Metabolic clearance of human growth hormone in patients with hepatic and renal failure, and in the isolated perfused pig liver.

Abstract In order to define the possible roles of the liver and kidneys in the metabolic disposal of human growth hormone (HGH), the metabolic clearance rate (MCR) of radioactively labeled HGH was determined in four patients with hepatic failure and in four with renal failure. To define the role of the liver in more detail, measurements of the MCR of labeled HGH were made in two intact pigs and in the isolated perfused livers of the same animals. In the patients with hepatic failure, MCR of HGH was 41–144.5 (mean 83.8) liters/day/sq m and in the patients with renal failure MCR was 58–93 (mean 79.1). The means of these values were 41% and 39% respectively, of the MCR of HGH determined in normal subjects. MCR of HGH by the isolated perfused pig liver was 24% of that observed in the intact pig. It was concluded that the liver and kidneys are important sites of metabolic disposal of HGH. These observations may be relevant in the understanding of the sites of action of growth hormone, and may provide some explantation for abnormalities of plasma HGH in renal and hepatic disorders.

Measurement of angiotensin II in blood by radioimmunoassay

Abstract Specific and reliable measurement of circulating angiotensin II levels was performed by collection of blood into alcohol and extraction of angiotensin II prior to measurement by radioimmunoassay. 125I-labeled angiotensin II was used to correct for the recovery of angiotensin II through the extraction procedure in each sample. The immunoassay utilized highly purified 131I-angiotensin II as assay tracer, and antisera from rabbits immunized with angiotensin II coupled to porcine gamma globulin; separation of antibody-bound and free tracer was performed with dextran-coated charcoal. The accuracy of the procedure was close to 100% and the precision as given by the coefficient of variation was ± 3.5 to ± 14.2%. The blank of the assay was negligible (0.025 ng/100 ml) and the level of immunoreactive material detected in blood samples from nephrectomized sheep and humans was extremely low (0.2 ng/100 The normal values for angiotensin II in human blood were 2.5 ± 1.4 ng/100 ml (arterial) and 1.9 ± 1.2 ng/100 ml (venous). Slightly lower arterial blood angiotensin II levels were found in sheep (1.4 ± 1.1 ng/100 ml) and dogs (1.6 ± 1.6 ng/100 ml). Significantly elevated blood angiotensin II levels were observed during sodium depletion, in malignant and renal hypertension, and in women taking estrogen-containing oral contraceptive preparations. Plasma renin activity and blood angiotensin II levels showed a significant positive correlation, and plasma renin concentration was positively correlated with angiotensin II during sodium depletion and negatively correlated with blood angiotensin II during oral contraceptive therapy. Measurement of immunoreactive angiotensin II in arterial or venous bloodprovides a direct indication of the circulating level of the active peptide generated by the interaction of the various components of the renin-angiotensin system in vivo.

Antibody-coated tube method for radioimmunoassay of human growth hormone

Abstract The ability of polymeric surfaces to adsorb gamma-globulin has been utilized in the development of an extremely simple method of radioimmunoassay. In this method, polystyrene or other plastic tubes are coated internally with specific antibody by exposure to suitable dilutions of antiserum. The coated tubes are then used for radioimmunoassay by incubation with standards or samples and radioiodinated tracer antigen, followed by aspiration of the solutions and counting of the washed tubes to determine the antibody-bound radioactivity. The method has been shown to be satisfactory for radioimmunoassay of a wide range of peptide and protein hormones, and is extremely suitable for assay of pituitary hormones during clinical and physiological studies. The assay of human growth hormone in plasma can be routinely performed by overnight incubation. Advantages of the procedure are its simplicity, economy, wide applicability and ability to use tracer of relatively low specific activity.

Metabolic clearance rate of radioiodinated human growth hormone in man

The nature of the disappearance of radioiodinated human growth hormone (HGH) from plasma has been reexamined. The metabolic clearance rate (MCR) was determined both from single injection and constant infusion studies. After single injection of highly purified radioiodinated HGH, the disappearance curve remained multiexponential during the period of study (4 hr). The shape of the curve was independent of the growth hormone preparation used. Similar disappearance curves were obtained with unlabeled HGH.MCR values calculated from constant infusion studies were 203 +/-7.8 liters/day per m(2) and values derived from single injection studies agreed closely with this. The multiexponential nature of the disappearance curve does not permit meaningful calculation of volume of distribution or half-time of disappearance.

SUPPRESSION OF ŒSTRADIOL SECRETION AND LUTEINISING-HORMONE RELEASE DURING ŒSTROGEN-PROGESTAGEN ORAL CONTRACEPTIVE THERAPY

Abstract Plasma-œstradiol and luteinising-hormone levels were measured in women during the normal menstrual cycle and during combined oral contraceptive treatment. During normal cycles, the plasma levels of cestradiol consistently reached peak values and began to fall immediately prior to the peak of plasma-luteinising hormone. This finding, together with the known action of oestrogen on stimulation of luteinising-hormone secretion, indicates that feedback from the ovary via œstradiol secretion is an essential component of the physiological mechanism for cyclic luteinising-hormone release and

Aspects of radio-immunoassay

The development of radio-immunoassay has allowed extensive investigation of the circulating levels of peptide hormones, particularly the pituitary hormones. The method is both sensitive and specific and measurement down to the picogram range is possible under ideal conditions. All radio-immunoassay procedures are based on the competition between labelled and unlabelled forms of the antigen to be measured for a limited amount of specific antibody. Several types of separation procedure are then used to separate the antibody-bound and the free forms of labelled tracer antigen. These methods can be classified as : differential migration, precipitation of bound tracer, and adsorption of free tracer. Each class of procedure has certain advantages relating to simplicity, sensitivity and precision of the assay. Once the separation of bound and free tracer has been achieved, the antigen mass can be determined from a standard curve simultaneously performed. Many current radio-immunoassay procedures operate satisfactorily over the range 0.5-10.0 nanogram/ml. in plasma, while some are more sensitive. The development of a simple and inexpensive procedure for the estimation of certain protein hormones in plasma has been achieved by coupling antibody to polymer in the form of powder, discs and tubes. This solid-phase method is suitable for the routine estimation of pituitary hormones and other antigens in blood, and allows plasma levels to be estimated in a total period of one to two days.