Radioimmunoassay specific for amino (N) and carboxyl (C) terminal portion of parathyroid hormone.

A radioimmunoassay specific for the amino (N) terminal portion of the parathyroid hormone (PTH) molecule (N-PTH radioimmunoassay) has been developed by iodinating synthetic 1-34bovine PTH (1-34bPTH) and using commercially available bPTH antiserum. A radioimmunoassay specific for the carboxyl (C) terminal (C-PTH radioimmunoassay) has been carried out by adding enough amount of 1-34bPTH to the PTH radioimmunoassay system. The data obtained from N- and C-PTH radioimmunoassay were compared with those obtained from the PTH radioimmunoassay. It was observed that plasma levels of N-PTH, indicating biologically active PTH, were only one 8th to 32th to those of PTH and those of C-PTH were almost equal to those of PTH. These data corresponded well with those reported previously by using the antiserum specific for each terminal of the PTH molecule from the other laboratory. The half life of plasma N-PTH and C-PTH determined following the removal of parathyroid adenoma was less than 10 min and about 45 min respectively. These data indicate that the N-PTH radioimmunoassay can be done by iodinating 1-34bPTH and using commercially available antiserum.     

Pharmacokinetics of levonorgestrel in 18 women after 1 month of treatment with a triphasic oral contraceptive.

A triphasic levonorgestrel (LNG)- and ethinylestradiol-containing oral contraceptive was administered to 18 women. Plasma samples were obtained throughout a treatment cycle just before drug administration and on the last treatment day (day 21), several plasma samples were collected from each individual up to 48 h postadministration. LNG was determined by radioimmunoassay in all plasma samples. In addition, the concentration of sex-hormone-binding globulin (SHBG) was determined in plasma samples collected from the same subjects during treatment, as well as during a pre- and a posttreatment cycle. During the treatment cycle, plasma levels of LNG determined just before drug administration increased and reached steady state at about day 16. This increase was due to an increased dose of LNG according to the triphasic dose regimen, a concomitantly ethinylestradiol-induced increase in SHBG and due to pharmacokinetic accumulation, since LNG had a terminal half-life of approximately 28.5 h and the dosing interval was 24 h. Steady-state levels and pharmacokinetic parameters of LNG determined on the last day of treatment were in good accordance with previously published results.     

Plasma immunoreactive calcitonin in lung cancer.

We have measured plasma calcitonin in 135 untreated eucalemic men with lung cancer and a control/smoker population. Calcitonin levels were determined by radioimmunoassay and validated by immunoextraction. Plasma immunoreactive calcitonin moieties were purified by immunoadsorbent chromatography, treated with mercaptoethanol and urea, and characterized by gel filtration. Artifacts in human calcitonin radioimmunoassays of cancer-patient plasmas were detected by parallel plasma incubations in a salmon calcitonin radioimmunoassay system which does not detect human calcitonin and by immunoprecipitation of tracer at the end of radioimmunoassay incubations. Heating fresh plasmas to 65 degrees C for 1.5 hours reduced radioimmunoassay artifacts without loss of calcitonin moieties. Such characterization of hypercalcitoninemia in each of the histopathological types of lung cancer has raised some important questions about the interpretation of plasma calcitonin radioimmunoassay measurements in lung cancer. Based on inhibition of tracer-antibody binding, plasma calcitonin seemed to be elevated in 18% (14/80) of basal plasma samples obtained from patients with epidermoid or with anaplastic lung cancer. Unequivocal hypercalcitoninemia (heat stable, causing no inhibition of antibody-tracer binding in the salmon calcitonin radioimmunoassays, and immunoextractable with human calcitonin antibodies) was not found in any of the apparently hypercalcitoninemic plasmas from persons with epidermoid or anaplastic lung cancer. By contrast, unequivocal hypercalcitoninemia was found in 27% (15/55) of plasmas from patients with small cell carcinoma or adenocarcinoma. Most of the immunoreactive calcitonin recovered from small cell and adenocarcinoma lung cancer plasmas with unequivocally elevated calcitonin is much larger than calcitonin monomer.     

