Competitive light-initiated chemiluminescent assay: using 5-α-dihydrotestosterone-BSA as competitive antigen for quantitation of total testosterone in human sera.

This paper described a homogeneous method, light-initiated chemiluminescent assay (LICA), for quantitation of total testosterone in human sera. The assay was bead based and built on a competitive-binding reaction format, in which 5-α-dihydrotestosterone (5-α-DHT) competed with the testosterone in serum samples in binding with biotinylated anti-testosterone antibody. The more testosterone in the serum sample, the less 5-α-DHT that bonded with biotinylated anti-testosterone antibodies. 5-α-DHT was coupled with emission beads (doped with thioxene derivatives and Eu(III) as a chemiluminescence emitter) via bovine serum albumin as a linker. Once streptavidin-coated sensitizer beads (modified with phthalocyanine as a photosensitizer) were added, the streptavidin/biotin reaction between 5-α-DHT-bound anti-testosterone antibody and sensitizer beads could bring emission and sensitizer beads together, which allowed energy transfer from sensitizer bead to emission bead. As such, an exciting light (680 nm) impinging on the sensitizer beads led to light emission at 520-620 nm by emission beads. The strength of the emitted light was inversely proportional to the testosterone in serum sample. The detection range of this assay was from 13.3 to 1200 ng/dL. The coefficient variation for intra- and inter-assay was lower than 15%. The recovery of this method ranged from 95.5 to 105.9% for different samples. Moreover, the LICA assay was highly specific with low cross-reactivity and interference. The concentration of testosterone from 58 serum samples analyzed by the LICA method significantly correlated (y = 0.97x + 1.87, R2 = 0.970, p < 0.001) with those obtained with the SIEMENS Centaur Xp System. Graphical abstract ᅟ.

Androgen and Androgen Receptor as Enhancers of M2 Macrophage Polarization in Allergic Lung Inflammation.

Allergic asthma is a disease initiated by a breach of the lung mucosal barrier and an inappropriate Th2 inflammatory immune response that results in M2 polarization of alveolar macrophages (AM). The number of M2 macrophages in the airway correlates with asthma severity in humans. Sex differences in asthma suggest that sex hormones modify lung inflammation and macrophage polarization. Asthmatic women have more M2 macrophages than asthmatic men and androgens have been used as an experimental asthma treatment. In this study, we demonstrate that although androgen (dihydrotestosterone) reconstitution of castrated mice reduced lung inflammation in a mouse model of allergic lung inflammation, it enhanced M2 polarization of AM. This indicates a cell-specific role for androgens. Dihydrotestosterone also enhanced IL-4-stimulated M2 macrophage polarization in vitro. Using mice lacking androgen receptor (AR) in monocytes/macrophages (ARfloxLysMCre), we found that male but not female mice exhibited less eosinophil recruitment and lung inflammation due to impaired M2 polarization. There was a reduction in eosinophil-recruiting chemokines and IL-5 in AR-deficient AM. These data reveal an unexpected and novel role for androgen/AR in promoting M2 macrophage polarization. Our findings are also important for understanding pathology in diseases promoted by M2 macrophages and androgens, such as asthma, eosinophilic esophagitis, and prostate cancer, and for designing new approaches to treatment.

Dihydrotestosterone levels at birth associate positively with higher proportions of circulating immature/naïve CD5+ B cells in boys.

Boys present with higher proportions of immature/naïve CD5+ B cells than girls up to 3 years of age. Boys also have higher fractions of regulatory T cells (Tregs) in early infancy, but the mechanisms for these sex-related differences are unknown. In the prospective FARMFLORA follow-up study of 23 boys and 25 girls, we investigated if these immunological differences remained at 8 years of age. We also examined if testosterone or dihydrotestosterone (DHT) levels at birth and at 8 years of age were associated with immune maturation. Immunological variables and androgen levels were examined and measured in blood samples obtained at birth, 3-5 days and at 8 years of age. Boys had higher proportions of CD5+ and immature/transitional CD24hiCD38hi B cells, whereas girls had higher fractions of B cells with a memory phenotype at 8 years of age. School-aged boys also presented with higher frequencies of Tregs, and a greater capacity to produce T-cell-associated cytokines. Among boys, higher cord blood DHT levels were associated with higher proportions of CD5+ B cells in early infancy and at 8 years of life. These results suggest that DHT actions in utero might be involved in the mechanism for delayed peripheral B-cell maturation in boys.

