A specific radioimmunoassay for PGE2 using an antibody with high specificity and a sephadex LH-20 microcolumn for the separation of prostaglandins.

Antiserum against PGE2 was raised in rabbits following immunization with prostaglandin-hen-gamma-globulin conjugate. The antiserum exhibited 14% cross reactivity with PGE1 and far less cross-reaction with heterologous prostaglandins. A microcolumn of Sephadex LH-20 was used for a partial, but sufficient separation of PGE2 from PGE1 and a complete separation from heterologous prostaglandins to ensure a specific RIA for PGE2. The precision of the method in the rage 10-500 picograms showed a coefficient of variation varying between 4 and 13%. The detection limit was 10 picograms corresponding to 15 pg/ml of PGE2 in serum. In order to demonstrate the validity of the method values obtained for non-diuretic rat renal venous serum were compared with those obtained using the isotope derivative method of Bojesen & Buckhave (1972) on the same samples. The concentrations of PGE2 obtained were 239 +/- 25 pg/ml and 250 +/- 58 pg/ml, respectively.     

Enzyme immunoassay for serum dexamethasone using 4-(carboxymethylthio)dexamethasone as a new hapten.

A sensitive and simple enzyme immunoassay for direct quantitation of serum dexamethasone was established. An antiserum with high specificity was produced by the immunization of rabbits with a newly synthesized 4-(carboxymethylthio)dexamethasone-bovine serum albumin conjugate. Alkaline phosphatase was used as a labeling enzyme. The minimum amount of dexamethasone detected was 2 pg per tube on the basis of B/Bo 100 - 2 SD (%) of standard curve. However, taking into account the cross-reaction with steroids such as cortisol in dexamethasone-free serum, the measurable range was from approximately 0.13 to 10 micrograms/dl. Intra- and interassay coefficients of variation were 1.5 - 5.4% and 0.6 - 6.5%, respectively. Serum levels of dexamethasone and cortisol in four normal subjects after an oral administration of 1 mg of dexamethasone are also reported.     

Inhibition of cortisol production in isolated guinea-pig adrenal cells

Cortisol, added to 1 ml incubation medium containing 3-4 X 10(5) isolated guinea-pig adrenal cells, provoked a decrease in basal and ACTH (250 pg)-stimulated cortisol production, in correlation with the amounts used (50 ng-2,000 ng). A decrease in aldosterone production could be seen when cortisol concentrations reached or exceeded 1,000 ng/ml. There were no variations in either androgens (delta 4-androstenedione, dehydropiandrosterone) or 17-hydroxyprogesterone. Only 11-deoxycortisol was slightly increased. Using increasing concentrations of ACTH (50-250 pg), both in the absence and in the presence of 1,000 ng cortisol, it was noted that the inhibition induced by cortisol was of a competitive type and could be overcome by ACTH. This decrease in cortisol was concomitant with an increase in 11-deoxycortisol. Neither corticosterone nor dexamethasone reduced cortisol production. In addition, it was shown that the conversion of tritiated 11-deoxycortisol to radioactive cortisol increased significantly under the influence of 250 pg ACTH (mean relative variation of 21.7% +/- 7.7 (SEM), n = 6, P less than 0.05); but decreased significantly under the combined effect of 1,000 ng exogenous cortisol and the same dose of ACTH: (mean relative variation of 4.3% +/- 1 (SEM), n = 8, P less than 0.005). There is therefore reason to believe that the concentrations of cortisol at the adrenal level modulate the stimulation induced by ACTH and that this self-adjustment forms part of the control mechanisms involved in corticosteroidogenesis.     

A highly specific heterologous enzyme-linked immunosorbent assay for measuring testosterone in plasma using antibody-coated immunoassay plates or polypropylene tubes.

