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

Steroids 69 (2004) 461–471

Development of a highly sensitive and specific new testosterone time-resolved fluoroimmunoassay in human serum Jean Fiet a,b,∗ , Frank Giton a , Ibrahim Fidaa c , Alain Valleix d , Hervé Galons e , Jean-Pierre Raynaud f a

c

Emi Inserm 03-37, Centre de Recherche Chirurgicale CHU Henri Mondor, Faculté de Médecine, 8 rue du Général Sarrail 94010 Créteil France b Laboratoire de Biochimie, Faculté de Pharmacie, 75006 Paris, France Laboratoire de Biochimie Hormonale, Hˆopital Saint-Louis, 1 Avenue Claude Vellefaux, 75475 Paris France d Service des Molécules Marquées CEA Saclay, 91191 Gif/Yvette, France e Laboratoire de Chimie Organique, Faculté de Pharmacie, 75006 Paris, France f Université Pierre et Marie Curie, 4 place Jussieu, 75252 Paris, France Received 26 February 2004; received in revised form 1 April 2004; accepted 14 April 2004 Available online 24 June 2004

Abstract 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. © 2004 Elsevier Inc. All rights reserved. Keywords: Time-resolved fluoroimmunoassay; Celite chromatography; Testosterone; Steroid

1. Introduction Synthesis of different biotinylated steroid tracers allowed us to develop several steroid immunoassays using time-resolved fluorometric detection of europium (Delfia Technology) [1–9]. These steroid immunoassays exhibited usually higher sensitivity than those obtained with tritiated tracers, enzyme conjugated tracers and challenged the immunoassays using 125 iodine labelled steroids with the advantages of stable, non-radioactive tracer.

∗

Corresponding author. Tel.: +33 1 4981 3558; fax: +33 1 4981 3552. E-mail address: [email protected] (J. Fiet).

0039-128X/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2004.04.008

Although numerous publications concerning testosterone immunoassay measurements have been reported, to our knowledge one publication using testosterone TR-FIA have been published [10]. We report a new testosterone TR-FIA with a new biotinylated tracer with the ambition to obtain a maximal sensitivity and specificity, thanks to biotinylated tracer with a long arm between biotin and the steroid and to new convenient minicolumns for celite chromatography before time-resolved fluoroimmunoassay (TR-FIA). Moreover, an extensively controlled excellent reproducibility was equally obtained. This new testosterone TR-FIA has been developed for the measurement of testosterone in untreated and treated hypogonadal men, and in women consulting for hirsutism, acne or menstrual cycle irregularities.

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2. Materials, apparatus, methods and patients

sued overnight. The mixture was concentrated under reduced pressure and extracted with CH2 Cl2 75 ml, washed twice with water and chromatographed on a silica gel column using CH2 Cl2 /EtOH/Et3 N 90:8:2 as eluent. Rf (CH2 Cl2 /EtOH/Et3 N 75:20:5 v/v/v) = 0.42; yield =46%; 1 H NMR (CDCl ) δ: 1.3 (m, 16H, 8CH ); 1.5 (s, 9H, Boc); 3 2 2.6 (t, 2H, CH2 –NH2 ); 3.0(q, 2H, CH2 –NH–Boc); 4.5 (bs, 1H, NHBoc); 7.2(bs, 2H, NH2 ).

2.1. Steroids and reagents 2.1.1. Commercial steroids, radioactive testosterone and chemical reagents All steroids used as calibrators and for the synthesis of biotinylated conjugates were purchased from Steraloids (RI, USA). Tritiated (1,2,6,7-3 H) testosterone came from Amersham Biosciences (91898 Saclay, France). Chemical reagents for conjugate synthesis were obtained from Sigma/Aldrich (BP 701, 38297 Saint-Quentin Fallavier, France).

2.1.2.2. 10-Boc-amino-decylbiotinylamide (2b). To a cold (5 ◦ C) solution of biotin (3.14 g, 11.5 mmol) in DMSO (12 ml), was added tributylamine (2 ml) and iso-butylchloroformate (1.50 ml, 11 mmol) in 3 ml dioxane. After 10 m stirring, 10-Boc-amino-decylamine (2.72 g, 10 mmol) and triethylamine (2 ml) in dioxane was added. After stirring overnight the mixture is poured in 100 ml of ice water. The precipitate is filtered and washed twice with 10 ml ice water. The solid is dried overnight in a desiccator containing P2 O5 . Rf (CH3 CN/EtOH/Et3 N 5:3:1 v/v/v) = 0.7; yield = 90%; m.p. = 120–125 ◦ C. 1 H NMR (CDCl3 ) δ: 1.2 (m, 16H, 8CH2 ); 1.5 (s, 9H, Boc); 1.6 (m, 6H, 3CH2 );

2.1.2. Synthesis of biotinylated testosterone tracers (Scheme 1) 2.1.2.1. 10-Boc-amino-decylamine (1). To a cold (5 ◦ C) solution of 1,10-diaminodecane (17.2 g, 100 mmol) in 90 ml dioxane di-tert-tbutyldicarbonate [(Boc)2 O] (5.04 g, 25 mmol) in 30 ml dioxane was added. Stirring was pur-

O S H

H HN

S

COOH

NH

1. iso-BuOCOCl

H

2. H2N(CH2)10NHBoc, 1

NH

H HN

O

(CH2)10

NH

NH 2b

O

O

O C(CH3)3

CF3COOH O S

NH H

H HN

(CH2)10

NH

+ NH3 _ CF3COO

O 3b

OH

OH 1. iso-BuOCOCl O 2.