Gender-specific expression of beta1 integrin of VLA-4 in myelin basic protein-primed T cells: implications for gender bias in multiple sclerosis.

Susceptibility to multiple sclerosis is higher in females than males. However, the underlying mechanism behind this gender difference is poorly understood. Because the presence of neuroantigen-primed T cells in the CNS is necessary to initiate the neuroinflammatory cascade of multiple sclerosis, we first investigated how these T cells interacted with astroglia, major resident glial cells of the CNS. Interestingly, we found that myelin basic protein (MBP)-primed T cells from female and castrated male mice, but not from male mice, produced proinflammatory molecules, such as NO, IL-1beta, and IL-6 in astroglia, and these responses were purely via contact between T cells and astroglia. Because T cell:glia contact requires several integrin molecules, we examined the involvement of integrins in this process. Both alpha4 and beta1, subunits of VLA-4 integrin, were found to be necessary for T cell contact-induced generation of proinflammatory molecules in astroglia. Interestingly, the expression of beta1, but not alpha4, was absent in male MBP-primed T cells. In contrast, female and castrated male MBP-primed T cells expressed both alpha4 and beta1. Similarly, we also detected beta1 in spleen of normal young female, but not male, mice. Furthermore, we show that male sex hormones (testosterone and dihydrotestosterone), but not female sex hormones (estrogen and progesterone), were able to suppress the mRNA expression of beta1 in female MBP-primed T cells. These studies suggest that beta1, but not alpha4, integrin of VLA-4 is the sex-specific molecule on T cell surface, and that the presence or absence of beta1 determines gender-specific T cell contact-mediated glial activation.

Analysis of anabolic steroids in the horse: development of a generic ELISA for the screening of 17alpha-alkyl anabolic steroid metabolites.

Due to the potential for misuse of a wide range of anabolic steroids in horse racing, a screening test to detect multiple compounds, via a common class of metabolites, would be a valuable forensic tool. An enzyme-linked immunosorbent assay (ELISA) has been developed to detect 17alpha-alkyl anabolic steroid metabolites in equine urine. 16beta-Hydroxymestanolone (16beta,17beta-dihydroxy-17alpha-methyl-5alpha-androstan-3-one) was synthesised in six steps from commercially available epiandrosterone (3beta-hydroxy-5alpha-androstan-17-one). Polyclonal antibodies were raised in sheep, employing mestanolone (17beta-hydroxy-17alpha-methyl-5alpha-androstan-3-one) or 16beta-hydroxymestanolone conjugated to human serum albumin, via a 3-carboxymethyloxime linker, as antigens. Antibody cross-reactivities were determined by assessing the ability of a library of 54 representative steroids to competitively bind the antibodies. Antibodies raised against 16beta-hydroxymestanolone showed excellent cross-reactivities for all of the 16beta,17beta-dihydroxy-17alpha-methyl steroids analysed and an ELISA has been developed to detect these steroid metabolites. Using this 16beta-hydroxymestanolone assay, urine samples from horses administered with stanozolol (17alpha-methyl-pyrazolo[4',3':2,3]-5alpha-androstan-17beta-ol), were analysed raw, following beta-glucuronidase hydrolysis, and following solid-phase extraction (SPE) procedures. The suppressed absorbances observed were consistent with detection of the metabolite 16beta-hydroxystanozolol. Positive screening results were confirmed by comparison with standard LCMS analyses. Antibodies raised against mestanolone were also used to develop an ELISA and this was used to detect metabolites retaining the parent D-ring structure following methandriol (17alpha-methylandrost-5-ene-3beta,17beta-diol) administration. The ELISA methods developed have application as primary screening tools for detection of new and known anabolic steroid metabolites.

Experimental autoimmune prostatitis: dihydrotestosterone influence over the immune response.

