Open-sandwich enzyme immunoassay for one-step noncompetitive detection of corticosteroid 11-deoxycortisol.

A noncompetitive immunoassay has the potential for improved sensitivity and working range compared with corresponding competitive assays. However, monovalent antigens with less than 1000 in molecular weight are not susceptible to sandwich assays due to their small size. As a noncompetitive immunoassay that can be performed with a clone of an antibody, an open-sandwich immunoassay (OS-IA) based on the antigen-dependent stabilization of the antibody variable region (V(H) + V(L)) was applied to the quantification of 11-deoxycortisol (11-DC; M(r) 346.5), a corticosteroid serving as a diagnostic index for pituitary-adrenal function, as a model target hapten. By one step OS-IA detection of enzyme-labeled V(H) fragment bound to immobilized V(L) in the presence of sample in microplate wells, 11-DC was measured with a femtomolar detection limit and the working range was wider than that with corresponding competitive assay. In addition, the selectivity against analogues was found almost identical to that of conventional assays. The effect of the mutagenesis of a V(H) residue at the V(H)/V(L) interface to reduce background signal was also shown, implying the wider application of OS-IA in small molecule analyses.

Cross reactions elicited by serum 17-OH progesterone and 11-desoxycortisol in cortisol assays.

BACKGROUND: Different pathophysiological situations such as congenital adrenal hyperplasia, adrenocortical carcinoma, metyrapone treatment, etc. elicit specificity problems with serum cortisol assay. METHODS: We assayed cortisol using 2 kits and performed cross reaction studies as well as multiple regression analysis using 2 other steroids: 11-desoxycortisol and 17-OH progesterone. RESULTS: Analysis showed the existence of an analytical bias. Importantly, significantly different biases were demonstrated in newborns or patients taking metyrapone. Multiple regression analysis and cross reaction studies showed that 11-desoxycortisol level significantly influenced cortisol determination. Moreover, despite using the normal ranges provided by manufacturers discrepant results occurred such as 17% discordance in the diagnosis of hypocorticism in infants. CONCLUSION: We wish to raise awareness about the consequences of the (lack of) specificity of cortisol assays with regard to the evaluation of hypocorticism in infants or when "unusual" steroids may be increased.

Dissociation-independent selection of high-affinity anti-hapten phage antibodies using cleavable biotin-conjugated haptens.

The engineering of hapten-specific antibodies with affinity constant higher (K(a) values >10(10)M(-1)) than those of conventional antibodies promises hapten immunoassays exhibiting sub-femtomole range sensitivity, based on the conventional competitive assay principle. Here we report a simple method to select phage particles displaying anti-hapten antibody fragments with exceptionally high affinity. 11-Deoxycortisol (11-DC), selected as a model target hapten, was covalently conjugated to biotin via a spacer that included a reductively cleavable disulfide bond. Phage particles displaying high-affinity, single-chain Fv fragments (scFvs) specific for 11-DC (K(a)1.3 x 10(10)M(-1)) were incubated with the "cleavable biotin"-conjugated 11-DC, and the resulting complexes was captured on immobilized NeutrAvidin. Mild reductive conditions that did not decrease phage infectivity easily cleaved the disulfide bond, allowing the recovery of target phage particles; this process is fully independent of the dissociation of the antigen-antibody interaction. Five serial rounds of selection enabled the isolation and enrichment of the anti-11-DC phage (specific phage ratio >90%) from among a 100,000-fold excess of nonspecific phage particles. This method will be applicable for selection of extra-high-affinity phage antibodies against a wide variety of haptens.

Monoclonal anti-idiotype antibodies recognizing the variable region of a high-affinity antibody against 11-deoxycortisol. Production, characterization and application to a sensitive noncompetitive immunoassay.