Detection and quantitation of low density lipoprotein (LDL) receptors in human liver by ligand blotting, immunoblotting, and radioimmunoassay. LDL receptor protein content is correlated with plasma LDL cholesterol concentration

Low density lipoprotein (LDL) receptor activity has been detected and identified in human liver samples by ligand blotting with biotinylated lipoproteins and by immunoblotting with a monoclonal antibody raised against the bovine adrenal LDL receptor. The molecular weight of the human liver LDL receptor, approximately 132,000 on nonreduced polyacrylamide gels, is identical to that of LDL receptors detected in normal human skin fibroblasts by the same methods. LDL receptor-dependent binding activity in human liver samples has been semi-quantitated by integrating the areas under the peaks after scanning photographs of ligand blots, and receptor protein determined by radioimmunoassay with purified bovine adrenal LDL receptor protein as the standard. There was a highly significant correlation between the values obtained by each method for seven different liver samples (r = 0.948). The LDL receptor protein content of liver membranes from 10 subjects as determined by radioimmunoassay was inversely related to the plasma LDL cholesterol concentration (r = 0.663, p = 0.05) but not to other plasma lipid values, including total plasma cholesterol, high density lipoprotein cholesterol, or plasma triglyceride concentrations.     

Radioimmunoassay of nafarelin ([ 6-(3-(2-naphthyl)-D-alanine)]-luteinizing hormone-releasing hormone) in plasma or serum

A procedure which is suitable for the radioimmunoassay (RIA) of nafarelin [( 6-(3-(2-naphthyl)-D-alanine)]-luteinizing hormone-releasing hormone) in plasma or serum at concentrations as low as 50 pg/ml is described. Antiserum was prepared by replacing the pyroglutamyl portion of nafarelin with glutaric acid, coupling the product to keyhole limpet hemocyanin, and immunizing rabbits with the resulting conjugate. At a dilution of 1:30,000 the binding was approximately 50%. The antibodies did not cross react with luteinizing hormone-releasing hormone. For RIA, 125I-labeled analyte was used as the tracer and charcoal was used to separate the free and the bound fractions. No purification of samples was required prior to RIA. Accuracy of the method was assessed by adding known quantities of nafarelin to nafarelin-free plasma and determining the ratio of measured to added analyte. Linear regression analysis for the concentration range 0.050-5.00 ng/ml yielded a regression equation of y = 1.01x - 0.066 and a correlation coefficient of 0.997. At 0.050 ng/ml the CV was 11.3% (interassay). Additional validation was obtained from an in vivo study in which [3H]nafarelin was administered to monkeys and plasma profiles were determined by RIA, by high-performance liquid chromatography (HPLC), and by an HPLC-radiochemical method. The results obtained by RIA agreed well with those obtained by the HPLC methods.     

A non-chromatographic radioimmunoassay for plasma aldosterone

A non-Chromatographic radioimmunoassay has been developed for determining plasma aldosterone. Endogenous plasma proteins were used to bind corticosteroids while aldosterone was adsorbed to fuller's earth in an initial purification step. An antiserum to an aldosteronebovine serum albumin conjugate was used for a second purification step. Finally, radioimmunoassay of the purified material was performed using an antiserum prepared against a different aldosterone-bovine serum albumin conjugate. Two ml plasma samples were used for duplicate determinations. Accuracy and precision were satisfactory throughout the analytic range of the method (2.5 to 50 ng/100 ml). Comparison of results using this method with those obtained using an established radioimmunoassay containing a chromatographic purification step indicates that there is a small but tolerable degree of non-specific interference. Twenty-four samples can be assayed in 1 1/2 working days.     

Radioimmunoassay of human plasma trypsin.

A radioimmunoassay has been developed for the determination of human trypsin (3.4.21.4) in plasma. It allows the measurement of trypsin concentration in spite of the presence of plasma or pancreatic inhibitors. The human trypsin used as a standard and for labelling was isolated from pancreatic tissue and purified by affinity chromatography. The antiserum was obtained from guinea-pigs immunized with partially purified human trypsin. In the radioimmunoassay, the values of trypsin in serial dilutions of plasma were parallel to those of the standard curves. The assay was shown to be reproducible, sensitive and specific. However, the two antisera used did not distinguish between the enzyme and its proenzyme. In normal subjects, plasma values were found to be around 400 ng/ml. They were 10-40 times higher in patients with acute pancreatitis. The method appears to be much more specific for the diagnosis of acute pancreatitis than the current determinations of amylase and lipase activity.     

