Plasma 11beta-hydroxy-4-androstene-3,17-dione: comparison of a time-resolved fluoroimmunoassay using a biotinylated tracer with a radioimmunoassay using a tritiated tracer

The plasma concentration of 11β-hydroxy-4-androstene-3,17-dione (11β) is very high in 21-hydroxylase deficiency, Cushing’s syndrome, and hyperandrogenism of adrenal origin, and very low in congenital 11-hydroxylase deficiency and adrenal insufficiency. Thus, when plasma 4-androstenedione is elevated, it is useful to measure the plasma 11β level in order to determine the adrenal or ovarian origin of the hyperandrogenism.

To eliminate disadvantages related to the 11β radioimmunoassay (RIA), which uses a tritiated tracer, as well as the high cost associated with scintillation proximity assay (SPA), we developed a non-isotopic 11β assay that utilizes an 11β-biotin conjugate synthesized in our laboratory to measure time-resolved fluorescence after addition of streptavidin-europium to microtitration wells.

The analytical qualities of this assay are very similar to those of the radioimmunoassay using a tritiated tracer, and an extraction step followed by celite chromatography (which separates 11β from interfering plasma steroids) prior to a final radioimmuno-competition step. The correlation coefficient between 11β levels measured by time-resolved plasma 11β fluoroimmunoassay (TR-FIA) and RIA was 0.965.

Finally, the TR-FIA technique was more sensitive and of greater precision than the RIA method.     

Preparation of 125I polypeptide hormones for radioimmunoassay using glucose oxidase with latoperoxidase.

The hydrogen peroxide-generating property of glucose oxidase was used in combination with the catalytic property of lactoperoxidase to radioiodinate polypeptide hormones with ¹²⁵-iodine. This rapid, gentle, enzymatic method reproducibly results in higher yields of iodinated hormones for use in radioimmunoassays.     

Development of a highly sensitive and specific new testosterone time-resolved fluoroimmunoassay in human serum

A new time-resolved fluoroimmunoassay (TR-FIA) of testosterone in serum is described, using a biotinylated testosterone tracer, with a long spacer arm between biotin and testosterone, coupled to the C3 of the testosterone: a biotinylaminodecane carboxymethyloxime testosterone. This tracer affords a great sensitivity of the standard curve, because a amount of 0.3 pg of testosterone can be significantly measured on the testosterone standard curve. The "functional" sensitivity is at least equal to 21 pg/ml of serum. The specificity of the assay is insured by a celite chromatographic step on new minicolumns before immunoassay. The variation coefficient of inter-series reproducibility measured on low and normal testosterone levels in untreated and testosterone treated hypogonadal men were between 2.17 and 5.07%. The accuracy test, (overload and dilution tests) gave satisfying results. Moreover, in a comparison with GCMS, it appeared that the correlation coefficient was 0.992 and no significant difference could be exhibited between the two methods. Consequently, this specific, sensitive reproducible and easy to use method is well suited to the measurement of testosterone in clinical and pharmacological conditions.     

Radioimmunoassay of unconjugated and total serum estetrol using a 125I-iodinated tracer

A radioimmunossay (RIA) for the measurement of both unconjugated and total serum estetrol has been developed, using an antiserum to an E4-3-conjugate and a 125I-radioiodinated E4 tracer. Assay of dried ethyl ether extracts was used for the determination of unconjugated E4, while a direct measurement of unextracted hydrolyzed serum in the presence of 0.3% 8-anilino-1-naphthalene sulphonic acid (ANS) proved adequate for total E4. Assay reliability was evaluated and the procedure standardized through a series of tests aimed at assessing accuracy, sensitivity and precision. No steroidal interference was found to practically affect the assay (0.3% estriol cross-reactivity), nor were solvent and sample blanks observed in the case of unconjugated E4. For total E4 assay, the sample blank effects were acceptably overcome by using hydrolyzed male serum and 0.3% ANS, as a standard diluent. An interassay variability amounting to approximately 10 and 6% resulted for unconjugated E4 and total E4 RIA, respectively. A number of serum samples (285 for unconjugated E4, 147 for total E4) randomly collected throughout normal pregnancy were assayed. The unconjugated E4 levels at 15th week and at term were 62.7 +/- 22.6 and 766.5 +/- 208.2 (SD) pg/ml, respectively. Total E4 was about 6--7 times higher than the levels of free E4 and increased 7 times from the 15th week to term.     

