The production and assessment of monoclonal antibodies to cortisol

In an extensive series of experiments, Balb/C mice and Lou rats were immunised with 3-O-(carboxymethyl)oximinocortisol conjugated to bovine serum albumin. The spleen cells from selected animals were fused with cells from mouse or rat plasmacytoma lines. Out of many hundreds of hybridomas screened, more than seventy produced antibody that bound 125I-labeled cortisol. These cultures were investigated further for stability of antibody production, affinity for cortisol and cross-reactivity with other steroids. An unexpected but consistent finding was that immunised rats produced antibody which cross-reacted with 11-deoxycortisol to a level greater than 100% and this characteristic was reproduced by rat-rat hybridomas. Strategies designed to improve the chances of generating non-cross-reactive anti-cortisol monoclonal antibodies did not appear to be successful. Nevertheless, several monoclonals were identified with properties that suggest they may be useful for the development of sensitive and specific cortisol assays.     

The production of monoclonal antibodies against aldosterone

We have prepared several monoclonal antibodies against aldosterone-3-carboxy-methyloxime-BSA conjugate by fusing spleen lymphocytes from an immunized mouse with the mouse myeloma line HL-1 Friendly. A total of 6 different clones were isolated and expanded. All of the antibodies exhibited low cross-reactivities against most of the compounds tested. Antibodies A5A3, A2E11, and C1E2 exhibited low cross-reactivity with 18-hydroxycorticosterone and 18-hydroxydeoxycorticosterone and showed no detectable displacement of tritiated aldosterone from the antibodies with cortisol, corticosterone, and related steroids. The only steroid that showed moderate cross-reactivity was 3 alpha,5 beta-tetrahydroaldosterone (around 3%). Clone A5H12 antibodies exhibited high cross-reactivity with tetrahydroaldosterone (19.3%) but otherwise was very similar to the above clones. Antibody of clone C1E4 showed high cross-reactivity to tetrahydroaldosterone (41.2%) and 18-hydroxyDOC (2%) with relatively low cross-reactivity to DOC (0.078%). Clone A2G9 antibodies were the only ones for which cortisol and corticosterone displaced tritiated aldosterone with cross-reactivities of 0.0042% and 0.125%, making them unsuitable for a direct radioimmunoassay of plasma aldosterone. The monoclonal antibodies were very sensitive to freezing and thawing. The cross-reactivities of the first three clones' antibodies compare favorably with those polyclonal antibodies that have been described to be suitable for use in direct radioimmunoassays of plasma aldosterone. Their advantage is the reliable supply of an antibody with consistent, predictable properties.     

The production and characterisation of antisera to 17-hydroxyprogesterone

Sheep were immunised with 11-deoxycortisol-21-hemisuccinate-bovine serum albumin (11-deoxycortisol-21-HS-BSA) or with 17-hydroxyprogesterone-7 alpha-carboxyethyl thioether-keyhole limpet haemocyanin (17-OHP-7 alpha-CETE-KLH) or with 17-OHP-3-(O-carboxymethyl)oxime-KLH (17-OHP-3-CMO-KLH). The resultant antisera were assessed using [3H]17-OHP and dextran-coated charcoal to separate the antibody bound and free fractions. All sheep produced antisera with an apparent affinity constant of from 1.4 to 6.6 X 10(9) 1/mol. Those raised against 11-deoxycortisol-21-HS-BSA had titres ranging from 1:12,000 to 1:78,000 but showed significant cross-reactivity with many of the steroids tested. Sheep immunised with 17-OHP-7 alpha-CETE-KLH had antisera titres of from 1:102,000 to 1:180,000 and only 17-hydroxypregnenolone cross-reacted significantly (10-20%). The best antisera were raised in sheep immunised with 17-OHP-3-CMO-KLH. Titres ranged from 1:168,000 to 1:390,000 and there were about 8 g/l of specific antibodies which cross-reacted 5.7% or less with 17-hydroxypregnenolone, and less than 0.5% with progesterone, 11-deoxycortisol and the other steroids studied. The antisera to 17-OHP-3-CMO-KLH were further assessed using [125I]17-OHP; titres ranged from 1:5,700,000 to 1:18,000,000 with affinity constants of from 1.67 to 2.5 X 10(10) 1/mol. They showed minimal or no cross-reactivity with the steroids studied. Reimmunisation after an 8-month interval produced antisera with a higher affinity constant and even lower cross-reactivity with other steroids.     