A refined blood collection method for quantifying corticosterone

In many rodent laboratories, blood samples are collected from rats using the tail vein nick procedure and analyzed to quantify blood corticosterone levels as an indicator of stress. The standard method of corticosterone quantification often requires the collection of a relatively large volume of blood, followed by the extraction of the blood plasma. An alternative blood sampling method requires the collection of only a drop of blood on paper (the 'drop' method), minimizing handling of the animals, and does not require plasma extraction. The authors aimed to validate the drop method of blood sampling for use in corticosterone quantification. They induced stress in rats by cerebral ischemia, collected blood samples at various intervals using both the drop method and the plasma extraction method and then quantified corticosterone by radioimmunoassay. Corticosterone levels of the ischemic rats were compared with those of sham-operated rats and those of ischemic rats that had been given metyrapone, a glucocorticoid synthesis inhibitor, prior to vessel occlusion. Blood corticosterone levels in the samples obtained from the same animal using the two different methods were highly correlated for all rats. The authors further provide a regression model that can be used to predict plasma corticosterone values from those obtained from the drop blood samples. Quantification of corticosterone from only a small drop of blood has many practical and ethical advantages and should be considered as an alternative to standard methods.     

Melanin concentrating hormone. IV. Development of a sensitive solid-phase radioimmunoassay for melanin-concentrating hormone

A two-step solid-phase radioimmunoassay for melanin-concentrating hormone (MCH) was developed for direct determination of the hormone in plasma samples. To this end, synthetic MCH was coupled to bovine thyreoglobulin and the complex was injected into rabbits. Specific antisera of high titer were obtained which did not crossreact with other hormones. The IgGs were chemically linked to immunobeads, an acrylamide/acrylic acid polymer matrix. In the first step, plasma MCH was immunoextracted by incubation of diluted plasma samples with anti-MCH immunobeads. In the second step, the washed polymer was incubated with radioiodinated MCH tracer for titration of non-occupied sites. This procedure made it possible to determine as little as 4 pg MCH per ml of plasma. Application of the radioimmunoassay to plasma levels of black or white background-adapted trout showed a marked difference in circulating MCH: while trout on a black background contained a mean value of 29 +/- 5.6 pg/ml, animals on a white background had 106 +/- 19 pg/ml. These findings strengthen the hypothesis that MCH is directly involved in the control of color change of teleost fishes. By contrast, there was no detectable salmonid MCH immunoreactivity in rat or human plasma.     

Human platelet-derived growth factor: radioimmunoassay and discovery of a specific plasma-binding protein

The platelet-derived growth factor (PDGF) is the principal mitogen in serum for cultured cells of mesenchymal origin. PDGF also is a potent chemotactic protein for inflammatory cells and for cells required for wound repair. Because activity levels of PDGF in biological fluids are difficult to measure, we attempted to develop a radioimmunoassay for PDGF. Rabbits were immunized with purified PDGF; the antiserum obtained was monospecific for PDGF in immunodiffusion analysis against concentrated platelet lysates, serum, and plasma. A radioimmunoassay for PDGF was developed with a sensitivity of congruent to 0.2 ng/ml. Levels of PDGF in plasma/serum were measured and compared with PDGF levels determined by a receptor-competition assay and by a standard biological assay measuring incorporation of [3H]thymidine into 3T3 cells. Radioimmunoassay showed apparent PDGF levels of 50 ng/ml in human plasma and 103 ng/ml in serum. The 50 ng/ml PDGF in plasma was unexpected because the plasma samples contained little or no platelet release products as determined by very low levels of platelet factor 4. We therefore sought an immunologically reactive PDGF molecule in human plasma. No immunologically reactive protein was detected by immunodiffusion analysis or when plasma was treated with an immunoaffinity gel. Subsequently, a 125I-PDGF-binding protein was identified; the 125I-PDGF-plasma-binding protein complex was not reactive with anti-PDGF immunoglobulin. Correction for 125I-PDGF bound by the plasma-binding protein established serum levels of PDGF of congruent to 50 ng/ml; congruent to 50 ng/ml PDGF was found in serum by radioreceptor-competition assays and by mitogenic assays as well. The plasma-binding protein may serve to clear PDGF released in the circulation, thereby limiting PDGF activity to its local interactions at the site of blood-vessel injury.     