PURPOSE: In experimental autoimmune prostatitis in a rat model of chronic prostatic inflammation of noninfectious origin the prostatic 5alpha-dihydrotestosterone (DHT) concentration decreases because of depressed 5alpha-reductase activity. This decrease in androgens in situ could favor the development of autoimmune status at the same time. We noted that a DHT increase could protect the gland from immune aggression and/or its consequences in regard to prostatic androgenic metabolism. MATERIALS AND METHODS: We analyzed in vitro the (3H)-DHT enzymatic bioconversion of prostate homogenates of male accessory sexual gland extract (MAG) immunized rats and MAG immunized plus DHT implanted rats (DSG rats), and performed ventral prostate histological observations. The specific cell immune response against MAG antigen(s) was studied by delayed type hypersensitivity. RESULTS: In DSG and MAG rats, and controls enzymatic activities (3alpha/3beta-hydroxysteroid oxidoreductases) were 112.7 +/- 11.3, 91.4 +/- 15.0 (not significant) and 147.0 +/- 12.8 pmol per minute per mg protein (p <0.025). Histological findings in DSG rat ventral prostates revealed infiltrating mononuclear cell foci in lower quantity and less magnitude than in MAG rat prostates. Delayed type hypersensitivity values were positive in MAG rats and lower in DSG rats in relation to kidney treated and untreated rats. CONCLUSIONS: Results suggest that constantly elevated DHT levels could decrease the cell immune response but not at significantly. In contrast, androgenic metabolism remains altered in the presence of exogenous androgens.

Immunization against 5alpha-androstane-3alpha,17beta-diol affects ovarian folliculogenesis in Merino ewes.

The 5alpha-reduced androgens have been implicated as antagonists of follicular development. In this experiment, we examined the effect of active immunization against 5alpha-reduced androgen on follicular development in ewes. During the breeding season, cyclic Merino ewes were either actively immunized three times against 5alpha-androstane-3alpha,17beta-diol (3alpha-diol) or served as controls. Six to nine weeks after the last immunization, they were treated with PGF(2alpha) analog (PG, 125mg cloprostenol i.m.) and luteolysis was induced. Fourteen days after the PG treatment, the ewes were either killed (mid-luteal phase) or treated a second time with PG and killed 24h later (early follicular phase). At slaughter, blood samples were collected and ovaries recovered. All CL and follicles larger than 2mm were dissected and their size and appearance were recorded. Follicular fluid was collected and concentrations of estradiol-17beta (E(2)), progesterone (P), androstenedione (A(4)), testosterone (T), dihydrotestosterone (DHT), 5alpha-androstane-3alpha-ol,17beta-one (androsterone: 3alpha-ol) and 3alpha-diol were determined by RIA. Immunization induced antibodies primarily to DHT and its 5alpha-reduced substrates 3alpha-diol and 3alpha-ol but not to E(2), P, A(4) or T. Immunization increased ovulation rate, size of ovulatory follicles and weight of CL. Immunization appeared to increase ovulation rate by decreasing the incidence of atresia in large preovulatory follicles. Regardless of their physiological status follicles contained only low levels of DHT; 3alpha-ol and 3alpha-diol were not detected in most follicles. Immunization did not appear to affect levels of DHT or other steroids in the follicular fluid. In conclusion, the induction of antibodies to 5alpha-reduced androgens increases ovulation rate by enhancing follicular viability during the preovulatory period in ewes. However, this effect is not brought about by the direct immune-neutralization of DHT or its 5alpha-reduced substrates 3alpha-ol and 3alpha-diol at the ovarian level.

Analysis of the binding energies of testosterone, 5alpha-dihydrotestosterone, androstenedione and dehydroepiandrosterone sulfate with an antitestosterone antibody.