A highly specific and sensitive enzyme-linked immunosorbent assay, using a heterologous combination of antiserum raised against testosterone-3-(O-carboxymethyl) oxime-bovine serum albumin and penicillinase-labeled testosterone-11 beta-carboxymethyl ether, was developed for measuring testosterone in human plasma. Immunoassay plates (96 wells) provided a sensitivity of 2.5 pg/well. This was achieved by maintaining the molar ratios of steroid to enzyme between 10 and 20. The assay was very specific for testosterone and did not show any cross-reaction with the related C19 steroids tested. Replacement of immunoassay plates with the locally available polypropylene tubes raised the detection limits to 25 pg/tube, but improved the range of doses of testosterone that could be measured up to 10,000 pg. The antiserum to testosterone derivative was linked to both immunoassay plates and polypropylene tubes through immunochemical bridges. Comparison of testosterone values of 52 plasma specimens obtained by both solid phase methods with those of radioimmunoassay showed good correlation.     

Affinity chromatography using competitive elution separates polyclonal glucocorticoid antisera into fractions of varying cross-reactivity

Affinity chromatography of glucocorticoid antisera using cross-reacting steroid-Sepharose columns and competitive elution with the immunising steroid has allowed the separation of polyclonal antibodies into fractions of varying cross-reactivity. Elution was at neutral pH in the presence of 20% acetonitrile followed by dissociation of the eluted immunoglobulin-steroid complex, by dialysis. A polyclonal cortisol antibody with an initial 70% cross-reactivity to 11-deoxycortisol yielded a fraction with 10% cross reactivity and improved affinity. This fraction was suitable for determining plasma cortisol on patients undergoing the metyrapone test whereas another fraction of similar affinity but higher cross reactivity to 11-deoxycortisol, as well as the intact antiserum, grossly over-estimated plasma cortisol on these patients. This technique should permit the use of antibody fractions for immunoassay when the intact antiserum may be unsatisfactory due to lack of specificity.     

A simple radioimmunoassay for plasma cortisol and 11-deoxycortisol (17, 21-dihydroxy-4-pregnene-3, 20-dione).

A simple procedure for the measurement of cortisol and 11-deoxycortisol in plasma or serum is described. One-half ml. of plasma is extracted with methylene chloride. Separation of cortisol and deoxycortisol is achieved by a Sephadex LH-20 mini-column. The assay itself is achieved by the use of a commercially available cortisol kit employing rabbit anti-cortisol coated tubes. This antibody exhibits a 20% crossreactivity with 11-deoxycortisol. This procedure is extremely useful in the assessment of a pituitary-adrenal reserve in the Metyrapone test.     

Properties of antisera to progesterone and to 17-hydroxy-progesterone elicited by immunization with the steroids attached to protein through position 7

Antibodies to progesterone (P) and to 17-hydroxyprogesterone (17-OHP) were raised by immunization of rabbits with progesterone-7α-carboxyethyl thioether--bovine serum albumin (P-7—BSA) or with 17-OHP-7α-carboxyethyl thioether--BSA (17-OHP-7--BSA). The antisera produced were of high affinity: K_a towards the homologous hapten was 3. 7 × 10¹⁰ 1./mol for the anti-P serum and 5. 9 × 10⁹ 1/mol for the anti-17-OHP serum. The antiserum to P-7—BSA displayed little or no cross reaction (?= 2%) with the 20α-, 20β- or 5β-dihydro-derivatives of progesterone, moderate cross-reaction with pregnenolone (4%), but considerable cross-reaction with 11-deoxycorticosterone (7%), 5α-dihydro-progesterone (11%) and 17-OHP (15%). The antiserum to 17-OHP-7--BSA showed very little cross-reaction (?= 2%) with progesterone and other steroids lacking a 17α-hydroxyl group, such as pregnenolone or 11-deoxycorticosterone, but reacted significantly with 17α, 21-dihydroxy-4-pregnene-3, 20-dione (8%) and 3β, 17-dihydroxy-5-pregnen-20-one (13%). None of the sera reacted with testosterone, cortisol or estradiol-17β. It appears that conjugation of progesterone to protein through carbon-7 affords antisera comparable in specificity to those raised with 11α-conjugates and superior to those raised with 3-, 6- and 20-conjugates. The antiserum to 17-hydroxyprogesterone described is the first one that specifically recognizes this metabolite.     

