Radioimmunoassay of ACTH in plasma

Techniques are described in detail for a radioimmunoassay of plasma adrenocorticotropin (ACTH) that is capable of detecting hormone in unextracted normal human plasma at 1:5 dilution under the conditions described. The sensitivity of the assay is at the level of 1 mumug/ml (equivalent to 0.014 mU/100 ml).In normal subjects ACTH concentrations averaged 22 mumug/ml (equivalent to 0.308 mU/100 ml) plasma at 8-10 a.m. In a smaller group the concentrations averaged 9.6 mumug/ml (equivalent to 0.134 mU/100 ml) at 10-11 p.m. Although a circadian rhythm in normal subjects was not always well marked throughout the daytime hours, plasma ACTH usually fell to its lowest value in the late evening. In hospital patients who were not acutely ill, concentrations were infrequently above 100 mumug/ml in the morning and usually fell to significantly lower levels in the late evening. Severely ill hospital patients occasionally exhibited a.m. concentrations above 200 mumug/ml.In a group of subjects showing frequent spiking of plasma 17-OHCS concentrations throughout the day parallel spiking of plasma ACTH as well was generally observed.Metyrapone produced marked increases in plasma ACTH within 24 hr in all cases and generally within 3-6 hr except when started late in the day. Dexamethasone brought about a persistent reduction in plasma ACTH in a patient under continued treatment with metyrapone.Hypoglycemia, electroshock, surgery under general anesthesia, histalog and vasopressin administration were usually followed by significant increases in plasma ACTH concentration. Prior administration of dexamethasone blocked the response to hypoglycemia.Marked elevations in plasma ACTH were observed in patients with adrenal insufficiency off steroid therapy, in Cushing's disease after adrenalectomy even in the presence of persistent hypercortisolemia, and in some untreated patients with Cushing's disease.Umbilical cord blood contained higher plasma ACTH concentrations than maternal blood at delivery in seven of eight cases.After suppression of ACTH secretion by dexamethasone or cortisol. ACTH disappeared from plasma with half-times ranging from 22 min to 30 min in three cases studied.     

Radioimmunoassay of secretin in plasma

A sensitive and specific radioimmunoassay for secretin has been developed. 125I synthetic porcine secretin was used as label. Antibodies were raised against synthetic pocrine secretin. Pure natural porcine secretin was used as standard and free and bound hormone was separated by plasma-coated charcoal. Nonspecific interference with the assay system by plasma factors was abolished by extraction of plasma samples. The detection limit was 1.3 pmol/l. Within and between assay reproducibility (five triplicate determinations in each of eight assays) was 0.7 and 0.9 pmol/l respectivel (1 SD) at a concentration of 4.7 pmol/1. Fasting level in seventy normal persons ranged from less than 1.3 to 5.3 pmol/1. High concentrations were found in two patients with Zollinger-Ellison syndrome. In six normal persons intraduodenal acidification was immediately followed by an increase in secretin concentrations in peripheral venous blood to 7.7-23.6 pmol/1 (range), with a median of 15.5 pmol/1. In six anaesthetized pigs a fifty-fold increase in portal plasma secretin concentration after duodenal acidification was followed by a thirty-fold increased in the flow rate of pancreatic juice.     

Radioimmunoassay of cholecystokinin in plasma

A sensitive and specific radioimmunoassay for cholecystokinin (CCK) has been developed. Synthetic unsulfated carboxy-terminal fragment, CCK-8, was radioiodinated by the conventional Chloramine-T method. Antibodies were raised against sulfated CCK-8 covalently coupled to bovine thyroglobulin via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. By purification, highly immunoreactive 125I-labeled CCK-8 was obtained. The antiserum was highly avid, and plasma could be assayed directly. The detection limit of the assay was 5 pmol of sulfated CCK-8 per liter. The assay measured fragments CCK-8, CCK-33, and CCK-39 with equimolar potency. CCK-4, gastrin, and vasoactive intestinal polypeptide were not detected, even at higher concentrations. The concentration of CCK, as the sum of these CCK peptides, in plasma during fasting was low (10.5 +/- 2.1 pmol/L, mean +/- SEM) but still detectable in all normal subjects examined (range, 6.4-20.1 pmol/L). After ingestion of a test meal, CCK in plasma increased rapidly, peaking at 41.3 (SEM 5.7) pmol/L at 40 min and remaining high for 3 h after the meal. This supports the concept that CCK has important roles in digestion and absorption.     