Molecular dynamics simulations and molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) free energy calculations were used to study the binding of testosterone (TES), 5alpha-dihydrotestosterone (5ADHT), androstenedione (AND), and dehydroepiandrosterone sulfate (DHEAS) to the monoclonal antitestosterone antibody 3-C(4)F(5). The relative binding free energy of TES and AND was also calculated with free energy perturbation (FEP) simulations. The antibody 3-C(4)F(5) has a relatively high affinity (3 x 10(8) M(-1)) and on overall good binding profile for testosterone but its cross-reactivity with DHEAS has been the main reason for the failure to use this antibody in clinical immunoassays. The relative binding free energies obtained with the MM-PBSA method were 1.5 kcal/mol for 5ADHT, 3.8 kcal/mol for AND, and 4.3 kcal/mol for DHEAS, as compared to TES. When a water molecule of the ligand binding site, observed in the antibody-TES crystal structure, was explicitly included in MM-PBSA calculations, the relative binding energies were 3.4, 4.9, and 5.4 kcal/mol for 5ADHT, AND, and DHEAS, respectively. The calculated numbers are in correct order but larger than the corresponding experimental energies of 1.3, 1.5, and 2.6 kcal/mol, respectively. The fact that the MM-PBSA method reproduced the relative binding free energies of DHEAS, a steroid having a negatively charged sulfate group, and the neutrally charged TES, 5ADHT, and AND in satisfactory agreement with experiment shows the robustness of the method in predicting relative binding affinities. The 800-ps FEP simulations predicted that the antibody 3-C(4)F(5) binds TES 1.3 kcal/mol tighter than AND. Computational mutagenesis of selected amino acid residues of the ligand binding site revealed that the lower affinities of AND and DHEAS as compared to TES are due to a combined effect of several residues, each contributing a small fraction to the tighter binding of TES. An exception to this is Tyr99H, whose mutation to Ala lowered the binding of DHEAS 0.7 kcal/mol more than the binding of TES. This is probably due to the hydrogen bonding interaction formed between the OH group of Tyr99H and the sulfate group of DHEAS. Computational mutagensis data also showed that the affinity of the steroids to the antitestosterone antibody 3-C(4)F(5) would be enhanced if Trp47H were repositioned so that it would make more extensive contacts with the bound ligands. In addition, the binding of steroids to antitestosterone, antiprogesterone, and antiestradiol antibodies is discussed.

Sex steroid hormones enhance immune function in male and female Siberian hamsters.

Immune function is better in females than in males of many vertebrate species, and this dimorphism has been attributed to the presence of immunosuppressive androgens in males. We investigated the influence of sex steroid hormones on immune function in male and female Siberian hamsters. Previous studies indicated that immune function was impaired in male and female hamsters housed under short-day photoperiods when androgen and estrogen concentrations were virtually undetectable. In experiment 1, animals were gonadally intact, gonadectomized (gx), or gx with hormone replacement. Females exhibited the expected increase in antibody production over males, independent of hormone treatment condition, whereas male and female gx animals exhibited decreased lymphocyte proliferation to the T cell mitogen, phytohemagglutinin (PHA) compared with intact animals, and this effect was reversed in gx hamsters following testosterone and estradiol treatment, respectively. In experiment 2, testosterone, dihydrotestosterone, and estradiol all enhanced cell-mediated immunity in vitro, suggesting that sex steroid hormones may be enhancing immune function through direct actions on immune cells. In experiment 3, an acute mitogen challenge of lipopolysaccharide significantly suppressed lymphocyte proliferation to PHA in intact males but not females, suggesting that males may be less reactive to a subsequent mitogenic challenge than females. Contrary to evidence in many species such as rats, mice, and humans, these data suggest that sex steroid hormones enhance immunity in both male and female Siberian hamsters.

Testosterone and/or low estradiol: normally required but harmful immunologically for males after trauma-hemorrhage.

BACKGROUND: Previous studies indicate that after severe hemorrhage, immune functions are markedly depressed in males, whereas females do not show any depression. Although androgen depletion by castration of mice before soft-tissue trauma and hemorrhagic shock prevents the depression of cell-mediated immunity, it remains unknown whether testosterone per se is responsible for producing immune depression. METHODS: Female C3H/HeN mice were pretreated with 5alpha-dihydrotestosterone (DHT) or vehicle for 20 days. The mice then underwent soft-tissue trauma (laparotomy) and hemorrhagic shock (blood pressure 35+/-5 mm Hg for 90 minutes) followed by adequate fluid resuscitation (shed blood and lactated Ringer's solution) or sham operation. Two groups of nontreated male C3H/HeN mice were included as controls: one group was subjected to hemorrhagic shock followed by resuscitation, and the second group underwent only sham operation. At 24 hours after trauma-hemorrhage and resuscitation, animals were killed, macrophages harvested from the peritoneum and spleen, and their ability to release interleukin (IL)-1 and IL-6 was evaluated. Plasma DHT, estradiol, and corticosterone levels were measured by radioimmunoassay. RESULTS: Treatment of female mice with DHT produces a significant increase in DHT levels that was comparable with those seen in nontreated male mice. Alternatively, estradiol levels in female mice were significantly depressed by DHT treatment to levels comparable with those observed in control males. In the vehicle-treated female mice, no depression of the macrophage function was evident after trauma hemorrhage. In contrast, testosterone-treated female mice that had experienced hemorrhage showed significant depression in splenic and peritoneal macrophage IL-1 and IL-6 production, comparable with the values seen in macrophages from male mice that had experienced hemorrhage. CONCLUSIONS: These findings indicate that pretreatment of female mice with DHT depresses macrophage function after trauma-hemorrhage, which mimics the changes seen in normal male mice subjected to trauma-hemorrhage. We propose, therefore, that high testosterone and/or low estradiol levels are responsible for producing the immune depression in male mice after trauma-hemorrhage. Testosterone receptor blocking agents, e.g., flutamide, and/or estradiol administration should thus be useful adjuncts for preventing immune depression in male trauma patients.