Determination of bufalin-like immunoreactivity in serum of humans and rats by time-resolved fluoroimmunoassay for using a monoclonal antibody

The existence of a mammalian natriuretic substance or endogenous digitalis-like factor, which inhibits Na+,K+-ATPase and thereby regulates body fluid volume, has been speculated for a long time but has yet to be defined. We established in the present study a simple and highly sensitive procedure to measure bufalin, a constituent of toad venom preparation and a specific inhibitor of Na+,K+-ATPase by time-resolved fluoroimmunoassay (TR-FIA) and using a monoclonal antibody. The antibody was specific to bufalin and resembled bufadienolides but showed no cross-reactivity with digitoxin and ouabain. A bufalin-like immunoreactivity was detectable in serum of humans and rats by the proposed TR-FIA. The levels of bufalin-like immunoreactivity in serum of healthy volunteers were significantly correlated with their systolic blood pressure. Moreover, bufalin-like immunoreactivity in serum of Dahl-S rats increased in parallel with a period of high-salt diet. These results suggest that increased bufalin-like immunoreactivity may be associated with certain types of hypertension.     

Development of a sensitive and specific new plasma 4-androstene-3,17- dione time-resolved fluoroimmunoassay (TR-FIA)

We describe, for the first time to our knowledge, the development of a new, non-isotopic time resolved-fluoroimmunoassay of 4-androstene-3,17-dione in plasma or serum. This steroid exhibits a key role in steroid metabolism and is often assayed in the investigation of various pathologic endocrine states. Most of the 4-androstene-3,17-dione immunoassays are performed using a radioactive tracer. We synthesized a biotinylated 4-androstene-3,17-dione tracer from 4-androstene-3,17-dione-3-carboxymethyloxime by acylation of biotinylaminopropylammonium trifluoroacetate. A specific rabbit anti 6-hemisuccinate-4-androstene-3,17-dione/BSA was indirectly bound via an anti-rabbit sheep antibody immobilized on microtiter plate wells. The amount of biotinylated-4-androstene-3,17-dione tracer was then measured by adding streptavidin-europium, and the europium fluorescence was quantified by time resolved-fluorescence (TR-FIA, Delfia System). The plasma 4-androstene-3,17-dione-levels measured with this non-isotopic assay were compared to those measured with a radioimmunoassay previously published. In both cases, the same anti-4-androstene-3,17-dione antibody was used, and the assays were performed after an extraction step and a chromatographic step. The results obtained by the two methods were virtually the same. However, the main advantages of the new plasma 4-androstenedione-3,17-dione time-resolved-fluorescence immunoassay were its greater sensitivity than radioimmunoassay and its higher precision.     

Measurement of cholecystokinin octapeptide using a new specific radioimmunoassay

A peptide analogue of CCK-8 (Tyroc) which has a tyrosine in place of the amide group in the C-terminal end, has been used both for raising antisera and for iodination. The antisera produced by immunisation with Tyroc are directed towards the N-terminal end of the CCK-8 molecule. The assay system appears totally specific for the CCK-8 sulphated molecule and shows no significant cross-reaction with other molecular forms of CCK, or with the gastrins. The assay can detect changes between adjacent tubes of 0.25 fmol/tube CCK-8 with 95% confidence. The assay is robust, reliable and reproducible and can be used to measure tissue and plasma levels of CCK-8.     