Anti-idiotype antibodies recognizing the variable regions of a particular anti-hapten antibody are valuable tools, which can be used in sensitive hapten immunoassays based on a noncompetitive format. Here, we describe the production and characterization of monoclonal anti-idiotype antibodies against idiotopes on the variable regions of an antibody showing high affinity and specificity to 11-deoxycortisol (11-DC). 11-DC is the biosynthetic precursor of cortisol and a diagnostic index for the assessment of pituitary-adrenal function. BALB/c or A/J mice were repeatedly immunized with the anti-11-DC antibody conjugated with keyhole limpet hemocyanin and their spleen cells were then fused with P3/NS1/1-Ag4-1 myeloma cells. Seven kinds of anti-idiotype antibodies were generated, one of which was a beta-type antibody recognizing the paratope and others which were alpha-type antibodies recognizing the framework region. A noncompetitive ELISA based on idiotype-anti-idiotype reactions was established using one of these alpha-type antibodies in combination with the beta-type antibody and with the anti-11-DC antibody. This noncompetitive assay system provided improved sensitivity (detection limit: 1.0 pg=2.9 fmol), which is approximately 10 times higher than the corresponding competitive enzyme immunoassay, and offered a practical specificity for clinical use. Appropriate serum 11-DC levels were obtained for normal subjects [0.16+/-0.09 (S.D.) microg/l (n=6), ranging from 0.086 to 0.316 microg/l] using the present assay system.

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.

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.

Plasma 21-deoxycortisol: comparison of a time-resolved fluoroimmunoassay using a biotinylated tracer with a radioimmunossay using (125)iodine.

Plasma 21-deoxycortisol (21DF) is an excellent marker of 21-hydroxylase deficiency. Currently, it is the only marker able to detect heterozygous carriers with 21-hydroxylase deficiency after ACTH stimulation. We have already developed radioimmunoassays for 21DF using first tritiated, then 125I-21DF which had a ten-fold higher sensitivity. However, because the lifespan of 125I-21DF is short, the tracer needs to be reprepared every two months and this multiplies the risk of contamination by radioactive 125I vapours. We therefore developed a non-isotopic 21DF assay that uses a 21DF-biotin conjugate with a original bridge, a diaminopropyl arm, linking the steroid to biotin. The 21DF-biotin conjugate was measured by time-resolved fluorescence after adding streptavidin-europium to the microtitration wells. The analytical qualities of this assay were very similar to those of the radioimmunoassay using 125I-21DF as tracer. The results obtained by the two methods, in either normal subjects or patients with 21-hydroxylase deficiency, were virtually the same.

Changes in the specificity of antibodies by site-specific mutagenesis followed by random mutagenesis.

The specificity for 11-deoxycortisol (11-DOC) of a monoclonal antibody (mAb), designated SCET, was changed to specificity for cortisol (CS) by site-specific mutagenesis followed by random mutagenesis. The Fab form of SCET was expressed on the surface of a phage. During the first step, mutations were introduced at 14 amino acid positions in three complementarity-determining regions (CDRs) of the VH domain that seemed likely to form the steroid-binding pocket. A clone, DcC16, was isolated from the resultant library with multiple mutations and this clone was shown to have CS-binding activity but also to retain high 11-DOC-binding activity. During the second step, mutations were introduced randomly into the entire VH-coding region of the DcC16 clone by an error-prone polymerase chain reaction, and CS-specific mutant antibodies were selected in the presence of 11-DOC as a competitor. Three representative clones were analyzed with the BIAcore instrument, and each revealed a large increase in the binding constant for CS and a decrease in that for 11-DOC. Structural models, constructed by computer simulation, indicated the probable molecular basis for these changes in specificity.

The application of a new highly-sensitive radioimmunoassay for plasma 21-deoxycortisol to the detection of steroid-21-hydroxylase deficiency.

21-deoxycortisol (21-DF) is a steroid of strictly adrenal origin formed by the 11-hydroxylation of 17-hydroxyprogesterone. This metabolic pathway is minor in normal subjects, in whom basal plasma concentrations range from 0.03 to 0.63 nmol/L and from 0.865 to 1.50 nmol/L after adrenocorticotrophic hormone (ACTH; Synacthène Immédiat, Ciba/Geigy, France). However, this metabolic pathway becomes major in 21-hydroxylase-deficient patients: in those who have the classical form of congenital adrenal hyperplasia (CAH) basal plasma 21-DF levels can attain more than 144 nmol/L. The synthesis of two isomers, E and Z, of the 21-deoxycortisol-3-carboxymethyloxime (CMO) hapten enabled us to prepare the corresponding E and Z immunogens by coupling them to bovine serum albumin (BSA), as well as the corresponding iodinated E and Z 21-DF-3-CMO-histamine tracers. We developed a very sensitive radioimmunoassay for 21-DF in plasma by associating an anti-21-DF-3-CMO-BSA-E isomer antibody to an iodinated 21-DF histamine-Z isomer (standard curve IC 50 = 8 pg/tube). This plasma 21-DF radioimmunoassay allowed diagnosis of the classical form of CAH in untreated newborn (basal 21-DF levels greater than 144 nmol/L), as well as the late-onset form (post-ACTH 21-DF levels greater than 11.54 nmol/L), and also permitted detection of 21-hydroxylase-deficient heterozygotes of both forms of CAH among the general population (post-ACTH 21-DF levels between 2.02 and 9.52 nmol/L).

