Sensitivity of steroid enzyme immunoassays. Comparison of four label enzymes in an assay system using a monoclonal anti-steroid antibody

The sensitivities of monoclonal antibody-based enzyme immunoassays for 11-deoxycortisol using alkaline phosphatase (AP), horseradish peroxidase (HRP), beta-galactosidase (beta-GAL) and glucose oxidase (GOD) as labels were compared. The anti-11-deoxycortisol antibody used was that produced in ascites by inoculating antibody-secreting hybridoma cells into mice. Enzyme labeling of 11-deoxycortisol was carried out by the N-succinimidyl ester method. The activated ester of 4-(2-carboxyethylthio)-11-deoxycortisol was treated with each enzyme to give a homologous enzyme-labeled antigen. In the competitive immunoassay, the bound and free enzyme-labeled antigens were separated by a double antibody method and the enzymic activity of the immune precipitate was determined by colorimetric and fluorimetric methods. The AP activity was measured in three ways, using p-nitrophenyl phosphate, nicotinamide adenine dinucleotide phosphate (NADP), and 4-methylumbelliferyl phosphate as substrates. o-Nitrophenyl beta-D-galactopyranoside and 4-methylumbelliferyl beta-D-galactopyranoside were used for beta-GAL, and 3,3',5,5'-tetramethylbenzidine (TMB) and 3-(p-hydroxyphenyl)propionic acid (HPPA) for HRP. In the case of GOD, TMB and HPPA were used in combination with HRP. A dose-response curve with a high sensitivity was obtained in each 11-deoxycortisol assay system by the use of a minimum amount of the enzyme-labeled antigen at an appropriate dilution of monoclonal anti-11-deoxycortisol antibody (Ka = 2 x 10(10) M-1). The amounts of 11-deoxycortisol needed to displace 50% of the bound label ranged from 5 to 15 pg in the colorimetric methods, and 4-9 pg in the fluorimetric methods.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reversed-phase high-performance liquid chromatography separation of adrenal steroids prior to radioimmunoassay: application in congenital adrenal hyperplasia

21-Hydroxylase deficiency (21-OHD) is the most common form of congenital adrenal hyperplasia (CAH), followed by 11beta-hydroxylase deficiency (11beta-OHD). Diagnostic serum markers for these conditions are 17-hydroxyprogesterone (17-OHP) and 11-desoxycortisol (S), respectively. In 21-OHD, the large amounts of 17-OHP are further 11beta-hydroxylated to form 21-deoxycortisol (21-DF), making it also an excellent marker of this disease. These steroids can be measured in blood by radioimmunoassay (RIA). In this paper, we report the use of high-performance liquid chromatography (HPLC) for steroid purification, prior to RIA determinations of 21-DF, S, 17-OHP, and testosterone (T) in ether-extracted serum. The chromatographic separation is developed in a BDS-Hypersil column using water-methanol (53:47, v/v) as the mobile phase. The method is applied to 35 patients with the classic form of 21-OHD (18 females, 17 males, 5.1-14.2 years old) and 2 with 11beta-OHD (1 female, 1 male, 9.5 and 12.6 years old). Thirteen control children (5 females and 8 males, 5.2-15.2 years) are also studied. The results obtained for all measured steroids are compatible with those reported in the literature. The method is precise, and recovery is adequate. The HPLC technique proved to be of value for the purification of several steroids from single serum samples prior to RIA in patients with CAH.     

Hypertensive congenital adrenal enzymatic defects detected by high-performance liquid chromatography of corticosteroids.