Production of highly specific polyclonal and monoclonal antibodies using estradiol-3-O-carboxymethyl ether as hapten

The preparation of high affinity and high specificity polyclonal and monoclonal antibodies to estradiol is described. Monoclonal antibodies were derived from BALB/c mice hyperimmunized with estradiol-3-O-carboxymethyl ether conjugated to bovine serum albumin. Spleen cells were hybridized with mouse myeloma cells. Quite a few monoclonal antibodies showed very good affinity for estradiol. Extended immunization and hyperimmunization were essential for producing a greater number of positive clones secreting high affinity antibodies. Binding constants of the antisera and their cross-reactivities with related steroids were calculated. Both polyclonal and monoclonal antibodies showed very high affinity for estradiol exhibiting little or no cross-reactivities with structurally related steroids indicating that this site of linkage is a good choice for discriminating between differences at the 16-17 position in the D-ring. This monoclonal antibody (44.28.6), having negligible cross-reactivity with estriol and estrone, can be used for diagnostic purposes.     

Bovine adrenal cytochrome P-450(11 beta)-mediated conversion of 11-deoxycortisol to 18- and 19-hydroxy derivatives; structural analysis by 1H-NMR.

Incubation of 11-deoxycortisol with a cytochrome P-450(11β)-reconstituted system yielded, in addition to cortisol, several new steroid products. In this study, the structures of the three steroid products were elucidated. Retention time of the first product (Peak 2 substance) coincided with that of authentic 18-hydrocortisol on reverse phase HPLC. To further confirm the chemical identity of this product, the purified sample was subjected to ¹H-NMR analysis. The spectrum was essentially identical to that of 18-hydrocortisol. The retention time of the second product (Peak 3 substance) did not coincide with those of commonly occuring steroids. The one- and two-dimension ¹H-NMR spectra provided strong evidence for its structure of 19-hydroxy-11-deoxycortisol. The retention time of the third product (Peak 4 substance) did not coincide with those of commonly occuring steroids. The ¹H-NMR spectrum showed the presence of signals of 19-CH₃ and 18-CH₂ protons. There was also evidence that this product is not hydroxylated at the 11-position. Further analysis of the COSY spectra identified its structure as 18-hydroxy-11-depxycortisol. From these results, we conclude that bovine P-450(11β)deoxycortisol can catalyze the hydroxylation of 11-deoxycortisol at 11β-18- and 19-positions and produce cortisol, 18-hydroxy-11-deoxycortisol, 18-hydroxycortisol and 19-hydroxy-11-deoxycortisol.     

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.     

Production and characterization of monoclonal antibodies to nivalenol tetraacetate and their application to enzyme-linked immunoassay of nivalenol

Three monoclonal antibodies were obtained by the fusion of mouse myeloma cells with splenocytes isolated from BALB/c mice that had been immunized with 8-hydroxy-3,4,7,15-tetraacetyl-nivalenol hemiglutarate covalently bound to bovine serum albumin. These anti-nivalenol tetraacetate monoclonal antibodies were of the IgG type and highly specific to nivalenol tetraacetate, with an apparent association constant of about 10(8)M-1. The relative cross-reactivities of one monoclonal antibody with nivalenol tetraacetate, acetyl T-2 toxin, and scirpenol triacetate were found to be 1.0, 0.02 and 0.03, respectively. Other derivatives showed no cross-reactivity at all. An indirect enzyme-linked immunosorbent assay (ELISA) based on the competitive binding principle was developed using the antibody from clone D18.102.59. The sensitivity of the system was about 0.1 ng of nivalenol tetraacetate per assay. Comparison of nivalenol levels detected in naturally contaminated barley samples by competitive indirect ELISA and gas chromatography (GC) showed good agreement, indicating that the antibody is useful for the measurement of nivalenol in naturally contaminated cereals and grains.     