The development and application of a direct radioimmunoassay for corticosterone

A direct, simple and highly specific radioimmunoassay for corticosterone has been developed. The assay does not require preliminary solvent extraction of the sample or any chromatographic step. The assay utilises a highly specific antibody raised in rabbits against corticosterone-3-(0-carboxymethyl)-oxime-BSA immunogen and gamma-labeled corticosterone of high specific activity. An excellent correlation was obtained between results of the direct assay and those measured after paper chromatography (r=0.99, P<0.001). The coefficients of variation for intra-assay and inter-assay determinations of samples from normal and high plasma pools were 4.6–6.2% and 6.4–8.2% respectively. The minimum limit of detection was 5 pg/assay tube (0.1ng/ml). The assay has been applied to assess plasma corticosterone levels in various physiological and pathophysiological studies. It is extremely practical to the extent that a single technician can assay up to 1000 samples in a working week. Finally, the direct assay has been validated and employed for in vitro adrenal superfusion studies using either rat or human adrenal cells. The large numbers of samples produced by these studies would have exceeded the capacity of earlier radioimmunoassays requiring initial extraction and chromatography.     

Evaluation of an on-farm blood progesterone test for predicting the day of parturition in cattle

An on-farm blood progesterone enzymeimmunoassay (EIA) was evaluated as a diagnostic test to predict the time of calving within a 24-hour period in near-term dairy cows. Blood samples were taken daily from 45 cows beginning 5 days prior to their expected due dates until calving, and plasma was stored at -20 degrees C until all cows had calved. The EIA test was performed on frozen-thawed plasma samples, and progesterone concentrations were determined to be low (positive test for calving within 24 hours) or high (negative test for calving within 24 hours). Sensitivity, specificity and predictive value of the EIA to accurately determine parturition within 24 hours were 86.7, 90.8 and 75.0%, respectively. The EIA correctly predicted the day of parturition in 168 of 187 (89.8%) plasma samples. Ten additional cows were similarly monitored except the EIA was performed on whole blood immediately after collection, and the sensitivity, specificity and predictive value of the test were 80.0, 97.6 and 88.9%, respectively. The day of parturition was correctly predicted in 49 of 52 (94.2%) whole blood samples. More than 95% of the cows calved within 24 hours when their plasma progesterone reached < 1.3 ng/ml. When results of the EIA were compared with those of a radioimmunoassay (RIA), the EIA findings were used to correctly classify 190 of 232 (81.9%) plasma samples as having low (< 2.0 ng/ml) or high (>/= 2.0 ng/ml) concentrations of progesterone. The EIA test was found to be a quick, practical means of estimating progesterone concentrations in bovine plasma or whole blood and was a useful test for predicting the day of parturition in cows.     

Progesterone determination in Iberian red deer by time-resolved fluorometry: an alternative method to RIA

The validation for Iberian red deer of a commercially available Time-resolved fluoroimmunoassay (TR-FIA) designed for analysis of progesterone in human beings was carried out. Intra-assay coefficients of variation ranged from 3.6% to 7.4%, while inter-assay coefficients of variation varied from 5.2% to 15.5%. Accuracy, evaluated by comparing results yielded by TR-FIA with those obtained from a validated radioimmunoassay (RIA) in the measurement of 14 samples, provided a high regression coefficient (R(2)= 0.93). Different progesterone concentrations added to pool plasma showed percentages of recovery that ranged between 102.6% and 82.48%. The limit of detection was 0.102 nmol/L. The results obtained indicate that the present method is suitable for the measurement of progesterone in female Iberian red deer.     