N 4

O O

S

H

H HN

OH

NH O

_ CF3COO

NH

N

(CH2)n

O

NH3 + O

2a : n = 3 2b : n =10

5a : n = 3 5b : n = 10 H N (CH2)n HN

S O H

H HN

NH O

Scheme 1. Preparation of two biotinylated tracers.

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2.8 and 3d and dd (CH2 –S); 4.2 and 4.4(2m, 2H, 2CH–N); 4.5 (t, 1H, NHCO); 5.5 and 5.9 (2bs, 2H, 2NH–biot). 2.1.2.3. Biotinylaminodecyl-ammonium trifluoroacetate (3b). To a solution of 2b in CH2 Cl2 30 ml was added anhydrous trifluoroacetic acid 10 ml. The mixture was stirred at rt for 2 h. After evaporation of the solvent, the remaining oil crystallized upon trituration twice with 20 ml of cyclohexane and 20 ml of Et2 O. The solid was filtered and washed with Et2 O. Yield = 95%; m.p. = 145–152 ◦ C; 1 H NMR (CDCl3 ) δ: 1.4–1.6 (m, 16H, 8CH2 ); 2.05 and 2.15 (2t, 4H, 2CH2 ); 2.8 (m, 6H, 4CH2 ); 3(m, 3H, CH2 S and CHS); 4.10 and 4.30 (2m, 2H, 2CH–N); 7.5 and 8.5 (2bs, 7H, 4NH and NH3 ). 2.1.2.4. Final synthesis of biotinylated tracers (5a–b). The preparation of biotinylaminopropylammonium trifluoroacetate 3a has been previously reported [1]. Iso-butylchloroformate (35 ␮l, 0.22 mmol) was added to a cold (5 ◦ C) solution of testosterone-3-carboxymethyloxime 4 (0.1 g, 0.25 mmol) and triethylamine (40 ␮l, 0.3 mmol) in dioxane. After 5 min of stirring, the solution was added to a solution of biotinylaminoalkyl–ammonium trifluoroacetate 3a–b (0.25 mmol) and triethylamine (50 ␮l, 0.45 mmol) in 2 ml DMSO. After 4 h of stirring, the mixture was diluted in 10 ml cold water. The tracers, which precipitated, were filtered and washed with 2 ml of cold water and purified by column chromatography using EtOAC/MeOH (99.5:0.5 v/v) as eluent. 5a: yield 46%; m.p. 147–152 ◦ C; 1 H NMR (CDCl3 , TMS) δ: 0.72 (s, 3H, 18-CH3 ); 0.97 and 1.02 (2s, 3H, 19-CH3 ); 2.62 and 2.82 (d and dd, 2H, CH2 –S); 3.09 (m, 1H, CH–S); 3.25 and 3.35 (2q, 2 × 2H, 2CH2 NCO); 4.25 and 4.45 (2m, 2H, CH–N); 4.41 and 4.48 (2s, 2H, O–CH2 CON); 5.18 and 5.95 (2bs, 2H, NH); 5.69 and 6.32 (2s, 1H, 4-H E and 4-H Z); 6.60 and 6.75 (2t, 2 CH2 –NH–CO). 5b: yield 63%; m.p. 112–116 ◦ C; 1 H NMR (CDCl3 , TMS) δ: 0.72 (s, 3H, 18-CH3 ); 0.97 and 1.02 (2s, 3H, 19-CH3 ); 2.62 and 2.82 (d and dd, 2H, CH2 –S); 3.09 (m, 1H, CH–S); 3.25 and 3.35 (2q, 2 × 2H, 2CH2 NCO); 4.25 and 4.45 (2m, 2H, CH–N); 4.41 and 4.43 (2s, 2H, O–CH2 CON); 4.70 and 5.32 (2bs, 2H, NH); 5.65 and 6.20 (2t, 2CH2 –NH–CO), 5.75 and 6.35 (2s, 1H, 4-H E and 4-H Z). These two biotinylated tracers 5a and 5b were a mixture of two E/Z stereoisomers. We separated the 5b stereoisomers using HPLC procedure: HPLC Shimadzu SCL 10AS Apparatus, Hypurity C18; 250 × 4.6 column; acetonitrile/H2 O: 45/55; flow, 1.5 ml/min, at 35 ◦ C; detection, UV240 nm. 2.1.3. Preparation of the biotinylated testosterone solutions (tracers) The stock solutions of biotinylaminopropane carboxymethyloxime testosterone (5a reagent, see above) and biotinylaminodecane carboxymethyloxime testosterone (5b reagent, see above) tracers were prepared by dissolving, respectively, 4mg of 5a reagent and 5 mg of 5b reagent in