The simultaneous measurement of the adrenal deoxycorticosterone (DOC), 18-OH-DOC, corticosterone (B), 18-OH-B, 11-deoxycortisol (S) and cortisol (F) present in human plasma in cases of adrenal dysfunction was accomplished using a high-performance liquid chromatographic (HPLC) system with a UV detector and with a radioimmunoassay (RIA). After a solid-phase extraction, plasma samples were separated by HPLC using a gradient of water-acetonitrile-ethanol on a radial compressed reversed-phase column. In a 70-min cycle, a complete separation of adrenal steroids was accomplished. The UV detector allowed direct measurement of F in each plasma sample while in selected cases B and S were directly determined. It was therefore possible quickly to identify patients with hypertensive congenital adrenal enzymatic defects with this method: the 17-alpha-hydroxylase deficiency characterized by the absence of measurable levels of F with an evident peak corresponding to B and the 11-beta-hydroxylase deficiency in which high levels of S without F are detected. The RIA of DOC, B, 18-OH-DOC and 18-OH-B complete the characterization of the adrenal defect. Therefore, with this HPLC method it is possible to recognize the major hypertensive adrenal enzymatic deficiencies such as the defect of 17-alpha-hydroxylase or 11-beta-hydroxylase. With "RIA" detectors an almost complete spectrum of adrenal steroid secretion can be obtained.     

Steroidogenesis in Patients with Various Adrenal Cortex Diseases as Studied by Reversed-Phase High-Performance Liquid Chromatography

A method is proposed for the quantitative determination of diagnostically important corticosteroids (cortisol, cortisone, corticosterone, 11-deoxycorticosterone, and 11-deoxycortisol) in blood serum and urine (cortisol and cortisone) in an isocratic mode of reversed-phase high-performance liquid chromatography (RP HPLC) with the use of -cyclodextrin as a component of the mobile phase (CH₃CN : H₂O). Biological fluids (blood serum and urine) from a group of healthy donors and patients with various endocrine diseases (Cushing's syndrome, congenital adrenal hyperplasia, aldosteroma, and adrenal cortex carcinoma) were examined, and characteristic chromatographic steroid profiles were obtained for these disturbances.     

Isocratic reversed phase high performance liquid chromatography determination of twelve natural corticosteroids in serum with on-line ultraviolet and fluorescence detection

An isocratic reversed phase high performance liquid chromatography procedure utilizing ultraviolet and fluorescence detectors linked in series is described for the analysis of cortisone (E), cortisol (F), corticosterone (B), 11-deoxycortisol (S), 11-deoxycorticosterone (DOC), androstenedione (A), testosterone (T), 17-hydroxyprogesterone (17-OHP), progesterone (P), estriol, estradiol, estrone, prednisone acetate and dexamethasone acetate in serum. Serum specimens were extracted with ethyl ether. The optimized mobile phase was methanol + tetrahydrofuran + water (26:18:56, v/v/v). A Shim-pack ODS column was used. The recoveries were 80 to 103%. Intra- and inter-day coefficient of variance were less than 8%. The detection limit is 0.5 pmol per injection volume for estriol, estradiol, E, F and B; 1 pmol for S, A, DOC and estrone; 2 pmol for T and 17-OHP; and 4 pmol for P. Serum from normal subjects and patients with congenital adrenal hyperplasia due to 21- or 17-hydroxylase deficiency were measured, as well as samples of maternal and umbilical cord serum.     

Selective determination of urinary free cortisol by liquid chromatography after solid-state extraction

We have developed a selective and precise high-performance liquid chromatographic method for urinary free cortisol with an improved and efficient sample clean-up using C18 Sep-Pak cartridges. The urine sample (2 ml), with 11-deoxycortisol as internal standard, is applied to the Sep-Pak, which is then sequentially washed with acetone-water (1:4, v/v), water and hexane. Cortisol is eluted with diethyl ether, evaporated to dryness and redissolved in 2 ml of water. The wash cycle is repeated once using the same Sep-Pak cartridge. This double extraction greatly improves sample clean-up and allows modification of the mobile phase (tetrahydrofuran-methanol-water) so that cortisol is rapidly eluted as a single well resolved peak at 13 min. Chromatography is performed isocratically on a reversed-phase column with detection at 254 nm. Detection limits for urinary free cortisol by this procedure were two or three times lower than those obtained with two commercial radioimmunoassay kits. The chromatographic method was used successfully in the diagnosis of patients with hypercortisolism and Cushing's syndrome.     