A radioimmunoassay for plasma 11-desoxycortisol and its use in the rapid metopirone test

A radioimmunoassay for the measurement of 11-deoxycortisol in plasma is described. Antiserum against 11-deoxycortisol was produced by immunizing rabbits with the 21-hemisuccinate of 11-deoxycortisol coupled to bovine serum albumin. The method does not require chromatography but instead makes use of a simple extraction procedure which, in combination with the antibody characteristics, is relatively specific for the 11-deoxycortisol determination. The smallest amount measurable is 5 pg. The intra-assay coefficient of variation was 6.3% before metopirone and 7.2% after metopirone. The inter-assay coefficient of variation was 12.5% before metopirone and 10.3% after metopirone. Pituitary-adrenal reserve was evaluated in control and hypopituitary subjects by a simple midnight metopirone test.     

A specific radioimmunoassay for PGE2 using an antibody with high specificity and a sephadex LH-20 microcolumn for the separation of prostaglandins

Antiserum against PGE₂ was raised in rabbits following immunization with prostaglandin-hen-γ-globulin conjugate. The antiserum exhibited 14% cross reactivity with PGE₁ and far less cross-reaction with heterologous prostaglandins. A microcolumn of Sephadex LH-20 was used for a partial, but sufficient separation of PGE₂ from PGE₁ and a complete separation from heterologous prostaglandins to ensure a specific RIA for PGE₂. The precision of the method in the range 10–500 picograms showed a coefficient of variation varying between 4 and 13%. The detection limit was 10 picograms corresponding to 15 pg/ml of PGE₂ in serum.In order to demonstrate the validity of the method values obtained for non-diuretic rat renal venous serum were compared with those obtained using the isotope derivative method of Bojesen & Buckhave (1972) on the same samples. The concentrations of PGE₂ obtained were 239 ± 25 pg/ml and 250 ± 58 pg/ml, respectively.     

Radioimmunoassay for 11-deoxycortisol using iodine-labeled tracer.

A simple and sensitive radioimmunoassay for 11-deoxycortisol was developed. The antiserum produced in rabbits by immunizing with a complex of 11-deoxycortisol-3-oxime and bovine serum albumin (BSA) has little cross-reactivity with other endogenous steroids. The immunoassay procedure requires only one-step ethanol denaturation of binding proteins in plasma and extraction by an organic solvent can be omitted. Furthermore, use of 125I-labeled tracer significantly simplify the counting procedure. The method is sensitive enough to detect 1 microng/100 ml of 11-deoxycortisol. Plasma 11-deoxycortisol levels measured by this method after the administration of a single dose of metyrapone ranged from 5.0 to 19.2 microng/100 ml, whereas they were 0 to 4.0 microng/100 ml in hypopituitary patients. It is concluded that this simple method is useful for the routine assay of plasma 11-deoxycortisol as a parameter of the metyrapone tests.     

A radioimmunoassay for human plasma corticosterone.

A radioimmunoassay for human plasma corticosterone has been developed. Antiserum against corticosterone was produced in rabbits immunized with corticosterone-21-hemisuccinate conjugated to bovine serum albumin. The antiserum cross-reacted with progesterone, DOC and dehydrocorticosterone more than 20%. After the extraction with ether, and the separation by Sephadex LH-20 microcolumn chromatography, recovery was 51.2 +/- 12.1% in 50 assays. The mean coefficient of variation between assays was 7.7% and within assays was 8.6%. Human plasma corticosterone is measured readily by assaying aliquots of an ether extract of 0.05 to 0.1 ml of plasma after microcolumn chromatography. The mean plasma corticosterone concentration at 9 a.m. was 7.1 +/- 3.2 ng/ml in 45 normal subjects. Plasma corticosterone increased 5.2 times as much as basal values after ACTH injection, whereas radioimmunoassayed cortisol increased 2.4 times. On the other hand, plasma corticosterone decreased to 22.6% of basal values at four hours after 1 mg dexamethasone, whereas radioimmunoassayed cortisol decreased to 12.3% of basal values.     