Murine monoclonal antibody against aldosterone: production, characterization and use for enzymoimmunoassay

Hybridomas secreting monoclonal antibodies to aldosterone were obtained by fusion of myeloma cells and spleen cells from Balb/c mice immunized with aldosterone-3-carboxylmethyloxime-bovine serum albumin. A monoclonal antibody was purified from ascites fluid and characterized. An affinity constant of 1.61 x 10(9) M-1 has been measured and no cross-reactivity with tetrahydroaldosterone (THA), cortisol, cortisone, corticosterone, deoxycorticosterone (DOC), dehydroepiandrosterone (DHA), progesterone and estrone, could be detected. A peroxidase conjugated-antibody (1.5 mole of enzyme per mole of antibody) was obtained and used for microwell enzyme immunoassay and Immun-Blot assay. The high affinity and specificity of this antibody should make the direct determination of aldosterone in biological fluids possible at concentrations as low as 5 x 10(-10) M.     

The development and application of a radioimmunoassay for 18-hydroxy-corticosterone.

A sensitive and specific radioimmunoassay has been developed for 18-hydroxy-corticosterone (18-OH-B) and applied to the measurement of this steroid in peripheral plasma. High specific activity label (3H-18-OH-B) was prepared using the incubation of 3H-corticosterone with duck adrenal mitochondria. Antisera were produced by immunisation with 18-OH-B gamma-lactone 3-oxime conjugated to bovine serum albumin. The antibodies examined showed 100% cross-reactivity with 18-hydroxy-deoxycorticosterone gamma-lactone (18-OH-DOC gamma-lactone), but minimal cross-reactivity with other steroids. Paper chromatography was used to separate 18-OH-DOC gamma-lactone from 18-OH-B gamma-lactone. The interassay precision was 7.6% and the intra-assay precision 11.0%. The accuracy of the method was confirmed by showing a linear relationship between amounts of 18-OH-B added and amounts of 18-OH-B gamma-lactone measured (y = 0.854 X +15.1, r = 0.9. p less than 0.001). The mean plasma level in normal subjects on an ad libitum sodium intake was 225 +/- 92.7 (SD) pg/ml (n = 17) when standing, and 99 +/- 38.3 (SD) pg/ml (n = 6) after lying down for 30 minutes.     

Antibodies against cortisol block suppressive effects of corticosteroids on lymphocytes in vitro.

Lymphocyte transformation assays were used to test the ability of antibodies against cortisol to reduce bioactivity of corticosteroids in vitro. Mononuclear cells were separated from whole bovine blood and cultured in the presence of PHA alone, PHA + steroid, PHA + steroid + anticortisol, or PHA + steroid + anti-bovine serum albumin. Tritiated thymidine uptake was determined for all groups during the last 24 hr of a 72-hr culture period by scintillation counting. Polyclonal anticortisol against cortisol-bovine serum albumin conjugated in the 21 position was more effective in blocking cortisol activity than monoclonal anticortisol built against conjugates in the 3 position. The steroids that suppressed PHA-induced lymphocyte proliferation in a concentration-dependent manner were: cortisol, corticosterone, dexamethasone, prednisolone, 11-deoxycortisol, and 11-deoxycorticosterone. Aldosterone, cortisone, cholesterol, estradiol, and progesterone did not exhibit concentration-dependent effects and, thus, were not considered suppressive. These concentration-independent steroids were also the least suppressive (with the exception of aldosterone). Anticortisol was able to reduce bioactivity of suppressive corticosteroids that had an 11-hydroxy group, suggesting the antibody was primarily made against this site. Anti-BSA was not effective in blocking corticosteroid activity, but it did enhance proliferation of lymphocytes if added in combination with weakly suppressive steroids. Anticortisol also had an enhancing effect when added with some weakly suppressive steroids. We conclude that antibodies against cortisol are capable of reducing bioactivity of steroids that strongly suppress lymphocyte proliferation. Additionally, the 11-hydroxy group may be an important antigenic determinant of steroid molecules.     

Aldosterone synthase activity in the Y-1 adrenal cell line

The Y-1 adrenal cell line was shown to produce 20-dihydroaldosterone from deoxycorticosterone. This compound was identified by GC-MS by comparison with the previously synthesized reference compound. Two other 18-hydroxylated metabolites were identified as 11β,18-dihydroxy-20-dihydroprogesterone from endogenous cholesterol and 18-hydroxy-20-dihydro-11-dehydrocorti-costerone from DOC. The conditions necessary for the synthesis of these compounds are culturing in 20% serum-supplemented medium and repeated incubations with the substrate. The production of 11β-hydroxylated steroids and that of 18-oxygenated steroids is stimulated differently by ACTH and angiotensin II suggesting the expression of two different enzymes, cytochrome P-450_11β and cytochrome P-450_aldo The Y-1 cell line can secrete either 11β-hydroxylated steroids characteristic of the glucocorticoid pathway or 18-oxygenated steroids characteristic of the mineralocorticoid pathway, which in vivo are generally produced in two different zones of the adrenal cortex. This cell line should be an interesting model for the study of the molecular mechanisms regulating the expression of these two enzymes involved in the final steps of the steroidogenic pathways.     