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100 ml of ethanol. The biotinylated tracer intermediary solutions were prepared by diluting the stock solutions 1/100 in ethanol. Stock and intermediary ethanolic solutions were kept at +4 ◦ C. 2.1.4. Preparation of the anti-testosterone antibody The anti-3-carboxymethyloxime-testosterone antibody was prepared in rabbits according to Vaitukaitis’ method and described previously [11]. Specificity was studied by assessing cross-reactivities for a displacement of 50% of the biotinylated tracers by various steroids. 2.1.5. Special reagents, devices and apparatus for TR-FIA (described previously) [5] Microtitration plates: 12 × 8 NUNC (Ref. 1244-550 Perkin-Elmer 91945 Courtaboeuf France). Goat anti-rabbit antibody: Valbiotech (75010 France; 4 mg/2 ml) for coating. Washing solution: physiological saline solution with Tween 20 (0.1%). Eu-labelled streptavidin: 0.1 mg/ml, 2.5 ml (Ref. 1244360, Wallac), diluted 1/1000 in the following buffer: BSA 5 g/l + Tween 40, in physiologic saline solution + Na azide (0.5%), diethylene triamine penta-acetic acid 15 mg/l, adjusted to pH 7.8 with Tris–HCl. Delfia Enhancement solution (Ref 1244-105, PerkinElmer). Coating buffer: 0.05 M, pH 9.6. Anhydrous Na carbonate (Na2 CO3 ) 1.55 g Na hydrogen carbonate (NaHCO3 ) 2.97 g qsp 1000 distilled water, adjusted to pH 9.6 with diluted HCl. Saturation solution: pH 7.4 0.05 M. Disodium hydrogen phosphate (Na2 HPO4 ·2H2 O) 7.2 g, sodium di-hydrogen phosphate (NaH2 PO4 ·2H2 O) 1.3 g, distilled water qsp 1000 ml. Dissolve bovine serum albumin 12 g (Sigma A-9647) and Na azide 2 g (Merck, Ref. 106-688), kept at +4 ◦ C. Assay buffer: pH 7.4 0.05 M. Sodium chloride (NaCl) 9 g, disodium hydrogen phosphate (Na2 HPO4 ·2H2 O) 7.2 g, sodium di-hydrogen phosphate (NaH2 PO4 ·2H2 O) 1.3 g, distilled water qsp 1000 ml. Dissolve bovine serum albumin 3 g (Sigma A-9647) Na azide 2 g (Merck, Ref. 106688). Fluorometer: time-resolved fluorescence was measured with a 1234 Delfia Fluorimeter (Perkin-Elmer, 91945 Courtaboeuf, France). A model 1296-024 platewash device (Perkin-Elmer, Wallac) was also used. GC/MS: GC/MS analysis were performed with HP6890 for the gas chromatography and with HP5973 for the mass spectrometry. 2.1.6. Coating and saturation of microtiter plates Microtiter plate wells were coated by adding 250 ␮l of goat anti-rabbit antibody, diluted 1000-fold in coating buffer. After washing three times with a washing solution, free well sites were saturated by adding 300 ␮l of the

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saturation solution. The microtitration plates were covered with sealing tape (Corning 430454) and kept at +4 ◦ C.

2.2. Procedures for testosterone assays in serum 2.2.1. TR-FIA procedure

2.1.7. Testosterone calibrator solutions for establishing two standard curves—a classical standard curve (CSC)—and a sensitized standard curve (SSC) Mother solutions containing 10 mg of testosterone per 100 ml of pure ethanol, were used to prepare alcoholic daughter solutions 100 times less concentrated. These ethanolic standard solutions were kept at +4 ◦ C and used to prepare extemporaneously the calibrator solutions in the assay buffer. The amounts of testosterone added into the wells of the microtitrate plates to establish the CSC were in pg/well (pmol): 600 (2.08), 300 (1.04), 150 (0.52), 75 (0.26), 37.5 (0.13), 18.75 (0.06), 9.38 (0.03), 4.69 (0.016). For establishing the SSC, the quantities of testosterone put into the wells were in pg/well (pmol): 75 (0.26), 37.5 (0.13), 18.75 (0.06), 9.38 (0.03), 4.69 (0.016), 2.34 (0.008), 1.13 (0.004), 0.58 (0.002). 2.1.8. Reagents solvents and devices for extraction and chromatography Iso-octane, cyclohexane, ethylacetate, dichloromethane and ethylene glycol were supplied by Merck (Nogent-surMarne, France), Carlo-Erba (Rueil Malmaison, France) and Prolabo. Celite, i.e. “Celite analytical filter aid” with very small pore size (median pore size 2.5 ␮m) was from Lompoc, California 93438-0518, USA. This celite was conditioned by treating with cyclohexane extensive washing, dried then kept at 100 ◦ C, before packing into chromatographic minicolumns. Minicolomns (Supelco, St. Quentin-Fallavier Cedex, France), Visiprep apparatus (St. Quentin-Fallavier Cedex, Ref. 57250-U).