Ontogeny of immunocytochemically demonstrable androgen- and androgen receptor-containing cells in the hypothalamus and adenohypophysis of the chick embryo.

Brain sections of male chick embryos, 6.5-18.5 days of age, were examined immunocytochemically for the presence of androgen- and androgen receptor-containing cells in the hypothalamus and adenohypophyseal pars distalis. Using antibodies (Ab) against both androgens (T-Ab) and the androgen receptor (AR-Ab), single- and double-immunostained cells were located in a total of five nuclei of the anterior-, mid-, and posterior-hypothalamus, as well as in the rostral and caudal lobes of the adenohypophyseal pars distalis. From Days 9.5-12.5, the mean number of androgen-immunostained cells within the hypothalamus and pars distalis increased significantly (P < 0.01), while from Days 12.5-18.5 there was no further statistically significant increase. The results of the present investigation support previous findings which suggest that in the chick embryo the negative feedback loop of the hypothalamo-adenohypophyseal-testicular (HATest) axis is functional by the 13th day of development (Woods et al., 1989a,b). They also agree with the observations of Wilson and Glick (1970) that in the male chick embryo testosterone organizes masculine mating behavior prior to Day 13.0.

Synthesis of cobalt carbonyl complexes of cortisol and testosterone. Study of their recognition by specific polyclonal antibodies.

We have synthesized organometallic complexes of steroids (cortisol, testosterone, dihydrotestosterone) for potential use as tracers in nonisotopic carbonyl-metal immunoassays (CMIA). An ethynyl/CO2(CO)6 fragment at the end of a five-atom spacer was coupled to position 3 of the steroid skeleton. In the case of cortisol, we exploited the difference in reactivity of the ketone and enone functions toward amines in order to form an enamine which was then made to react with carboxymethylamine to yield 3-[(carboxymethyl)oxime] steroid. Activation of the carboxylic acid function with N,N'-dicyclohexylcarbodiimide in the presence of propargylamine introduced an acetylenic function at the end of the spacer. The triple bond was then complexed by CO2(CO)8 to form complexes 5a-c. Complexes for use in CMIA should be stable in biologic media and effectively recognize specific antibodies. Complexes 5a-c were stable in the buffers we use in biochemical tests. Their cross reactivities for anti-cortisol and anti-testosterone antibodies ranged from 50 to 110% according to batch, indicating, first, that the addition of an organometallic complex in position 3 of the steroid skeleton does not hinder recognition between the organometallic steroid and antibody and, second, that their individual behavior differs substantially according to antibody batch. Although all of these complexes could be used as tracers in CMIA, it is necessary, in each case, to establish which tracer-antibody duo gives rise to the most sensitive immunoassay.

Estradiol and testosterone in minor salivary glands of Sjögren's syndrome.

Nine patients with Sjögren's syndrome were studied in terms of estradiol, testosterone, and dihydrotestosterone in the labial minor salivary glands using the peroxidase-antiperoxidase method. In normal controls in women, estradiol was positive in the epithelial cells of duct, but testosterone and dihydrotestosterone were negative or doubtfully positive by case. Thus, it seems that there is a sex difference of receptors in the ductal epithelia. In the labial minor salivary glands of the patients, all estradiol, testosterone, and dihydrotestosterone were positive. As the background of Sjögren's syndrome, it seems that there is an influence of sex hormones.

Influence of different combinations of antibodies and penicillinase-labeled testosterone derivatives on sensitivity and specificity of immunoassays.