Production and characterization of monoclonal antibodies to 17 alpha-hydroxyprogesterone

Hybridoma clones producing antibodies to 17 alpha-hydroxyprogesterone (17-OHP) were established by using a 17-OHP-bovine serum albumin conjugate as an immunogen. Six representative IgG-class monoclonal antibodies of high affinity (10(8)-10(9) M-1) showed differential reactivities with several structurally related steroids. Two enzyme immunoassay (EIA) systems (fluorescence EIA and micro-EIA) for 17-OHP using OHP 4B2.2.3, which showed the lowest cross-reactivity with other steroids, were established. The micro-EIA system was shown to be applicable to the mass-screening of congenital adrenal hyperplasia.     

An adrenocortical tumor secreting weak mineralocorticoids

An adrenocortical carcinoma (15.5 g) secreting excessive amounts of steroids with weak mineralocorticoid activity in a 25-year-old woman was studied with particular reference to its in vivo and in vitro secretions of steroids. Severe hypertension, occasional low serum potassium and suppressed PRA were the major clinical findings, and were improved with removal of the tumor. In the preoperative stage, plasma levels of 11-deoxycorticosterone, 18-hydroxy-11-deoxycorticosterone, corticosterone and 18-hydroxycorticosterone were all increased. However, the plasma level of aldosterone was repeatedly normal. Although plasma levels of pregnenolone, 17-hydroxypregnenolone, progesterone and 17-hydroxyprogesterone were very high, those of other late step steroids, i.e. 11-deoxycortisol, cortisol, dehydroepiandrosterone, androstenedione and testosterone were almost normal. From these findings, a major etiological role of weak mineralocorticoids such as 11-deoxycorticosterone, 18-hydroxycorticosterone and corticosterone in her hypertension was suggested. Pregnenolone and 17-hydroxypregnenolone in tumor tissue were increased, but 11-deoxycorticosterone, corticosterone, aldosterone, cortisol and adrenal androgens such as dehydroepiandrosterone, androstenedione and testosterone were below normal or low normal. In vitro production of 11-deoxycorticosterone, aldosterone or cortisol by the tumor tissue slices was very low and scarcely responded to synthetic ACTH.     

18-Hydroxy-11-deoxycortisol: a new steroid isolated from incubations of the adrenal with 11-deoxycortisol

Cortisol has been shown to be metabolized in the zona glomerulosa of the adrenal gland through the same pathway involving the cytochrome P-450, corticosterone methyl oxidase by which corticosterone is transformed to 18-hydroxycorticosterone and aldosterone. When cortisol is the precursor, 18-hydroxycortisol and 18-oxocortisol are formed. 18-Hydroxycortisol can also be made at a similar rate in the bovine zona fasciculata and reticularis as in the zona glomerulosa. We studied the possibility that the formation of 18-hydroxycortisol in the zona fasciculata and reticularis might be through a different pathway involving initial 18-hydroxylation of 11-deoxycortisol before 11 beta-hydroxylation. Rat adrenal capsules or cores were incubated with 10 micrograms of cortisol or 11-deoxycortisol and the formation of 18-hydroxycortisol was measured by radioimmunoassay. Both capsules and cores transformed 11-deoxycortisol to 18-hydroxycortisol, but cortisol was only transformed in the capsular portion. Sixty-two rat adrenals were incubated with 10 mg of 11-deoxycortisol and the putative steroid, 18-hydroxy-11-deoxycortisol, was purified by TLC and HPLC and subjected to gas chromatography mass spectrometry. The mass spectra indicated that the steroid isolated was indeed 18-hydroxy-11-deoxycortisol. The function of this steroid is still unknown.     

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.     