2.2.1.1. Steroid extraction step from serum. In order to monitor losses occurring during the extraction, chromatography and redissolution steps before immunoassay, 0.1 ml of a aqueous tritiated testosterone solution (containing 4000 dpm) was added to 0.1–1 ml of serum in 16 ml propylene tubes (Sarstedt Ref. 55515). After 30 min of incubation with intermittent shaking, extraction was carried out with 10 ml of a mixture of ethylacetate/cyclohexane 50/50 (v/v), by mixing for 2 min with a multivortex. Centrifugate at 2500 t/min × 5 min. Freeze the aqueous lower phase and transfer the upper organic layer to 14 ml glass tubes. Evaporate at 37 ◦ C under a stream of filtered air. We obtained a dry extract containing the steroids. 2.2.1.2. Chromatographic separation steps (Fig. 1). The dry extracts were redissolved in isooctane (1.5 ml), vortexed 30 s, ultrasonicated 5 min and vortexed again for 30 s. This organic solution was layered onto celite chromatographic minicolumns as already reported [9] and passed through with the aid of a negative pressure (−5 kg Pa), using Visiprep apparatus. This negative pressure was constant during passing the elution chromatographic solvents. Each addition of solvents into the chromatographic columns was performed while the vacuum pump was stopped. Two milliliter of pure isooctane was added into the column and this volume was not collected. Five milliliter of pure isooctane were added and collected if necessary (this fraction contains androstenedione.). Five milliliter of a mixture of isooctane + dichloromethane 88/12 (v/v) were added

Fig. 1. Celite-ethylene glycol separation chromatography of testosterone from other steroids, using solvents of increasing polarity. Percentage of recoveries of the following 3 H tritiated steroids: ( ) DELTA4 = ∆4-androstenedione; ( ) ANDRO = androsterone; ( ) DHEA = dehydroepiandrosterone; ( ) DHT = 5α-dihydrotestosterone; ( ) TESTO = testosterone; ( ) 17OHP = 17-OH-progesterone; ( ) 11BETA = 11βOH-∆4-androstenedione; ( ) ADIOL = 5α-androstan-3α,17β-diol; ( ) DELTA5 = 5-androsten-3β,17β-diol.

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and collected or not (this fraction contains DHT). At last the addition of 5 ml of a more polar mixture of isooctane + dichloromethane 80/20 (v/v) allowed the elution of testosterone. This collected organic chromatographic elution fraction was evaporated to dryness. The dry residue is redissolved in assay buffer (0.5–1 ml) with the aid of vortexing and sonication. In this aqueous solution, testosterone was immunoassayed by TR-FIA.

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men before and after testosterone treatment, and secondly to study women suffering from hirsutism, acne or menstrual cycle irregularities. The clinical and pharmacological results will not be reported in the present paper.

3. Results 3.1. Synthesis of the tracers

2.2.1.3. TR-FIA of testosterone. The assays were done in duplicate. The assay was carried out by adding to each coated well, 0.1 ml of the testosterone aqueous chromatographic solution or 0.1 ml of standards, then 0.05 ml of the appropriate dilution of the labelled biotin-testosterone tracer and 0.05 ml of adequate anti-3-carboxymethyloximetestosterone/BSA. Microtitration plates were incubated while being agitated 350 rev/min, at room temperature, for 2 h. The immunoreaction was stopped by washing the wells three times with washing solution. Then, 0.2 ml of a Eu-labelled steptavidin was added and the plates were agitated for 20 min at 350 rev/min before washing three times. The Eu was dissociated by adding 0.2 ml of enhancement solution to each well, then agitated 250 rev/min for 20 min before measuring time-resolved fluorescence. (the appropriated dilution of the biotin-testosterone tracer on one hand and of the anti-3-carboxymethyloxime-testosterone/BSA on the other hand were reported in results, § 3.2.1). 2.2.2. RIA procedure 2.2.2.1. Competition RIA of testosterone with tritiated testosterone as tracer [11]. After extraction followed by celite chromatographic on 5 ml Kimble glass pipettes as columns, the aqueous solution of testosterone was assayed by RIA using the same anti-3-carboxymethyloximetestosterone, tritiated testosterone as tracer and a scintillation proximity reagent (RPN 140) (Amersham/Biosciences, 91898, Orsay, France) for separation and counting as previously described [11]. 125