Radioimmunoassay for neopterin in body fluids and tissues

Specific antibodies against D-erythroneopterin have been prepared in rabbits using a conjugate of D-erythroneopterin to bovine serum albumin (D-erythroneopterinylcaproyl-bovine serum albumin). The antiserum distinguished D-erythroneopterin from other pteridines, i.e., three stereoisomers of neopterin, L-erythrobiopterin, folic acid, xanthopterin, and four other synthetic pteridines. Using this specific antiserum, a radioimmunoassay for D-erythroneopterin has been developed to measure the neopterin concentrations in urine and tissues. The conjugate of D-erythroneopterin with tyramine (NP-Tyra) was synthesized and labeled with 125I as the labeled ligand NP-[125I]tyra for the radioimmunoassay. The minimal detectable amount of neopterin was about 0.1 pmol. The concentration of total neopterin (neopterin, 7,8-dihydroneopterin, quinonoid dihydroneopterin, and tetrahydroneopterin) in the biological samples was obtained by iodine oxidation under acidic conditions prior to the radioimmunoassay, and that of neopterin plus 7,8-dihydroneopterin by oxidation under alkaline conditions. Total neopterin values in human urine obtained by this new radioimmunoassay showed a good agreement with those obtained by high-performance liquid chromatography with fluorescence detection. With rat tissue samples which contained very low concentrations of neopterin as compared to biopterin, biopterin was simultaneously determined by our previously reported radioimmunoassay, and neopterin values were corrected for the cross-reactivity (0.1%). The neopterin concentrations obtained by this method agreed with the values obtained by the radioimmunoassays for neopterin and biopterin after their separation by high-performance liquid chromatography. This very small amount of neopterin, as compared with biopterin, in rat tissues could not be determined by high-performance liquid chromatography-fluorometry alone due to the masking of the neopterin peak by a large biopterin peak.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasma and pituitary concentrations of LH, FSH and prolactin in aged female C57BL/6 mice

Plasma and pituitary concentrations of LH, FSH and prolactin were determined by radioimmunoassay in 2-month-old (young) and 16-20-month-old (old) C56BL/6 mice. There were no statistical differences in hormonal levels between aged females in oestrus (those exhibiting a copulatory plug) and those in constant dioestrus. In the old females plasma levels of LH (P < 0.002) and FSH (P < 0.001) were significantly elevated, while levels of prolactin (P < 0.001) were significantly depressed when compared with those from young animals. Pituitary homogenates from old females also contained more gonadotrophins (P < 0.001) and less prolactin (P < 0.001) than those of the young females. A radioreceptor assay utilizing a plasma membrane of luteinized rat or mouse ovaries indicated that LH from 2-month-old animals bound better to ovarian receptors (P < 0.05) than did LH from old mice, although radioimmunoassay of the same samples gave higher (P < 0.01) plasma LH levels for the old mice. Since the radioreceptor assay is considered to be a more sensitive test for biologically active LH, the results from these two types of assays suggest that there may be an alteration in the mouse LH molecule with age.     

Development of a sensitive enzymeimmunoassay (EIA) for progesterone determination in unextracted bovine plasma using the second antibody technique

A simple direct enzymeimmunoassay (EIA) on microtiter plates for plasma progesterone using the second antibody coating technique and horseradish peroxidase (HRP) as the enzyme label (EIA-HRP) is described and compared with an identical EIA procedure which employed alkaline phosphatase (AP) as the enzyme label (EIA-AP). The assays used antiserum raised against progesterone-7-carboxyethlthioether-BSA in rabbits. Both systems were further compared with the conventional direct progesterone radioimmunoassay (RIA) in regular use. The enzymes HRP and AP were coupled to progesterone-6 beta-hydroxy-hemisuccinate by a mixed anhydride method. While the precision of EIA-HRP was comparable to RIA, the sensitivity in terms of the lowest detection limit obtained in EIA-HRP was about 10 times better than that seen in RIA. Progesterone estimates from plasma samples in EIA-HRP showed good correlation (r = 0.94) with the RIA values and the levels measured in the two systems were identical. Progesterone estimates from plasma samples in EIA-AP were at least three times higher than those obtained by either EIA-HRP or RIA. Thus, only the EIA-HRP but not the EIA-AP was suitable for the reliable direct measurement of progesterone in plasma.     

A specific, non-chromatographic radioimmunoassay for human plasma cortisol.