Molecular characterization of monoclonal anti-steroid antibodies: primary structures of the variable regions of seven antibodies specific for 17 alpha-hydroxyprogesterone or 11-deoxycortisol and their pH-reactivity profiles.

The variable region nucleotide sequences of seven monoclonal anti-steroid antibodies that are specific for the closely related progesterone derivative, 11-deoxycortisol or 17 alpha-hydroxyprogesterone (17-OHP), have been determined by genomic cloning and DNA-sequencing or by direct mRNA-sequencing. As for their heavy chain variable regions, the nucleotide sequences of the SCET.M8.1 (SCET) and OHP 4B2.2.1 (4B2) antibodies were classified into the VH-9 family, while OHP 7D7.2.3 (7D7), 1E9.3.1 (1E9), 57.G6.1 (57) and 138.H8.1 (138) used VH-3 family genes. OHP 101.B11.1 (101) used a gene of the VH-1 family. For their light chain variable regions, SCET and 57 used VK-28 group genes, while 4B2, 7D7, 1E9, 101 and 138 antibodies used genes of the VK-21 subgroups (21A, 21B or 21C). All of the antibodies used different combinations of genes in the VH families and VK groups or subgroups. This indicates that the antibody response against the steroid hapten, 17-OHP, is fairly polyclonal, and several VH/VL combinations show high affinity for progesterone-related steroids. Although the primary structures of hypervariable loop regions of the mAbs were relatively diverse, generally, hydrophobic and aromatic amino acids were rich in these regions. Moreover, the length of heavy chain CDR3 was constant in all the antibodies investigated in this paper as well as the previously reported anti-progesterone monoclonal antibodies (mAbs). This suggests that the length of VH CDR3 in these mAbs has a considerable influence on the formation of antigen-combining pockets. The pH-reactivity profiles for the anti-17-OHP mAbs indicated that the the steroid-mAb binding was independent of pH between pH 4 and 11 in most of the mAbs. The results suggest that the steroid-mAb interactions are not largely affected by the electrostatic environments near the combining sites of these mAbs. Taken together, these data imply that the shape of hydrophobic depressions in the combining sites is important for the binding of relatively large, hydrophobic and rigid haptens like steroids.

Effects of glucocorticoid and antiglucocorticoid hormones on leukocyte numbers and function.

Hormones were administered to mice in seven daily intraperitoneal injections of saline suspensions. Progesterone and cortexolone, which often fail to act as antiglucocorticoids in vivo, were found to have antiglucocorticoid effects on the immune system under these conditions. The effects seen were increases in numbers of lymphocytes, monocytes, neutrophils and total leukocytes in the blood, increases in the number of peritoneal exudate cells and splenic plaque-forming cells, and increased splenocyte responses to the mitogen phytohemagglutinin. Deoxycorticosterone, sometimes also considered to be an antiglucocorticoid, acted only as a glucocorticoid here. Both deoxycorticosterone and the glucocorticoid corticosterone had effects opposite to those produced by progesterone and cortexolone on these parameters.

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.

Radioimmunoassay for 21-deoxycortisol: clinical applications.

A radioimmunoassay for 21-deoxycortisol is described. The immunogen, 21-deoxycortisol-3-(0-carboxymethyl) oxime-bovine serum albumin, was prepared, the antisera raised against it were studied and the reliability of the assay was checked. The antiserum selected cross-reacted with 11-deoxycortisol (0.08%), corticosterone (0.25%), cortisol (0.6%) and 17-hydroxyprogesterone (1.6%). 21-deoxycortisol was separated by celite partition chromatography and eluted in the 70/30 (v/v) isooctane/ethyl acetate fraction together with 11-deoxycortisol and corticosterone. The radioimmunoassay was used to measure 21-deoxycortisol in the plasma of normal subjects and patients with androgen excess. In normal subjects, men (0.19 ng/ml +/- 0.08) and women (0.18 ng/ml +/- 0.09) had similar basal levels (mean +/- SD). One hour after ACTH stimulation, these levels were increased by a factor of 3.5. In 7 patients treated for classical congenital adrenal hyperplasia associated with 21-hydroxylase deficiency, basal values varied between 9.1 and 39.9 ng/ml (measured at 8 a.m.). In 7 untreated women with late-onset congenital adrenal hyperplasia (with 21-hydroxylase deficiency), ACTH-stimulated levels were increased to between 9 and 25.5 ng/ml. In 14 heterozygous carriers of 21-hydroxylase deficiency, diagnosed by HLA genotyping, all ACTH-stimulated levels were well above the highest corresponding levels in normal subjects, whereas 17-hydroxyprogesterone levels remained within the normal range in 9 of the cases.