Radioimmunoassay of somatostatin in human plasma

A radioimmunoassay procedure for determination of somatostatin immunoactivity in human plasma is described. Labelling of tyrosine-1-somatostatin is performed by chloramine T. Specific activity of the tracer is 1.34 mol iodine for 1 mol of somatostatin. The immunoreactivity is 85%. Antisera are produced in rabbits at a titre of 179 X 10(-12) mol somatostatin per ml antiserum. Human plasma is extracted by n-butanol/2 mol/l acetic acid. Recovery rate is 85-105%. The intra-assay CV varies from 12% to 1.7%, the inter-assay CV from 16% to 7.5%, depending on the concentration of somatostatin.     

Radioimmunoassay of oestriol in pregnancy plasma

A method for the estimation of oestriol in pregnancy plasma is described. The principal steps of the method are addition of internal standard, precipitation of proteins, acid hydrolysis, solvent partition and estimation by radioimmunoassay using an oestriol antiserum. The radioimmunoassay is based on the precipitation of the “bound” tritiated oestriol with ammonium sulphate. Details of the precision, accuracy, sensitivity and specificity of the method are presented.     

Radioimmunoassay of vasopressin in unextracted plasma.

A radioimmunoassay (RIA) for arg8-vasopressin (AVP) in unextracted human plasma was based on a sensitive anti-AVP rabbit antiserum, inhibition of enzymatic damage to [125I]AVP and AVP, and the use of an individual plasma blank, to correct for interference of plasma factors with the RIA. Sensitivity was 0.4 pg of synthetic AVP detected, corresponding to 1.2 pg/ml of AVP in human plasma. Recovery of AVP added to pooled plasma was 94 +/- 9.3% (mean +/- S.D.) in the low range (AVP, 2.8 pg/ml added) and 106 +/- 11.7% in the high range (45.0 pg/ml added). In 26 healthy, ambulatory subjects on ad lib, water intake, plasma AVP concentration was 2.0 +/- 1.22 pg/ml in the supine position and in 28 healthy subjects, 6.2 +/- 4.3 pg/ml in the upright position. Water loading suppressed the plasma AVP concentration. Smoking caused increased plasma AVP in 3 subjects despite water loading.     

Radioimmunoassay of thyroxine-binding globulin in human plasma

A highly specific and precise radioimmunoassay for thyroxine-binding globulin (TBG) has been developed. Crossreactivity with albumin and prealbumin was excluded. The normal range in young adults was 0.97 mg/100 ml. In childhood TBG was elevated (1.34 mg/100 ml) and also in old age (1.28 mg/100 ml). In normals and in childhood there was a good correlation of TBG with thyroxine (T₄). T₄ did not correlate with TBG with regard to age. Triiodothyronine (T₃) did not correlate with TBG in any group.T₄and T₃ exhibited a progressive decrease with age. No correlation between age and TBG was found. In mild thyrotoxicosis (T₃ : 4.5 ng/ml, with normal T₄ and negative TRH-test) TBG was slightly increased (1.20 mg/100 ml), whereas in more severe hyperthyroidism (T₃ : 6.4 ng/ml, T₄ : 16.3 Mg/100 ml) TBG concentration was not significantly different from normals. In hypothyroidism TBG was elevated (1.26 mg/100 ml).The conclusion from these data is that TBG does not follow the progressive decrease of T₄ and T₃ with age. Thus, age-dependent euthyroid ranges for thyroid hormone concentration, including TBG concentration, must be established for better clinical discrimination of thyroid function. Possible dependence of TBG on the nature of thyroid hormone concentration must be considered in the production and peripheral kinetics of thyroid hormones.     