2.2.2.2. Competition RIA of testosterone with iodine testosterone as tracer (IM 1087). Commercial Kit of Beckman/Immunotech, Marseille Cedex 09, France: briefly, a chromatographic step was carried out before testosterone immunoassay, only in women. 2.2.3. GC/MS procedure. [12] Briefly, 1 ml of serum was extracted, using deuterated testosterone as internal standard. The derivatisation reagent was pentafluorobenzoyl chloride (Sigma–Aldrich). 2.3. Patients Testosterone TR-FIA was developed in the aim, firstly to investigate the androgenic hormonal status of hypogonadal

Acylation of biotinylaminopropylammonium and biotinylaminodecylammonium with testosteronene-3-CMO afforded two biotinylated tracers 5a and 5b. The E and Z stereoisomers of the 5b tracer were separated in two distinct peaks with a good resolution. All the analytical validation and clinical assays were carried out using the mixture of the 5b tracer (E and Z). 3.2. Characterization of the anti-testosterone antiserum 3.2.1. Titers of the antiserum anti-testosterone-3CMO-BSA used Dilution of the anti-T-3-CMO/BSA used in the RIA was 2 × 10−4 [11]. In the standard TR-FIA methodology, we obtained a CSC, using a final anti-testosterone antiserum dilution of 0.16 × 10−4 and a quantity of the biotinylaminodecane-3-CMO-testosterone tracer (5b Scheme 1, E and Z stereoisomeres mixture) of 192 pg/well. In the sensitized TR-FIA methodology, we chose a greater dilution of antiserum, 0.66 × 10−5 and 55 pg/well of the same tracer. With the biotinylaminopropane-3-CMOtestosterone tracer (5a Scheme 1), the dilution of antiserum was 0.5 × 10−5 , and the quantities of tracer was 50 pg/well. 3.2.2. Specificity of the anti-testosterone-3-CMO/BSA The specificity of the anti-testosterone-3-CMO/BSA was previously reported using tritiated testosterone as tracer [11]. The use of biotinylated tracer, biotinylaminodecane carboxymethyloxime testosterone (5b tracer) or biotinylaminopropane carboxymethyloxime testosterone (5a tracer) did not modify significantly the main cross-reactivities studied (delta-4-androstenedione 1.5%, dihydrotestosterone (DHT) 29%, dehydroepiandrosterone (DHEA) 0.5%, 5␣-androstane, 3␣,17␤-diol 0.1%, 5␣-androstane, 3␤,17␤-diol 0.1%, 17-OH progesterone <0.001%, androsterone <0.1%. Equally the use of one of the two stereoisomeres E and Z of 5b tracer, did not modify the cross reactivities obtained with the mixture of the E and Z isomers. 3.3. Establishement of testosterone standard curves The amounts of T (expressed in pg) put into the tubes and into the wells of the microtitration plates necessary for establishing, respectively, the immunocompetition standard curves for RIA and TR-FIA method were: 600, 300, 150,

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75, 37.5, 18.75, 9.38 and 4.69, which correspond to 2.08, 1.04, 0.52, 0.26, 0.13, 0.065, 0.032 and 0.016 pmol of T. The quantities of testosterone in the wells for establishing the TR-FIA sensitized standard curve were 75, 37.5, 18.75, 9.38, 4.69, 2.34, 1.17 and 0.59 pg. 3.3.1. Testosterone standard curves 3.3.1.1. Percentages of mean bindings. The RIA and TR-FIA standard curves are presented in Fig. 2. They were established from 12 intra-assay determinations. The means (±SD) of the binding for the following calibrators (pg/well) 4.69, 9.38, 18.75, 37.50, 75, 150, 300 and 600 were, respectively, 91.2 (6.9), 85.6 (3.5), 78.1 (3.4), 66.5 (2.2), 54.1 (2.6), 44.7 (2.7), 30.1 (1.5) and 18.8 (1.1) for the RIA standard curve (with a tritiated tracer) and 80.5 (1.1), 67.7 (1.6), 48.2 (1.3), 27.6 (0.6), 14.2 (0.4), 8.2 (0.2), 4.5 (0.1) and 3.05 (0.1) for the classical TR-FIA standard curve (CSC; established using the 5b tracer) and 85.5 (1.5), 78 (1.5), 62.2 (1.1), 42.1 (0.9), 27.2 (0.9), 19.1 (0.5), 16.4 (0.2) and 15.6 (0.3) (established using the 5a tracer). The means (±SD) of the binding for the following calibrators (pg/well) 0.56, 1.17, 2.34, 4.69, 9.38, 18.75, 37.50 and 75 of the sensitized TR-FIA standard curve (SSC) were 92.2 (1.2), 87.5 (0.9), 76.1 (1.0), 59.3 (0.8), 36.6 (0.5), 20.6 (0.3), 11.4 (0.2) and 6.7 (0.1). The means (±SD) of the binding for the Beckman-Immunotech Commercial Kit calibrators (pg/well) 4.70, 17.5, 75, 250 and 1000 were 97.1 (2.31), 81.1 (1.9), 50.9 (1), 31.1 (0.8) and 15.1 (0.5).