A simplified radioimmunoassay for plasma 11-deoxycortisol.

A simple, specific, accurate, precise and sensitive radioimmunoassay procedure developed for plasma 11-deoxycortisol is described. 1. The assay employs an anti-11-deoxycortisol serum generated against 11-deoxycortisol-3-(0-carboxymethyl) oxime coupled to bovine serum albumin, crystalline 11-deoxycortisol as standard, and [3H] 11-deoxycortisol as the radioactive ligand. 2. Cross-reactivity studies performed with structurally related steroids indicated cross reactivities with 17 alpha-hydroxyprogesterone, deoxycorticosterone and progesterone of 2.0%, 1.3% and 0.4% respectively; cortisone, corticosterone, cortisol, testosterone, less than 0.1%; and estrone, 17-beta-estradiol, estriol, and metyrapone less than 0.001%. Due to the high specificity of the anti-11-deoxycortisol serum, the method is simplified by the lack of need for chromatographic purification of the organic solvent extract of the plasma prior to the radioimmunoassay. The procedure was validated by comparing values for plasma 11-deoxycortisol with and without preliminary purification by chromatography on Sephadex LH-20 columns (y = 0.99 R/-x + 4.0, r = .98). Pretreatment of the plasma with n-hexane was found to eliminate interferences from high concentrations of 17 alpha-hydroxyprogesterone or progesterone. 3. Parallel dose-response curves were demonstrated between dilutions of plasma with elevated 11-deoxycortisol concentrations and the standard reference preparation. A non-specific binding less than 4% of the total [3H] 11-deoxycortisol was routinely observed. The detection limit of the assay was approximately 10 pg of 11-deoxycortisol which corresponds to a plasma concentration of approximately 0.7 micrograms/L 4. The analytical recovery of 11-deoxycortisol added to human plasma varied from 88 to 108%, with a mean recovery of 100%. The inter-assay variation was determined by assaying (n = 30) three different quality control pools. The following data were obtained: x 1 = 3.8 +/- 0.6 micrograms/L (CV = 15.8%); x 2 = 18.5 +/- 2.0 micrograms/L (CV = 10.8%); x 3 = 43.0 +/- 3.7 micrograms/dl (CV = 8.6%).

7 alpha- and 7 beta-carboxymethyl-derivatives of 17-hydroxyprogesterone and 11-deoxycortisol. Synthesis and immunogenic properties.

7 alpha- and 7 beta-carboxymethyl-derivatives of 17-hydroxyprogesterone and 11-deoxycortisol have been synthesized. After coupling to bovine serum albumin, they were used to elicit antibodies in rabbits. No major difference in the steroid specificity of the antisera was observed when either 7 alpha- or 7 beta-epimers were used for immunization. In both cases, highly specific antisera were obtained which may possibly be used to assay human plasma 17-hydroxyprogesterone and 11-deoxycortisol without chromatographic purification.

Compounds in the urinary pH 1 extract reacting with corticosteroid and tetrahydrocorticosteroid antisera. Separation of a nonpolar substance with affinity to a tetrahydroaldosterone antiserum.

In human urinary pH 1 extracts prepared for the aldosterone-18-glucuronide estimation, several other substances are present, crossreacting not only with aldosterone antisera, but also with various corticosteroid and tetrahydrocorticosteroid antisera. Aldosterone was measured before and after chromatographic purification. Further characterization of the non-aldosterone immunoreactive material was made by immunological analysis of paper chromatogram eluats. Pregnancy, and administration of ACTH, dexamethasone, and metopirone led to a change of excretion in the antigenic equivalents. A method for the separation of the antigenic material is described. For structural elucidation the gaschromatography-mass spectrometry (GC-MS) method was applied.