Radioimmunoassay of human plasma retinol-binding protein

A radioimmunoassay for human plasma retinol-binding protein (RBP) has been developed utilizing a double antibody precipitation technique. RBP was purified 1500- to 2000-fold by procedures described previously. A specific anti-human RBP antiserum was prepared in rabbits by three once-weekly injections of purified RBP emulsified with Freund's adjuvant. RBP was iodinated with (131)I and the RBP-(131)I was purified by gel filtration on Sephadex G-100 after complex formation with human plasma prealbumin. The RBP-(131)I was completely (> 95%) immunoprecipitable in the presence of an excess of specific antiserum, it was not (< 5%) immunoprecipitable in the absence of specific antiserum, and it could be completely displaced from antibody by excess unlabeled RBP. The standard curve obtained in the immunoassay with normal plasma was identical to that with pure RBP. Duplicate samples differed from their mean by 5 +/-5% (+/-SD). There was a quantitative recovery of pure RBP added in varying amounts to normal plasma. The immunoassay accurately measured RBP in amounts of 10-100 ng per assay tube. There was no significant difference in the immunoreactivity of apo-RBP as compared to holo-RBP. The mean plasma values (+/-SEM) for a group of 76 normal subjects were 47.2 +/-1.6 mug/ml for males and 41.6 +/-1.6 mug/ml for females. Plasma RBP levels were markedly depressed (15 +/-2.3 mug/ml) in 14 patients with acute viral hepatitis. There was a highly significant correlation between the plasma levels of RBP and of vitamin A in both normal subjects and patients with hepatitis. In all subjects plasma RBP was generally saturated with retinol. The data suggest that under normal circumstances RBP circulates almost exclusively as the holoprotein.     

Radioimmunoassay of plasma renin activity.

We describe a sensitive, simplified radioimmunoassay method for determination of plasma renin activity. Plasma was acidified to the optimal pH (6.0) of angiotensin l generation with the least possible dilution, by using a single addition of hydrochloric acid and the enzyme inhibitor hydroxyquinoline. Recovery of unlabeled angiotensin l added to plasma was 92-97%; that of monoiodinated angiotensin l exceeded 90%, indicating satisfactory protection from proteolytic enzymes. Plasma constituents interfered little with the radioimmunoassay. Bland values for plasma kept at 0 degrees C were 10.7 +/- 2.3 (mean +/- SD) percent of the activity values for samples kept at +37 degrees C (n equals 63). In the routine setting, 6.25 pg of angiotensin l or 10(-6) Goldblatt units of Standard Human Renin was detected. We report results of plasma renin activity measurements and a comparison with seven renin kits, and with bioassay for plasma renin activity.     

Radioimmunoassay of atrial natriuretic peptides in human plasma

The concentration of atrial natriuretic peptide (hANP) in plasma from venous blood of healthy subjects was measured by radioimmunoassay. hANP from 5 mL of EDTA-treated plasma was adsorbed onto Sep-Pak C18 cartridges, which were eluted with methanol/trifluoroacetic acid (5 mL/L), 90/10 by volume. The eluates were concentrated by evaporation under nitrogen and lyophilized. After redissolving the samples in 0.5 mL of sodium phosphate buffer, we incubated 100-microL aliquots with anti-alpha-hANP for 24 h, then added 125I-labeled alpha-hANP tracer; 24 h later, we separated the bound and free fraction by adding an antibody/polyethylene glycol complex as the second antibody. The sensitivity of the assay was 2 pg per tube (B0-3 SEM). In the useful range of B = 15 to 85% of B0, CVs for within-run and between-run precision did not exceed 8 and 12%, respectively. The 50% intercept of the standard curve was at 12 pg per tube. hANP concentrations for 36 healthy adults ranged from 8 to 68 ng/L.     