A fluorimetric assay for cortisol

A simple, rapid and sensitive fluorimetric assay for the quantitative determination of cortisol is reported. The assay is based on the formation of a fluorescent dye when cortisol is incubated with a mixture of sulfuric acid and acetic acid. The fluorescence spectrum recorded for the resulting dye shows a maximum extinction at 475 nm and a maximum emission at 525 nm. The solvent 2-methyl-4-pentanone was used for extraction and was found to act as a fluorescence amplifier. A limit of detection of 2.7 μM was achieved, making it possible to forego solvent evaporation. The assay suffers minor interference from 11-deoxycortisol which exhibits low fluorescence at λ _ex: 460 nm; λ _em: 505 nm. Typical standard deviations were below 4%. We validated the assay using a biotransformation with recombinant Schizosaccharomyces pombe which regioselectively hydroxylates 11-deoxycortisol to cortisol. The method described herein is suitable for preliminary screening of microorganisms capable of steroid hydroxylation.     

The Estimation of Serum Digoxin by Combined HPLC Separation and Radioimmunological Assay

Abstract

A specific method for the quantitation of digoxin in serum has been developed and applied to determination of drug concentrations in serum from digitalized patients. One ml of the supernatant from acetonitrile denatured serum, subsequently diluted with water, was chromatographed on a reversed phase HPLC column. The eluent corresponding to the retention time of digoxin was collected and analysed using a commercial radioimmunoassay kit. The recovery at 3ng/ml was 96.1 ± 3.0%. Endogenous substances, drugs and metabolites of digoxin do not interfere with the method. The mean value of the samples estimated by the present method was 84% of those determined by direct R.I.A.     

Determination of steroids in biological samples by micellar electrokinetic chromatography

The possibility of determining eight adrenocorticotropic steroid hormones (cortisol, cortisone, corticosterone, 11-dehydrocorticosterone, 17-hydroxyprogesterone, 11-deoxycortisol, and progesterone) by micellar electrokinetic chromatography (MEKC) using urea as an organic additive to the working electrolyte (a 25 mM phosphoric acid solution and 10 mM sodium dodecyl sulfate) was demonstrated. The use of online preconcentration (stacking and sweeping) allowed us to lower the detection limit for steroids to ～3 ng/mL. The total analysis time was 15 min.     

Direct radioimmunoassay of testosterone in human saliva

A simple direct radioimmunoassay for testosterone in 400 μl of whole human saliva is reported. Neither extraction nor chromatography is needed. The direct assay involves a commercially available, highly selective antiserum raised against testosterone-19-(O-carboxymethyl)-ether-BSA, an iodinated tracer, a serum added to give parallel inhibition of analyte and tracer binding to serum proteins and a double antibody/polyethylene glycol separation technique. The lower limit of determination is about 3 pM salivary testosterone and the calibration curve covers the range of clinical interest both for males and females (0–868 pM). The assay is specific as judged from recovery and dilution tests, from the cross-reactivity of the antiserum used and from comparisons with an extraction procedure. Within-assay and between-assay relative standard deviations are 4.1, 1.9, 10.5% and 6.3, 2.8, 15.3% for saliva samples with testosterone concentrations of 310, 117 and 52.7 pM, respectively.     

Development of a direct radioimmunoassay for serum progesterone

A direct radioimmunoassay for the measurement of progesterone in human serum is described. Progesterone 11-hemisuccinate was conjugated to tyrosine methyl ester (TME) by the mix anhydride method and then iodinated using chloramine-T. The radiochemical purity of different batches of¹²⁵I-progesterone was greater than 95% and showed 70–75% binding with excess antibody. Progesterone 11-hemisuccinate was coupled with BSA and injected to rabits. Antisera collected after three booster injections and having aK value of 1·10⁹1/M was selected for the assay. Significant reduction in binding with antibody was seen when hormone free serum was used in the assay system. Various blocking agents were tried to reduce the serum effects and none of them were found satisfactory. From a series of optimization experiments, an assay was developed without the use of blocking agents. This assay used a much higher concentration of antibody along with lower amount of serum sample (50 l). The optimized assay has a sensitivity of 0.5 nj/ml and a working range of 0.5 to 100 ng/ml. Serum samples were analyzed analysis showed good correlation between the results obtained from the present system and the DPC kit. (Y=0.93X+0.5,r=0.93, forn=25).     