125I]iodohydroxynitrobenzylthioinosine: a new high-affinity nucleoside transporter probe

[125I]iodohydroxynitrobenzylthioinosine ([125I]IH-NBMPR), a new gamma-labeled nucleoside transport inhibitor, has been prepared at a theoretical specific activity of 2000 Ci/mmol (1 Ci = 37 GBq). IH-NBMPR was more acidic than hydroxynitrobenzylthioinosine (H-NBMPR), having a pKa of 4.6. Site-specific binding of [125I]IH-NBMPR to membrane-enriched fractions (MEF) from S49 mouse lymphoma cells was pH dependent, increasing with the fraction of undissociated molecules present; it was maximal at pH 4.5 and negligible at pH 7.0. Scatchard analysis of specific binding to MEF from S49 cells under equilibrium conditions at pH 5.0 yielded a Kd of 15 nM (equivalent to 4.0 nM for the undissociated fraction of inhibitor molecules) and maximum number of binding sites (Bmax) of 4.9 pmol/mg protein. Specific binding of IH-NBMPR could not be demonstrated in MEF from AE1 cells, a nucleoside transport-deficient mutant of S49 cells. Influx of uridine into mouse erythrocytes at pH 5.0 in the presence of 5 microM IH-NBMPR (1.4 microM undissociated IH-NBMPR) was reduced to about 7% of the control value, indicating that this compound is an effective nucleoside transport inhibitor. Photoactivation of site-bound [125I]IH-NBMPR, following equilibration of the ligand with MEF from S49 cells at pH 5.0, resulted in specific covalent labeling of a polypeptide with a relative molecular mass of 52,000-63,000, identified on sodium dodecyl sulfate-polyacrylamide gels. These results indicate that the new, iodinated ligand is an inhibitor of nucleoside transport and that it binds specifically and with high affinity to nucleoside transporter polypeptides in mammalian cells.     

Development of a plasma 17alpha-hydroxyprogesterone time resolved-fluorescence immunoassay involving a new biotinylated tracer

A biotinylated 17alpha-hydroxyprogesterone probe (3) was prepared from 17alpha-hydroxyprogesterone-3-carboxymethyloxime and conjugate obtained by acylation of biotinylaminopropylammonium trifluroacetate. This new tracer was used in the development of a 17alpha-hydroxyprogesterone time-resolved fluoroimmunoassay using streptavidin-europium. The new method was compared to a long-standing radioimmunoassay method and found to be more sensitive and economical.     

Measurements of tumor tissue oxygen tension using a time-resolved luminescence-based optical oxylite probe: comparison with a paired survival assay

Recently, a system that measures tissue oxygen tension using time-resolved luminescence-based optical sensors has become available commercially (Oxford Optronix, Oxford, England). Two experiments were conducted using this system. First, the oxygen tension distribution was measured in two tumor lines: a spontaneous mouse fibrosarcoma, FSa-II, and a human squamous cell carcinoma xenograft, FaDu. The area in which the pO(2) was equal to or lower than 2.5 mmHg was defined as the hypoxic lesion, and the hypoxic cell fraction was taken as the fraction of these measurements in a tumor. The measured hypoxic cell fractions were compared with those determined by the paired cell survival assay for tumors of various sizes. Second, the tumor tissue pO(2) was measured continuously after administration of two different anesthetics to evaluate the effect of these drugs on tissue pO(2). Results indicated a good agreement between the hypoxic cell fractions measured by this system and those determined by the paired cell survival curve assay for tumors smaller than approximately 500 mm(3). For tumors larger than approximately 500 mm(3), the hypoxic cell fractions measured by the oxygen probe system were higher than those measured by the paired cell survival assay. This may suggest that the hypoxic cell fraction measured by the oxygen probes included both hypoxic and necrotic areas in large tumors where necrotic lesions occupied a significant portion of the tumor. Continuous measurements of pO(2) after anesthesia (Nembutal, or ketamine plus xylazine) showed a consistent rise in the pO(2) during the first 20-30 min of measurement. Subsequently, the pO(2) values became constant or continued to rise slowly. For comparison, the tumor cell survivals were assayed after a dose of 20 Gy given in air at 5, 20 and 60 min after anesthesia. The result showed a decrease in cell survival only in tumors irradiated 20 min after an injection of Nembutal.     