Radioimmunoassay of angiotensin II in unextracted plasma.

A reliable, direct radioimmunoassay of plasma angiotensin II (PAII) is described. No cross-reactivity of human renin, sheep renin substrate, saralasin acetate and angiotensin I with the anti-angiotensin II antiserum was found. However, des-¹Asp-angiotensin II has a stronger binding affinity for the antiserum than angiotensin II. A significant correlation was found between PAII determined in plasma samples without and with extraction on a Dowex 50W-X₂ resin.The combination of EDTA and o-phenanthroline has been found to be a very efficient angiotensinase inhibitor in plasma and is of equal potency as EDTA and diisopropylfluorophosphate in inhibiting the angiotensinase and converting enzyme activity. A significant correlation was found between PAII measurements performed in plasma samples in the presence of both inhibitor solutions.The intra-assay variations were checked by multiple analysis of a plasma pool and the inter-assay variations by measuring the angiotensin II concentration in different plasma samples on two occasions.The mean PAII in 10 healthy, male subjects on an ad libitum diet was 25.1 ± 13.5 (S.D.) pg/ml (range: 10.1 to 51.4). PAII correlated also better with plasma renin activity (active renin) than with plasma renin concentrations, measured after acidification of plasma (total renin).     

Radioimmunoassay of human plasma lecithin-cholesterol acyltransferase.

A sensitive and precise competitive-displacement double-antibody radioimmunoassay was developed for the human plasma enzyme lecithin-cholesterol acyltransferase (LCAT; Ec 2.3 1.43). The ability of plasma from various animal species to displace labeled human LCAT from goat anti-human LCAT could be ranked in the following order: man and sheep > nonhuman primates > cat or dog > pig > rabbit or guinea pig > mouse > rat. Normolipidemic subjects had levels of LCAT of 6.14 +/- 0.98 micrograms/ml (mean +/- SD, n = 66). Subjects with dysbeta-lipoproteinemia had the highest plasma LCAT levels (7.88 +/- 0.39 micrograms/ml, n = 7, P < 0.05), followed by hypercholesterolemic subjects (7.00 +/- 1.30, n = 41) and hypertriglyceridemic subjects (6.96 +/- 1.3, n = 10). LCAT-deficient subjects had the lowest enzyme levels (0.89, 0.83, and 0.05 micrograms/ml, respectively, and two subjects with no detectable enzyme). Males had lower LCAT levels (6.42 +/- 1.05 micrograms/ml, n = 90, for all subjects; 5.99 +/- 1.03, n = 44, for normolipidemics) than females (7.01 +/- 1.14, n = 34, for all subjects P < 0.01; 6.44 +/- 0.79, n = 22, for normolipidemics, P < 0.01). LCAT levels correlated significantly with total cholesterol (males, r = 0.384, P < 0.001; females, r = 0.519, P < 0.002); and total triglyceride (only in females, r = 0.512, P < 0.002). LCAT levels in females correlated inversely with HDL cholesterol (r = 0.341, P < 0.05) and apoprotein D (r = 0.443, P < 0.02), but no such relationship existed in males.     

Radioimmunoassay of atrial natriuretic polypeptide in heat-treated human plasma

In this simple, sensitive radioimmunoassay (RIA) of atrial natriuretic polypeptide (hANP) in human plasma, nonspecific interference is minimized by deproteinizing the plasma by heat treatment at 85 degrees C for 10 min. We directly measure alpha-hANP in the supernates by RIA, with use of antiserum that recognizes the N-terminal region of alpha-hANP. The minimal detectable value was 0.4 pg per tube. The intra-assay CV was 6.6% (n = 8). The mean concentration of hANP in plasma of 54 healthy volunteers was 41 (SD 29) ng/L. Concentrations of hANP in plasma increased after saline infusion and high salt intake for one week in patients with essential hypertension. High concentrations were also measured in patients with renal failure and congestive heart failure. This method, which requires no extraction or purification with column chromatography, is especially useful for simultaneous measurement of several samples.