Identification of 17-hydroxyprogesterone and other steroid hormones in saliva from a normal child and patients with congenital adrenal hyperplasia by plasmaspray liquid chromatography/mass spectrometry

A very sensitive, selective and simultaneous method for the analysis of salivary steroid hormones was examined by discharge-assisted thermospray liquid chromatography mass spectrometry (plasmaspray LC/MS). Plasmaspray LC/MS gave [M + H]+ or [MH - H2O]+ as a predominant ion in most of the steroids in this work and in some cases fragment ions were also observed. Eight salivary steroid hormones, pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone, 11-deoxycortisol, cortisol, aldosterone, and testosterone were identified within 10 minutes using the selected ion monitoring technique in conjunction with plasmaspray LC/MS. Progesterone, 17-hydroxyprogesterone, cortisol, aldosterone and testosterone were detected in 1 mL of saliva from a normal child and two patients with congenital adrenal hyperplasia.     

Enzyme labeling in steroid enzyme immunoassays. Comparison of the p-nitrophenyl ester and N-succinimidyl ester methods.

Enzyme labeling of steroids by the p-nitrophenyl ester method was investigated in comparison with the N-succinimidyl ester method. The active ester of a testosterone or 11-deoxycortisol derivative was treated with beta-galactosidase and horseradish peroxidase to give labeled antigens. Various molar ratios of steroid to enzyme and pH conditions were tested. Satisfactory immunoreactivities with an anti-steroid antibody in each enzyme immunoassay system were obtained with the labeled antigens prepared at pH 8.5 by the use of molar ratios higher than 30. The enzyme labeling method should be useful in the case of polar steroids or drugs, since the p-nitrophenyl ester is relatively stable when compared with the N-succinimidyl ester.     

Comparison of the use of anionic and cationic surfactants for the separation of steroids based on MEKC and sweeping-MEKC modes

In attempts to improve the selectivity and sensitivity of steroid separation and to determine their migration order, a comparison of the use of anionic and cationic surfactants based on the MEKC and sweeping-MEKC modes was made. A mixture of six steroids (progesterone, 17-hydroxy progesterone, 11-deoxycortisol, corticosterone, cortisone, and cortisol) could be separated and detected by means of the CE/UV-absorption method. The order of migration time for these steroids was compared under various conditions, including acidic/alkaline buffers, anionic/cationic surfactants, and positive/negative applied voltage, causing the direction of the EOF and the migration of micelles to change. The major rules for generally predicting the migration order of steroids are summarized. The detection limits were significantly improved when the sweeping-MEKC mode was applied.     

Synthesis of a dibenz[b,e]oxepin-bovine serum albumin conjugate for radioimmunoassay of KW-4679 ((Z)-11-[3-(dimethylamino)propylidene]-6,11- dihydrodibenz[b,e]oxepin-2-acetic acid hydrochloride).

(Z)-11-[3-(Dimethylamino)propylidene]-2-(methoxycarbonyl)methyl-6, 11- dihydrodibenz[b,e]oxepin-9-acrylic acid (5) was prepared for application to the radioimmunoassay of KW-4679 (1, (Z)-11-[3-(dimethylamino)propylidene]-6,11-dihydrodibenz[b,e ] oxepin-2-acetic acid hydrochloride). The acrylic acid moiety in the 9-position of 5 was employed for coupling with an amino group of bovine serum albumin (BSA) to provide 17. Subsequently, the conjugate 17 was treated with aqueous NaOH to hydrolyze the terminal methoxycarbonyl group in the 2-position of the BSA conjugated 5. Antiserum raised against the antigenic BSA-conjugate 4 finally obtained was specific for 1.     

Optimization of an isocratic reversed phase liquid chromatographic system for the separation of fourteen steroids using factorial design and computer simulation

An optimization strategy for an isocratic reversed phase high performance liquid chromatographic system (RP-HPLC) is described. Factorial design and a computer program are used to predict the retention time and resolution of fourteen steroids. An optimized rapid (less than 25 min) isocratic RP-HPLC system for the satisfactory separation of cortisone, cortisol, corticosterone, 11-deoxycortisol, 11-deoxycorticosterone, 17 alpha-hydroxyprogesterone, progesterone, androstenedione, testosterone, estrone, estradiol, estriol, prednisone acetate and dexamethasone acetate has been developed using this strategy through eight experiments.     