3.3.1.2. Radioactivity and fluorescence unit ranges. The mean range of the radioactivity of the RIA standard curve (with tritiated tracer) was between 5440 dpm for the zero standard calibrator and 1012 dpm for the 600 pg calibrator (with a mean background of 180 dpm) [11]. The mean range of the arbitrary fluorescence units of the TR-FIA standard curve was between 220,200 for the zero calibrator (absence of analyte), and 6650 for the last calibrator (600 pg; with a background of less than 600 fluorescence units). The range of the sensitized TR-FIA standard curve was between 37.200 units of fluorescence for the zero calibrator and 2500 for the last calibrator (75 pg; with a background of less than 250). 3.3.1.3. Sensitivity of the testosterone standard curves. They were established as the quantities of testosterone per tube or per well which displaced 20, 50 and 80% of the tracers. These displacements were obtained for the RIA standard curve by 21.5, 110 and 478 pg of testosterone; for the classical TR-FIA standard curve by 4.70, 16.8 and 65.0 pg of testosterone; and for the TR-FIA sensitized standard curve by 1.5, 6.9 and 17 pg of testosterone. In the standard curve of the Beckman-Immunotech Testosterone Kit, the 20, 50 and 80 displacements of the 125 iodine testosterone tracer were obtained with 18, 75 and 650 pg, respectively. 3.3.1.4. Lowest detection limits of testosterone from the standard curves. The least quantities of T that significantly displaced the tracers, i.e. equivalent to B0 or mean

Fig. 2. TR-FIA sensitized standard curve ( ); TR-FIA classical standard curve (䊉); RIA (125I testosterone as tracer) standard curve of Beckman-Immunotech Kit ( ); RIA (3H testosterone as tracer) standard curve (䉲); IC50 of the standard curves.

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binding of tracer in the absence of analyte-3SD were found to be 4.10 pg/well on the RIA standard curve (with a tritiated tracer), 0.7 pg/well on the classical TR-FIA standard curve and 0.35 pg/well on the sensitized TR-FIA standard curve. 3.4. Validation of the testosterone TR-FIA method

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We obtained a mean level of 44 pg/ml (CV = 11.2%) for serum containing theoretically 40 pg/ml using the classical methodologic conditions (consequently the LLOQ was <44 pg/ml) and a mean value of 21 pg/ml for a serum containing 20 pg/ml (CV 7.6%) using the sensitized methodologic conditions (consequently the LLOQ was <21 pg/ml). 3.4.4. Reproducibility

3.4.1. Specificity of the testosterone TR-FIA DHT which cross-reacts with anti-testosterone antiserum was completely eluted in an iso-octane/DCM 12% chromatographic fraction and no DHT is present in the more polar chromatographic elution fraction, iso-octane/DCM 20%, in which the testosterone was eluted. Consequently, DHT does not interfere at all in the testosterone assay. Moreover, the 4-androstenedione is eluted before DHT, the DHEA and androsterone are eluted in the DHT elution fraction, the more polar steroids 11-keto, 11-hydroxy-testosterone, 11␤-hydroxy-4-androstenedione and androstanediols were eluted in more polar elution fractions after the testosterone elution fraction. 17-OH progesterone, a pregnene steroid present in the testosterone elution fraction do not cross react with the anti-testosterone antibody. 3.4.2. Recovery of the minute doses of tritiated testosterone added to the serum samples for monitoring the extraction + celite chromatography steps The mean recovery of minute doses of tritiated testosterone added to each serum samples to monitor the recovery after the two-step extraction + chromatography was equal to 82.8% ± 6.7 (n = 350). The individual result of recovery of each assay was taken into account to calculate the serum testosterone concentration. 3.4.3. Detection limits 3.4.3.1. Detection limits in serum calculated from the lowest detection limits of testosterone on the standard curves. The theoretical detection limit of testosterone in the serum depends on the methodology used. With 1 ml of extracted serum, 1 ml of assay buffer used to redissolve steroid after chromatography, 0.1 ml of aqueous chromatographic solution assayed by TR-FIA (§ 2.2.1), a mean recovery of testosterone of 82.8% (§ 3.4.2), and the lowest detection limits of testosterone from the TR-FIA standard curves (§ 3.3.1.4), the theoretical calculated detection limits of testosterone in serum was 8.5 pg/ml using the methodological conditions to obtain the classical testosterone standard curve and 4.3 pg/ml using the sensitized methodological conditions and the sensitized testosterone standard curve. Comparatively, the theoretical detection limit using the RIA was 49 pg/ml. 3.4.3.2. The quantification limit or “lower limit of quantification” (LLOQ) or “functional” sensitivity. LLOQ was determined in assaying 20 times, four sera whose T concentrations were, respectively, 60, 40, 20 and 15 pg/ml.