Single-chain Fv fragments derived from an anti-11-deoxycortisol antibody. Affinity,specificity, and idiotype analysis

Single-chain Fv fragments (scFvs) against a corticosteroid, 11-deoxycortisol (11-DC), have been generated as a template antibody fragment from which a comprehensive mutated antibody library containing various anti-steroid antibodies could be constructed. The cDNAs encoding variable heavy (V(H)) and light (V(L)) domains of a mouse anti-11-DC antibody (CET-M8), were amplified by RT-PCR, combined via a common linker to construct the sequence of 5'-V(H)-(Gly(4)Ser)(3)-V(L)-3', and cloned into a phagemid vector, pEXmide 5. The phage clones exhibiting binding activity to 11-DC were isolated after single panning against a hapten-immobilizing immunotube. The scFv gene in one of these clones was reamplified to introduce the ochre codons, and then expressed in the bacterial periplasm as the soluble antibody fragment. Two different scFvs (#6 and #12) were cloned, whose binding characteristics were examined by a radioimmunoassay using a tritium-labeled 11-DC. Both of them showed high affinity (K(a)=1.3x10(10)M(-1)) and practical specificity (cross-reactivity: cortisol, <0.2%; cortisone, <0.3%) to 11-DC, and furthermore, strong reactivity with an anti-idiotype antibody which recognizes the paratope of CET-M8. These results suggest that the present scFvs retain the three-dimensional structure of the paratope of the original monoclonal antibody.     

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.     

Reductively aminated D-xylose-albumin conjugate as the immunogen for generation of IgG and IgE antibodies specific to D-xylitol, a haptenic allergen

Sugar alcohols are widely used as food additives and drug excipients. Xylitol, a five-carbon sugar alcohol, and a low-calorie alternative sweetener to sucrose (approx 40% fewer calories), has enjoyed an enviable record of safety, and allergic reactions to xylitol are very rare. A case of oral erosive eczema to xylitol has been reported recently [Hanakawa, Y., Hanakawa, Y., Tohyama, M., Yamasaki, K., Hashimoto, K. (2005) Xylitol as a causative agent of oral erosive eczema. Brit. J. Dermatol. 152, 821-822]. Xylitol does not contain any reactive groups; hence, it is nonimmunogenic. In order to explain the immunogenicity of xylitol, polyclonal antibodies to xylitol have been raised using the reductive aminated product of D-xylose conjugated to bovine serum albumin (BSA) as the immunogen. Rabbits immunized with xylitol-BSA conjugate (52 haptens/molecule) gave a good antibody response. Purification of antixylitol antibodies was carried out using hapten-affinity chromatography on xylitol-keyhole limpet hemocyanin-Sepharose CL-6B; the yield was approximately 40 microg/mL of rabbit immune serum. Purified xylitol-specific antibodies appeared to be homogeneous by native PAGE with a pI of approximately 7.2 by isoelectric focusing. Although the purified antibodies are specific for the xylitoyl moiety of xylitol-protein conjugates, they reacted equally well with the Schiff base conjugate of xylosyl-protein conjugates (68% cross-reactivity) indicating that carbons 2 to 5 of xylitol act as an epitope. Xylitol antibodies showed excellent specificity towards xylitol and <4.4% cross-reactivity with D-xylose and various sugar alcohols except ribitol and galactitol, which showed approximately 11% and 8% cross-reactivity, respectively. D-Xylitol-BSA conjugate was used to raise IgE antibodies in BALB/c mice by repeated intradermal administration. Passive cutaneous anaphylaxis using the immune sera confirmed the haptenic nature of xylitol.     

Monoclonal antibodies to queuine

Monoclonal antibodies specific for queuine have been prepared. Synthetic 9-(5-carboxypentyl)queuine (cp9Q) was conjugated with bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH), and the conjugate was used to immunize BALB/c mice by intraperitoneal and subcutaneous injection. Monoclonal antibodies were subsequently obtained by fusion of spleen cells and the mouse myeloma cell line X63Ag8U1. An enzyme-linked immunoabsorbent assay (ELISA) using o-phenylenediamine as peroxidase substrate was used for screening of clones and characterization of antibodies. Inhibition experiments with various homologous nucleosides revealed that the monoclonal antibody designated as 2D8E6 has no cross-reactivity with guanosine, adenosine or 7-methylguanosine.     

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.