Direct radioimmunoassay of serum progesterone using heterologous bridge tracer and antibody

The standardisation of a direct radioimmunoassay for progesterone using an¹²⁵I labeled progesterone prepared by iodinating the tyrosine methyl ester (TME) conjugated to a progesterone hemiphthalate derivative and an antibody prepared using progesterone linked to bovine serum albumin through 11α hemisuccinate derivative is described. The hemiphthalate derivative of progesterone was prepared by reacting 11α-hydroxy progesterone with phthalic anhydride which was then conjugated to TME by using isobutyl chloroformate. The conjugate was iodinated with¹²⁵I using chloramine-T as oxidising agent and purified by thin layer chromatography. Radiochemical purity of the tracer was >95% in all batches. The tracer gave 70–75% binding with excess antibody. Assays were optimised with 8-anilino-1-naphthalene sulphonic acid (ANS) and sodium salicylate as blocking agents to release the progesterone from binding proteins. The assay optimised with sodium salicylate as blocking agent has a sensitivity of 0.25 ng/ml and a working range of 0.25–50 ng/ml, whereas the assay with ANS has a sensitivity of 0.75 ng/ml and a working range of 0.75–100 ng/ml. Serum samples were analysed and compared with the values obtained with a homologous bridge assay.     

Generation of a novel monoclonal antibody against cortisol-[C-4]-bovine serum albumin conjugate: application to enzyme-linked immunosorbent assay for urinary and serum cortisol.

Measurement of cortisol levels in body fluids is important for monitoring pituitary gland and adrenal functions. To develop a specific and standardized enzyme-linked immunosorbent assay (ELISA), a novel monoclonal anti-cortisol antibody has been generated using a reasonably designed haptenic derivative. Spleen cells were prepared from the BALB/c or A/J mouse, which had repeatedly been immunized with a conjugate of 4-(2-carboxyethylthio)cortisol (CET) and bovine serum albumin, to be fused with P3/NS1/1-Ag4-1 myeloma cells. After four fusion experiments, one hybridoma clone secreting a practical antibody has been established. The resulting monoclonal antibody CS#38 (isotype gamma1, kappa) showed an affinity constant (K(a)) for cortisol of 1 x 10(9) M(-1) and provided a practical calibration curve (detection limit, 0.26 ng per assay) in a homologous ELISA system employing horseradish peroxidase-labeled CET as a labeled antigen. Cross-reactivities with related C-21 steroids were acceptably low: 11-deoxycortisol (4.3%), cortisone (4.0%), corticosterone (1.9%), progesterone (1.6%), 17alpha-hydroxyprogesterone (12%), 6beta-hydroxycortisol (8.4%), and tetrahydrocortisol (< 0.1%). Urinary and serum cortisol levels of healthy volunteers were determined by this method after methylene chloride extraction to be 39.0 +/- 17.0 microg/day (n = 7) and 80.8 +/- 38.9 ng/mL (n = 10), respectively, both of which are in the reference range.     

Ein Radioimmunoassay (RIA) zur Bestimmung von menschlichem Lysozym (Muramidase) im Serum, Urin und Liquor

Ohne Zusammenfassung

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A human lysozyme radioimmunoassay for serum, urine and cerebrospinal fluid

     

Production and specificity of anti-22-oxacalcitriol antisera.

22-Oxacalcitriol 3-hemiglutarate, a haptenic derivative of 22-oxacalcitriol, was synthesized to obtain a specific antibody for use in radioimmunoassay. Three antisera were elicited in rabbits against the hapten conjugated with bovine serum albumin, and their specificity was examined by cross-reaction study. One of the antisera was found to be satisfactorily specific, and expected to provide a radioimmunoassay useful for the pharmacokinetic study of 22-oxacalcitriol.