3.4.4.1. Reproducibility of duplicate. The CV of duplicate TR-FIA measurements were always below 2%. 3.4.4.2. Intra-series precision. Four pools of sera in which the testosterone concentrations (ng/ml) were 0.15, 0.30, 0.60 and 1.20 were aliquoted in 10 samples for each level (consequently 40 samples in all). Each sample (in which the concentration was unknown to the technician) was assayed in the same run according to the sensitized methodology (n = 40 samples). The same experience, but using the standard methodology, was performed on sera pools in which testosterone levels (ng/ml) were 2, 4, 8 and 16. The results were reported in Table 1. 3.4.4.3. Inter-series precision. The testosterone interassay precision was determined in 80 serum assay series of hypogonadal men (three control sera (ng/ml): 0.75, 1.50 and 3.00 were assayed in each series) and in 17 assay series of testosterone treated hypogonadal men sera (control sera (ng/ml): 2.50, 4.00 and 6.00). The means ± SD (ng/ml), (CV, %) were, respectively, 0.755 ± 0.037 (4.883); 1.53 ± 0.078 (5.070); 3.024 ± 0.126 (4.179) and 2.456 ± 0.112 (4.580); 4.084 ± 0.145 (3.555) and 6.004 ± 0.131 (2.174). In each run, a steroid-free charcoal-stripped serum gave undetectable testosterone concentration. 3.4.5. Accuracy 3.4.5.1. Recovery experiments. Four sera (two male and two female sera) were overloaded with four concentrations of testosterone. The testosterone concentrations of the overloaded sera were assayed by TR-FIA and the recoveries

Table 1 Intra-assay precision Theoretical testosterone levels (ng/ml)

Measured testosterone levels (mean ± S.D.; ng/ml)

0.15 0.30 0.60 1.20 2.00 4.00 8.00 16.00

0.16 0.313 0.613 1.145 2.016 4.00 8.056 15.523

± ± ± ± ± ± ± ±

0.01 0.02 0.02 0.04 0.06 0.10 0.33 1.00

CV (%) 6.84 5.01 4.00 3.22 3.16 2.46 4.15 6.47

TR-FIA measurement of 80 samples containing 8 levels of testosterone. Each level was assayed in 10 samples in the same run.

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Table 2 TR-FIA measurements of testosterone (ng/ml) in undiluted and diluted sera (three female serum-levels 1.2.3 and three male serum-levels 4.5.6) Serum dilutions

T level 1

T level 2

T level 3

T level 4

T level 5

T level 6

Undiluted Dilution 1/2 Dilution 1/4 Dilution 1/8

0.66 0.68 0.64 0.64

0.76 0.72 0.64 0.80

1.06 1.00 1.04 0.96

2.83 2.72 2.92 2.96

4.88 4.82 4.72 4.80

5.36 5.32 5.52 5.20

calculated. The analytical recoveries of T added to sera were between 90 and 110%. 3.4.5.2. Dilution test. Testosterone concentrations were measured in the serum of six patients, three hyperandrogenic women, and three testosterone treated hypogonadal men. Testosterone was assayed at various dilutions ranging from undiluted to eight-fold diluted. The woman sera were assayed using sensitized methodology and the men sera with the classical methodology. The results are reported in Table 2.

The two compared methods included a chromatographic step. The equation of the regression curve was: Y = 0.961X + 0.032 (Y: TR-FIA; X: RIA), with R2 = 0.971 (Fig. 3).

3.4.6. Comparison of results with RIA method and with GC/MS

3.4.6.2. Comparison with GC/MS. Sample sera of untreated hypogonadal men (n = 13) sera samples of testosterone treated hypogonadal men (n = 13) and sera samples of hyperandrogenic women (n = 10) were simultaneously assayed by TR-FIA and GC/MS (GCMS; LLOQ = 50 pg of testosterone per ml of serum). The equation of the regression curve was Y = 1.0644X − 0.0358 (Y: TR-FIA; X: GCMS), with R2 = 0.9832. There is no significant difference between the two paired series of GCMS and TR-FIA results.

3.4.6.1. Comparison of woman testosterone levels using TR-FIA sensitized methodology and RIA [11]. The testosterone levels were assayed simultaneously in 67 samples of consecutive women consulting for hirsutism or acne.

3.4.7. Stability of the tracer The stock solution and intermediary alcoholic tracer, biotin-aminodecane 3-CMO-testosterone, have been stored for 24 months so far at +4 ◦ C with no loss of activity.

8

7 Y = 1.064 . X - 0.036 R 2 = 0.983

TR-FIA (ng/ml)

6

5

4

3

2

1

0

0

1

2

3

4

5

6

7

8

GC-MS (ng/ml) Fig. 3. Comparison of testosterone serum levels measured by GCMS (X-axis) and by TR-FIA (Y-axis) in 13 untreated and 13 testosterone-treated hypogonadal men, and in 10 women suffering from hirsutism, acne or period disturbances. Regression curve, square of the correlation coefficient R2 .