Three antisera raised against bovine serum albumin (BSA) conjugates of testosterone-3-(O-carboxy-methyl)-oxime (T-3-CMO), 11 beta-hydroxytestosterone-11-carboxymethyl ether (T-11 beta-O-CME) and 19-hydroxytestosterone-19-carboxymethyl-ether (T-19-O-CME) were evaluated in enzyme immunoassays (EIAs) in combinations with penicillinase-labeled T-3-CMO, T-11 beta-O-CME, T-19-O-CME, and testosterone-17 beta-hemisuccinate (T-17 beta-HS) for their influence on the sensitivity and specificity of EIAs. Of the various combinations, anti-T-3-CMO antiserum along with T-11 beta-O-CME-penicillinase showed no cross-reaction with any of the closely related steroids, although the same antibody had 21.6% binding to 5 alpha-dihydrotestosterone (5 alpha-DHT) in radioimmunoassay. All the homologous combinations appeared to be less sensitive due to their low affinity for testosterone. It was also apparent that of all the heterologous systems tested, only two combinations, (a) anti-T-19-O-CME antiserum and T-3-CMO-penicillinase and (b) anti-T-3-CMO antiserum and T-11 beta-O-CME-penicillinase, were found to be more sensitive. The former was less specific; it showed 70% cross-reaction with 5 alpha-DHT. The ability of testosterone to displace the hapten-enzyme conjugate and the specificity of the assay appear to depend on the position of the enzyme label on the steroid molecule as well as on the availability of antigenic sites in particular combinations of antibody and hapten-enzyme conjugates.

New steroid haptens for radioimmunoassay: synthesis of steroids substituted with thioether or ester linkages at the 2 alpha-position.

Haptens with bridge at the 2-position have not yet been explored. Radioimmunoassays with antibodies directed against 2 alpha-alkyl bridged steroid haptens are expected to be highly specific due to greater topographical exposure and similarity in conformation to the native steroid. The 2 alpha-alkyl bridged haptens were synthesized by first adding a cyclopropane ring to 2-methylene-4-en-3-one. Selective opening of the three-membered ring with trimethyl silyl iodide and transformation of the iodo group gave a carbocyclic acid, the desired analog for conjugation with protein.

The development of a radioimmunoassay system for testosterone (T) and dihydrotestosterone (DHT). Part 2. The preparation of antisera to T.

Testosterone-3-O-carboxymethyl-oxime derivative was synthetized and coupled to bovine serum albumin (BSA). The T-3-CMO-BSA conjugate homogenized with Freund's adjuvant used as immunogen was injected multiple sites in rabbits. The antisera collected were characterized in a radioimmunological system, separation with dextran-charcoal using 125I-Testosterone as tracer. The antibody titres varied from one animal to another. The titre of anti-T serum selected for RIA was 1: 10(4)-1: 2 X 10(4) (initial dilution). All anti-T sera 100 percent crossreacted with 5 alpha-dihydrotestosterone but nonsignificant interference was observed with other C19, C21 and C18 steroids. The affinity constant of the selected anti-T serum was in the range 1.4-1.9 X 10(9) litres/mole. The data so far published on the antisera toward testosterone are reviewed. We conclude that the selected anti-T-3-CMO-BSA serum may provide assays for testosterone with potential for clinical applications.

The development of a radioimmunoassay system for testosterone (T) and dihydrotestosterone (DHT). Part 3. The preparation of radioiodinated testosterone.

A two step-method was applied for the preparation of a tracer adequate for a radioimmunoassay (RIA) system for testosterone. Histamine was radioiodinated by the Chloramine-T method and then coupled to testosterone-3-carboxymethyl-oxime (T-3-CMO) derivative. After purification by TLC, the steroid tracer was stable in ethanol for at least four months. Using an anti-T-3-CMO-BSA serum, a (bridge) homologous RIA system for testosterone was developed. The reagents (antiserum, tracer and standard) were incubated 2 hrs at 37 degrees C and then the free radioactivity was removed by a dextran-charcoal suspension. The testosterone RIA system, with the sensitivity of 30 pg/tube, is suitable for the measurement of the steroid hormone in biological fluids and tissues.

Characterisation of monoclonal and polyclonal antibodies to 19-linked conjugates of testosterone with corresponding radioligands.