A radioimmunoassay technique has been developed for the measurement of cortisol in a single methylene chloride extract of human plasma without chromatography. The antiserum, obtained by immunizing rabbits with cortisol-3-carboxymethyl-oxime conjugated to bovine serum albumin, had a high affinity (KA = 1.8 X 10(9) 1/mole) and capacity (2.3 X 10(-6) moles/L undiluted serum) for cortisol. The minimum detectable amount determined at the lower 95% confidence limit of the buffer control tubes was 8.3 +/- 4.7 pg/tube and a log dose - logit response standard curve was linear between 20 pg and 20 ng/tube. The antiserum was highly specific for cortisol with only corticosterone, cortisone, 11-deoxycortisol and 21-deoxycortisol showing significant cross-reaction (12.4, 6.6, 3.8 and 3.7%, respectively). The cross-reaction for the other tested naturally occurring and synthetic steroids did not exceed 1%. Regression analysis of cortisol concentration estimates obtained on 20 samples before and after Sephadex LH-20 column chromatography gave a coefficient of correlation (r) of 0.995 and a regression coefficient (b) of 1.04. Recovery of cortisol added to plasma samples was quantitative. The intra-assay error was 8.5% and the inter-assay error averaged 5.7%. The method is simple requiring a single solvent extraction of plasma, therefore permitting large numbers of samples to be handled efficiently by a single technician.     

Effects of pregnancy and labor on oxytocin levels in human plasma and cerebrospinal fluid

In order to investigate the significance of oxytocin in pregnancy and labor, oxytocin concentrations in plasma and cerebrospinal fluid (CSF) were determined using the specific radioimmunoassay. Plasma and CSF samples were obtained from 23 pregnant women (11 pre labor, 12 in labor), 15 nonpregnant women and 4 men at spinal puncture for anesthesia. In males and nongravidas, CSF levels of oxytocin were significantly higher than plasma levels. Plasma levels in pregnant patients pre or in labor were significantly higher than those in nongravidas. No significant difference between CSF levels in prelabor gravidas (mean +/- SE, 9.7 +/- 1.5 mu u/ml) and nongravidas (10.1 +/- 1.2 mu u/ml) was found. However, CSF levels in gravidas in labor (18.6 +/- 2.3 micromicrons/ml were significantly higher than the levels in prelabor gravidas. These results strongly suggest that oxytocin levels in human plasma and CSF are controlled by different mechanisms and that the increased oxytocin could have some specific central actions.     

Interrelationship between plasma estradiol concentration and oxytocin-induced PGF2alpha release in heifers

The objective of this study was to determine if the increase in responsiveness to oxytocin toward the time of luteolysis was correlated with an increase in plasma estradiol in the cow. Six heifers each had a cannula placed in the jugular vein on Day 14 of the estrous cycle. Then, beginning on Day 15, growth of the largest follicles was determined by ultrasonography, and a blood sample was taken via the cannula for the measurement of progesterone and estradiol by radioimmunoassay (RIA). After the first blood sample, 3 more samples were taken at 10-min intervals, 100 IU oxytocin were injected into the vein, and a further 3 blood samples were taken at 15, 30 and 60 min after injection. The concentration of 13,14-dihydro-15-keto prostaglandin F2alpha (PGFM) was measured in these frequent samplings and was used to determine the ability of oxytocin to stimulate PGF2alpha release from the uterus. This procedure was repeated daily for at least 7 d. The results showed that the response to oxytocin increased before luteolysis and that there was a significant increase in the response to oxytocin (P<0.05) before any changes in plasma estradiol or progesterone were detected. These data show that an increase in estradiol secretion from the ovulatory follicle does not appear to initiate luteolysis.     

Determination of plasma cyclic AMP with automated radioimmunoassay system (Gamma-flo)

We have adapted the Gamma-flo automated radioimmunoassay system to analyze for cyclic 3',5'-AMP in plasma. Direct analysis of plasma samples was not possible due to an interfering substance(s) in plasma. Evidence suggested that the interfering substance is human serum albumin. An efficient, relatively simple method was developed by applying the acetylation procedure to ultrafiltrates of plasma samples and utilizing the Gamma-flo system for rapid radioimmunoassay (50 samples per hour) of cyclic AMP content in the filtrates. Coefficient of variation within assays averaged 3% and interassay coefficient of variation was 4.7%. Recovery of cyclic AMP from plasma into the ultrafiltrate ranged from 93 to 100%. Increments of cyclic AMP added to plasma were recovered linearly up to approximately 60 pmol/ml, the highest concentration tested.