Development of a sensitive enzymeimmunoassay for oxytocin determination in bovine plasma

A highly sensitive and specific second antibody enzymeimmunoassay (EIA) on microtiterplates for oxytocin determination in bovine plasma using the biotin–streptavidin amplification system was developed. Biotin was coupled to oxytocin and used to bridge between streptavidin-peroxidase and the immobilized oxytocin antiserum in the competitive assay. The assay was carried out directly in 200 μl of bovine plasma. Oxytocin standards prepared in hormone-free plasma were used. The sensitivity of the assay was 0.25 pg/well which corresponded to 1.25 pg/ml plasma; the 50% relative binding was seen at 2.8 pg/well. Plasma volumes for the assay ranging from 50 to 200 μl did not influence the shape of the oxytocin standard curve; however a distinct drop in the OD₄₅₀ was observed with higher plasma volumes. The oxytocin antiserum used in the assay showed no significant cross-reaction with other octapeptides tested. The assay was compared with a radioimmunoassay (RIA) procedure employing prior solvent extraction of plasma samples. The oxytocin concentrations assayed by EIA and RIA in plasma samples obtained from four cows before, during and after milking were highly correlated and very similar (r=0.97). Hence the assay developed offers an attractive alternative to the RIA since no prior laborious plasma extraction is needed. Further, the assay has the distinct advantage of being non-radioactive in nature.     

Sample preparation and liquid chromatography-tandem mass spectrometry for multiple steroids in mammalian and avian circulation

Blood samples from wild mammals and birds are often limited in volume, allowing researchers to quantify only one or two steroids from a single sample by immunoassays. In addition, wildlife serum or plasma samples are often lipemic, necessitating stringent sample preparation. Here, we validated sample preparation for simultaneous liquid chromatography--tandem mass spectrometry (LC-MS/MS) quantitation of cortisol, corticosterone, 11-deoxycortisol, dehydroepiandrosterone (DHEA), 17β-estradiol, progesterone, 17α-hydroxyprogesterone and testosterone from diverse mammalian (7 species) and avian (5 species) samples. Using 100 μL of serum or plasma, we quantified (signal-to-noise (S/N) ratio ≥ 10) 4-7 steroids depending on the species and sample, without derivatization. Steroids were extracted from serum or plasma using automated solid-phase extraction where samples were loaded onto C18 columns, washed with water and hexane, and then eluted with ethyl acetate. Quantitation by LC-MS/MS was done in positive ion, multiple reaction-monitoring (MRM) mode with an atmospheric pressure chemical ionization (APCI) source and heated nebulizer (500°C). Deuterated steroids served as internal standards and run time was 15 minutes. Extraction recoveries were 87-101% for the 8 analytes, and all intra- and inter-run CVs were ≤ 8.25%. This quantitation method yields good recoveries with variable lipid-content samples, avoids antibody cross-reactivity issues, and delivers results for multiple steroids. Thus, this method can enrich datasets by providing simultaneous quantitation of multiple steroids, and allow researchers to reimagine the hypotheses that could be tested with their volume-limited, lipemic, wildlife samples.     

Changes in oestrone sulphate concentrations in peripheral plasma of Pony mares associated with follicular growth, ovulation and early pregnancy

A simple and rapid (less than 2 h) immunoassay method has been developed based upon a novel separation technique called LIDIA (Ligand Differentiation Immunoassay), enabling direct estimation of the concentration of oestrone sulphate in ethanolic extracts of blood plasma. An antiserum raised against oestrone-3-glucuronyl-BSA was used which showed a higher cross-reaction with the sulphate than the glucuronide metabolite. The assay had a sensitivity of 5.2 pg/tube and acceptable inter-(less than 18%) and intra-(less than 8.5%) assay precision. Analysis of samples of peripheral venous plasma obtained daily from Pony mares showed that the mean concentration of oestrone sulphate started to rise from a baseline value (less than 300 pg/ml) at 6 days and reached a peak (greater than 850 pg/ml) at 2 days before follicular rupture as determined by rectal palpation. Progesterone concentrations only started to rise above baseline (less than 0.5 ng/ml) on the day of ovulation and reached a peak 8 days later. Analysis of samples obtained during the first 30 days of pregnancy showed that there was no increase in oestrone sulphate at the time oestrus would have been expected had the mares not conceived.     