Mini-RIA: adaptation of conventional 125I-labeled radioimmunoassay to a 96-tube format

Radioimmunoassay (RIA) employing iodinated ligands represents a popular measurement method for small molecules due to its excellent sensitivity and specificity. Yet performing RIAs of large numbers of tubes remains a tedious laboratory chore due to the need to individually handle tubes multiple times. We here present a method in which conventional 125I-labeled RIA ([125I] RIA) is adapted to a microtiter plate format, termed mini-RIA. Tubes are handled in batch for centrifugation or during the separation of antibody-bound ligand from free ligand. A simple draining device for batch decantation of free ligand from 96 minitubes is used. Optimal conditions for the mini-RIA were established using two workup methods-double-antibody immunoprecipitation and direct polyethylene glycol precipitation. Use of the mini-RIA method was found to result in a considerable savings in assay times; in addition, the sensitivity of the mini-RIA was improved over conventional RIA. The mini-RIA is particularly useful for assay of large numbers of samples derived from chromatographic methods, since aliquots can be transferred directly from the fraction collector into the minitubes using multiple channel pipettors. Because the method is flexible with regard to assay workup, we predict that most conventional [125I] RIAs can be adapted to the mini-RIA format.     

Characterization of antibodies against oestrone-azo-protein conjugates using 3H and 125I radioligands

This report describes the synthesis of six oestrone-azo-protein immunogens differing in the position of the azo group in the steroid skeleton and in the nature of carrier protein. By nitration of oestrone, 2-nitro- and 4-nitro-isomers were prepared and converted to amino-derivatives by reduction of the nitro group. The diazotized amino-oestrones were coupled to albumin, thyreoglobulin and haemocyanin. The immunogens thus prepared were administered to rabbits to assess the effect of position of the bridge, carrier protein and adjuvant on antibody production and specificity. Very weak immune responses were obtained with both types of albumin immunogen. The presence of the azo group at C-2 position led to a lower antibody production as compared with C-4 derivatives. The relatively high antibody production was achieved by immunization with oestrone-4-azo-thyreoglobulin or oestrone-4-azo-haemocyanin, with the oil component of adjuvant omitted. The antibodies combined with tritiated and iodinated radioligands yielded typical curves showing the sensitivity of 10-20 pg for 3H labelled oestrone and that of 3-10 pg for 125I labelled oestrone. The specificity of the 6 antibodies was tested using 25 related structures. In all these cases there was low cross-reaction with oestradiol, oestriol and their derivatives. On the other hand, the discriminating ability of the antibodies with regard to the opposite end of the molecule, i.e. with regard to the A-ring and partly the B-ring was very low. This makes it possible to use these antibodies for the radioimmunoassay of oestrone alone or some of its metabolites modified in the A-ring. The results of cross reactivity are generalized to the effect that in the oestrone-azo-hapten structure the antigenic determinant area can be ascribed to the size not exceeding 2-3 steroid rings.     

Response of a brachytherapy model using 125I in a murine tumor system

The effects of low-dose-rate irradiation (brachytherapy) were investigated in vivo using a murine mammary adenocarcinoma (MTG-B) growing in the flank of C3H mice. For local tumor irradiations, a noninvasive cap was devised to cover the tumor and house three 125I seeds (average apparent activity 5.2 mCi each) located at 120 degree intervals around the circumference of the hemispherical cap (13 mm i.d.). Mice were secured during treatment in a tube allowing limited mobility while restricting access to the seeds. Tumors were exposed to a series of dose rates ranging from 14-40 cGy/h, and the total dose over the treatment interval (48 or 72 h) ranged from 830 to 2378 cGy. A total of nine experiments were conducted using the caps over a 10-week interval. In each experiment three groups (irradiated tumors, sham controls, and untreated controls) were analyzed, each containing 8-15 mice (N = 34, untreated control; N = 46, sham control; N = 91, brachytherapy irradiation). The brachytherapy results are compared to the effects of external beam irradiation in the same tumor system. A linear relationship was observed between the total radiation dose and doubling volume growth delay (GDDV) or treatment volume growth delay (GDTV) for the brachytherapy and external beam irradiation. The slopes of the dose-response curves are steeper for the acute dose (517 cGy/min) external beam irradiation (0.0072 day/cGy, GDDV; 0.00695 day/cGy, GDTV) than for the brachytherapy (0.0050 day/cGy, GDDV; 0.0057 day/cGy, GDTV) using both GDTV and GDDV end points. Comparison of the tumor volume regrowth slopes indicates that the tumor bed effect is larger for external beam irradiation than for brachytherapy, suggesting that the tumor bed effect may be dose-rate dependent.     