Monoclonal antibodies to testosterone T were produced using testosterone 19-O-carboxymethyl ether (T19C) and testosterone 19-hemisuccinate (T19H) immunogens. All antibodies were characterised with iodinated derivatives of both T19C and T19H. Monoclonal antibodies derived from the T19C immunogen had similar titres and assay sensitivities with both T19-tracers. In contrast antibodies derived from the T19H immunogen bound the homologous but not the heterologous tracer. Individual antibodies showed a wide variation in cross-reactivity with 5 alpha-dihydrotestosterone, DHT (4.4-100%), androstenedione AN (0.5-100%) and progesterone, Po (0.08-5.4%). One antibody 3F11 derived from a T19C immunogen gave 50% displacement of tracer with 180 pgT/tube and low cross-reactivity of 12% with DHT, 3.0% with AN and 1.1% with Po. In general, assay sensitivity and antibody specificity were poorer with an [125I]-histamine conjugate of T-3-carboxymethyloxime than with T19 tracers. Radioimmunoassays for T in extracted human serum were developed with [125I]T19C as tracer and monoclonal antibody 3F11 (T19C immunogen) and rabbit antiserum T19H3R1 (T19H immunogen). Sensitivities of the extracted assays were 43 and 20 pg/tube respectively and results correlated well with those obtained after chromatographic separation of testosterone (r = 0.97 for both antibodies). We conclude that 19-linked derivatives of T are highly immunogenic for the production of specific testosterone antibodies. Selection of the appropriate iodinated tracer is essential to achieve optimal titre, assay sensitivity and specificity, since these characteristics vary widely with individual monoclonal antibodies, and classical bridge recognition is not observed.

Monoclonal antibodies to testosterone: the effect of immunogen structure on specificity.

A 15 beta-thioalkyl derivative of testosterone conjugated to bovine serum albumin (BSA) was synthesised and used to produce monoclonal antibodies. These antibodies were evaluated using 15 beta-(2-carboxyphenylthio)-testosterone [125I]histamine as radioligand. Out of 1368 hybrids, 5 secreted anti-testosterone antibodies. These were compared with monoclonal antibodies derived from immunisation with testosterone-3-carboxymethyloxime-BSA. The first group of monoclonal antibodies all showed very low cross-reactivity with 5 alpha-dihydrotestosterone (less than 2.8%) indicating that this site of linkage is a good choice for discriminating between differences at the 4-5 position in the A-ring on the testosterone molecule. However they generally showed much higher cross-reactivity with progesterone and androstenedione than monoclonal antibodies raised to the 3-linked immunogen. Nevertheless within each fusion there were monoclonal antibodies with markedly different specificities. None of these antibodies could be considered suitable for use in a testosterone immunoassay, but it does suggest that an antibody with an improved specificity profile could be found using the monoclonal antibody approach.

Plasma testosterone levels in rats under various conditions.

A radioimmunoassay (RIA) for testosterone (T) was found to be suitable for determination in plasma of rats. Since the cross-reactive dihydrotestosterone levels were extremely low both in males (130.8 +/- 27.6 pmol/l) and females (189.4 +/- 41.3 pmol/l), the procedure measured T equally well, whether or not chromatography preceded the RIA. The method met all requirements of precision, sensitivity, accuracy and specificity. Basal levels of 5 male control groups (n approximately equal to 10) showed considerable variations from a low of 6.86 +/- 4.68 nmol/l to a high of 16.26 +/- 5.06 nmol/l. This high variability may be the reason, at least in part, for the fact that moderate stress did not produce always a significant decrease of T levels in males. However, after forced stress or administration of adrenaline plasma T was significantly (p less than 0.01) lower than in controls. Gonadectomy lowered extremely plasma T levels both in males and females, whereas adrenalectomy had no significant effect. These data demonstrate that the gonads are the main sources of T in both sexes. The significant (p less than 0.01) decrease of T levels after administration of adrenaline, dexamethasone and ACTH in adrenalectomized males indicated that these substances affect mainly the testicular synthesis of T. In cyclic females, administration of ACTH produced no significant change in plasma T levels. During pregnancy, there was a significant (p less than 0.01) increase of plasma T values from day 10 to day 18-19, followed by a decrease to day 22.