A new specific and sensitive time resolved-fluoroimmunoassay of 11-deoxycortisol in serum

A biotinylated 11-deoxycortisol tracer was synthesized from 11-deoxycortisol-3-carboxymethyloxime and the conjugate obtained by acylation of biotinylaminopropylammonium trifluoroacetate. This biotinylated tracer was used to develop an 11-deoxycortisol time-resolved-fluoroimmunoassay (TR-FIA). The tracer was quantified after adding streptavidine-Europium. A TR-FIA sensitive standard curve, with displacement of 20, 50, and 80% of tracer was obtained with 12.4, 70.7, and 512.8 pg of 11-deoxycortisol, respectively. After extraction followed by Celite chromatography, purified serum samples were simultaneously assayed by RIA and TR-FIA. The results obtained by the two methods were practically identical, however, this new specific, non-isotopic 11-deoxycortisol assay has the advantage of being more sensitive than RIA, thus well-suited to accurate measurement in endocrinological studies, particularly when serum 11-deoxycortisol levels in patients are just above the highest normal values. Moreover, this non-isotopic assay is cheaper than RIA.     

Solid phase fluoroimmunoassay for 11-deoxycortisol in serum using 21-amino-17-hydroxyprogesterone

Solid phase fluoroimmunoassay of serum 11-deoxycortisol (17,21-dihydroxy-4-pregnene-3,20-dione) was established using fluorescein isothiocyanate-labelled 11-deoxycortisol and anti-11-deoxycortisol antibody-conjugated polyacrylamide beads. 21-Amino-17-hydroxyprogesterone (21-amino-17-hydroxy-4-pregnene-3,20-dione) was synthesized as a useful derivative for preparing the fluorescent dye conjugate. Serum 11-deoxycortisol was measured with this assay system after extraction and purification by Sephadex LH-20 column chromatography. The minimal amount of 11-deoxycortisol detected was 40 pg/tube and the measurable range was from 0.04 to 5.0 microgram/dl. Intra- and inter-assay coefficients of variation were 8.3% (n=6) and 9.8% (n=5), respectively. 11-deoxycortisol values determined by the present assay correlated well with those determined by radioimmunoassay. The present assay is particularly suitable for estimating the conditions of the pituitary and adrenocortical functions.     

A radioimmunoassay for the regulatory allylic steroid, 3 alpha-hydroxy-4-pregnen-20-one (3 alpha HP)

The allylic steroid, 3 alpha-hydroxy-4-pregnen-20-one (3 alpha HP), found in gonadal and brain tissues by radiotracer and chemical methods, had been shown to play a role in gametogenesis, gonadotropin secretion and brain excitability. Since no simple assay was available, a radioimmunoassay for 3 alpha HP was developed using [3H]3 alpha HP and an antiserum raised against 3 alpha HP-20-CMO conjugated to bovine serum albumin. The specificity of the assay for the 3 alpha allylic configuration of 3 alpha HP was confirmed by examining 32 other steroids; cross-reaction with steroids containing different configurations (including metabolites of 3 alpha HP such as progesterone) was less than 0.9%. A Scatchard plot indicated a Ka of 1.56 X 10(9) M-1. Inter- and intra-assay coefficients of variation were 13.1 and 4.5%, respectively. The sensitivity of the assay was 6 pg and the 50% intercept of the standard curve was approx. 123 pg. The measurement by RIA of 3 alpha HP from standard solutions and HPLC purified tissue extracts was confirmed qualitatively and quantitatively by GC/MS methods. The RIA method was employed to determine 3 alpha HP levels in cultured Sertoli cells and in serum of intact and ovariectomized adult rats. Although for most uses, chromatography would not be necessary, two possible methods are presented to enable the separation of 3 alpha HP from other interfering steroids prior to RIA.     