(125)I]EYF: a new high affinity radioligand to neuropeptide FF receptors

[(125)I]EYF ([(125)I]EYWSLAAPQRFamide), a new radioiodinated probe derived from a peptide present in the rat Neuropeptide FF precursor (EFWSLAAPQRFamide, EFW-NPSF) was synthesized and its binding characteristics investigated on sections of the rat spinal cord and on membranes of mouse olfactory bulb. In both tissues, [(125)I]EYF binding was saturable and revealed a very high affinity interaction with a single class of binding sites in rat and mouse (K(D) = 0.041 and 0.019 nM, respectively).Competition studies showed that [(125)I]EYF bound to one class of binding sites exhibiting a high affinity for all the different peptides the precursor could generate (NPA-NPFF, SPA-NPFF, NPFF, EFW-NPSF, QFW-NPSF) with the exception of NPSF which displayed a low affinity.Autoradiographic studies demonstrated that [(125)I]EYF binding sites were fully inhibited by a synthetic Neuropeptide FF agonist (1DMe) in all areas of the rat brain. The density of [(125)I]EYF binding sites was high in the intralaminar thalamic nuclei, the parafascicular thalamic nucleus and in the superficial layers of the dorsal horn.Non specific binding reached 5-10% of the total binding in all brain areas. Similarly, in mouse brain experiments, the non-specific binding was never superior to 10%.These findings demonstrate that putative neuropeptides generated by the Neuropeptide FF precursor and containing the NPFF or NPSF sequences should bind to the same receptor. Furthermore, these data indicate that [(125)I]EYF is a useful radiolabeled probe to investigate the NPFF receptors; its major advantages being its high affinity and the very low non-specific binding it induces.     

(125I)iodo-zacopride: new ligand for the study by autoradiography of central 5-HT3 receptors

This paper describes the synthesis and the pharmacological characteristics of the first radioiodinated ligand of central 5-HT3 receptors: [125I]iodo-zacopride. Specific sites having a high affinity (Kd = 4.3 nM) for [125I]iodo-zacopride have been found in membranes from the rat entorhinal cortex. In addition, a highly significant correlation (r = 0.995) existed between the Ki of several 5-HT-related drugs for displacing both [125I]iodo-zacopride from its specific binding sites, and [3H]zacopride from 5-HT3 receptors. Finally, [125I]iodo-zacopride was successfully used for the autoradiographic mapping of 5-HT3 receptors in the rat central nervous system.     

125I](+/-)FISCH: a new CNS D-1 dopamine receptor imaging ligand

Radiolabeling and in vitro and in vivo evaluation of an iodinated benzazepine: [125I] FISCH 7-Chloro-8-hydroxy-1-(4'-iodophenyl)-3-methyl-2,3,4,5- tetrahydro-1H-3-benzazepine, as a potential imaging agent for CNS D-1 dopamine receptors in animals, were investigated. After an iv injection, this benzazepine derivative showed good brain uptake in rats (2.70, 1.28, 0.48 %dose/whole brain at 2, 15 and 60 min, respectively). The striatum/cerebellum ratio was 2.50 at 60 min after the injection. The regional distribution in rat brain, as measured by ex vivo autoradiography, displayed highest uptake in the regions of the striatal complex and the substantia nigra, regions known to have a high concentration of D-1 dopamine receptors. Furthermore, this localized regional cerebral distribution was blocked by pretreatment with SCH-23390, a selective D-1 dopamine receptor antagonist. The in vitro binding affinity of this agent in rat striatum tissue preparation displayed a Kd of 1.43 +/- 0.15 nM. Competition data (in vitro) showed the following rank order of potency: SCH-23390 greater than (+/-)IBZP much greater than apomorphine greater than WB 4101 greater than ketanserin approximately spiperone. The preliminary data suggest that this analog of SCH-23390 shows similar selectivity for the CNS D-1 receptor.