An enzyme immunoassay for plasma betamethasone

A sensitive enzyme immunoassay for plasma betamethasone was developed using betamethasone-3-(O-carboxymethyl)oxime-β-D-galactosidase conjugate as a labelled antigen and 4-methylumbelliferyl-β-D-galactoside as a fluorescence substrate. The performances of the enzyme immunoassay were compared with that of a radioimmunoassay using ³H-betamethasone and the same antiserum. The minimal detectable level for the enzyme immunoassay was 0.15 pg/tube or 0.15 ng/ml of plasma, which was remarkably more sensitive than the radioimmunoassay level of 10 pg/tube or 2 ng/ml of plasma. The specificity was sufficient, in particular, the cross reactivity of cortisol was 0.008%. However, the precision of the enzyme immunoassay was inferior to that of the radioimmunoassay.     

Comparison of serum ethinyl estradiol, sex-hormone-binding globulin, corticoid-binding globulin and cortisol levels in women using two low-dose combined oral contraceptives

The study included 69 women taking a desogestrel (n = 30)- or gestodene (n = 39)-containing low-dose combined oral contraceptive for at least 3 months. Group size was calculated to detect a difference in mean values of 80% of 1 standard deviation (alpha = 0.05, beta = 0.1). Seven serum samples were obtained up to 4 h, and 1 sample 24 h, after drug intake on 1 day between the 10th and the 21st day of the cycle. The concentrations of sex-hormone-binding globulin (SHBG), corticoid-binding globulin (CBG) and cortisol were measured in a 0- to 4-hour serum pool by radioimmunoassay. Ethinyl estradiol (EE2) levels were analyzed in single and pooled samples using anti-EE2-6 beta-carboxymethyloxime-bovine serum albumin antiserum. The area under the curves (AUC) up to 4 and 24 h and Cmax and tmax were evaluated. Statistical analysis (analysis of covariance) did not reveal a dependence of values on duration of treatment or day of cycle. Both treatments resulted in almost identical values for all parameters evaluated. The mean levels of SHBG, CBG and cortisol were in the range of 186-226 nmol/l, 89-93 mg/l and 280-281 micrograms/l, respectively. Mean maximum EE2 levels of 106-129 pg/ml were found 1.6-1.8 h after pill intake and AUC0-4 h accounted for 329-374 pg.h.ml-1. The recently reported differences in serum EE2 and CBG levels between two groups of 11 women each treated with desogestrel- and gestodene-containing pills, respectively, could not be confirmed.     

A radioimmunoassay for rat ghrelin: evaluation of method and effects of nonylphenol on ghrelin secretion in force-fed young rats

Antiserum YJC 13-31 against the rat ghrelin conjugated to bovine serum albumin (BSA) was produced in the rabbit and a double antibody radioimmunoassay (RIA) for ghrelin has been developed. Characterization results of this antiserum revealed no cross-reaction with human growth hormone and somatostatin. Weak cross-reactions with insulin (0.1%), rat growth hormone (0.1%) and glucagon (0.3%) were observed, which scarcely interfered the assay system. The sensitivity of this RIA was 5 pg per assay tube. With the rat serum samples, the within-assay precision was 7.1% and the between-assay precision was 12.3%. The RIA was also available to detect the ghrelin in rat tissue extracts with good parallelism to the rat ghrelin standard. In application, the serum ghrelin and corticosterone levels in weaned rats were measured by RIA. Gavage of saline was sufficient to raise serum ghrelin from 2.6 +/- 0.18 to 6.7 +/- 0.7 ng/ml (P < 0.01). Gavage with nonylphenol (NP) suppressed the elevation of serum ghrelin levels in a dose-dependent manner. Besides, gavages of saline elevated the serum levels of corticosterone from 108.8 +/- 13.5 to 188.7 +/- 23.5 ng/ml (P < 0.01) but the elevation effects of corticosterone from gavages were overcome by NP in the low dose of 50 mg/kg. It can be speculated that ingestion of NP is harmful to young animals during growth and environmental adaptation.