J. Fiet et al. / Steroids 69 (2004) 461–471

4. Discussion The aim of this work was to establish a sensitive, specific and accurate non-isotopic method to assay serum testosterone in testosterone treated and untreated hypogonadal men and in hyperandrogenic women, to compare with GCMS and with a previously published RIA method applied in hyperandrogenic women [11]. According to some steroid non-isotopic assay methods using biotinylated tracer [1–9], we synthesized two biotinylated testosterone tracers with different length of the spacer arm between biotine moiety and steroid (tracer a with 3C and tracer b with 10C). The two tracers were studied and using HPLC, we separated the two stereo isomers E and Z of the tracer b. The use of E or Z stereoisomer of biotinylaminodecane carboxymethyloxime testosterone (tracer b) instead of the mixture of the two, did not increase significantly the sensitivity of the testosterone standard curves and did not significantly decrease the cross-reactivities of the anti-testosterone immunserum. This was at variance with previously reports for 21-deoxycortisol [13] radioimmunoassays, in which the sensitivity of the standard curve was depending on the geometric isomerism of the 125 iodine 21-deoxycortisol tracers. The aim of the synthesis of the two tracers a and b was to reach the maximal sensitivity of the testosterone standard curves. It was reported that the chemical structure of the spacer arm between, biotin and steroid has a relevant influence on the process of steroid antibody recognition [14], and that generally the sensitivity increases as the length of the spacer arm increases [15]. Indeed it was clearly shown in the chemiluminescence immunoassay of testosterone using a biotinylated tracer [16], that the sensitivity of the testosterone standard curve greatly increased with the length of the spacer arm between biotin and the steroid, and that only a C11 alkyl spacer arm allowed to obtain a satisfying standard curve. Spacer arms of three or six carbon atoms attached to carbon 7 of the testosterone were not suitable for establishing testosterone standard curves. In our work, we report a slightly greater sensitivity of the standard curve using the biotinylated tracer with the longer spacer arm (10 carbon atoms in tracer b) compared to the biotinylated tracer with three carbon atoms (tracer a). However, this difference of sensitivity was not as striking as those described previously in testosterone chemiluminescence assay. This discrepancy may be attributed to the different sites of attachment of the spacer arm on testosterone backbone-on the seven carbon in the testosterone chemiluminescence immunoassay, and on the three carbon in our report. Indeed, another testosterone TR-FIA using also biotine-testosterone tracer with a spacer arm of six carbon atoms attached on C3 [10] exhibited also fair sensitivity performances in saliva testosterone immunoassay. In this work, all the validation and clinical assays were carried out using the tracer b.

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The TR-FIA testosterone assay sensitivity was adjusted by varying the tracer b concentration and anti-testosterone antibody dilution. Thus, we obtained on one hand, a CSC with a significant least detectable dose of 0.70 pg/well, and a 50% tracer displacement (IC50 ) of 18 pg/well (Fig. 2), and on the other hand, a SSC with a significant least detectable dose of 0.35 pg/well and an IC50 of 6 pg/well. This sensitivity appeared superior to those given by testosterone RIA using tritiated testosterone tracer [11], and even 125 iodine labelled testosterone tracer (Fig. 2). In taking into account these least detectable doses on the testosterone standard curves, we can calculate detection limits in serum. They were 9.3 and 4.7 pg/ml using methodologies with the CSC and the SSC, respectively. These detection limits were either lower or in the same range of those of other non-isotopic testosterone assays previously published [16,10]. In practice, the functional sensitivity was strictly lower (respectively, 44 pg/ml with CSC (with a CV = 11.2%), and 21 pg/ml with the SSC (CV = 7.6%), than those of the only reported in chemiluminescence testosterone assay [16] (equal to 230 pg/ml with a CV = 20%). The CVs of intra-series precision measured 10 times in 4 low level control sera using the more sensitive methodology (with SSC) and in 4 high level control sera using the classical methodology (with CSC) were between 3.16 and 6.84% consequently lower than those usually reported, intra-series CV between 7.7 and 9.1% [16], and between 8.9 and 14.6% [10]. The inter-series precision measured in 80 series of hypogonadal sera with low level of testosterone and in 17 series of testosterone treated hypogonadal men sera with high or normal level of testosterone was expressed by CVs between 2.17 and 5.07%. The CV reported in previous publications of non-isotopic methods, were rather higher, with CV between 7.9 and 8.7% [10] and between 12.8 and 15.9% [16]. The specificity of our testosterone immunoassay was based on a celite-ethylene glycol chromatographic step carried out before the immunoassay. The use of celite-ethylene glycol as a chromatographic phase has been developed since long time ago [17]. Testosterone was eluted in a solvent volume whose polarity was chosen so as to completely separate testosterone from DHT which was eluted in a less polar elution fraction. As reported in § 3.4.1., the less polar steroids androsterone, 4-androstenedione, DHEA were eluted before testosterone elution fraction while more polar such as androstanediols, 11-OH and 11-ketotestosterone and 11␤-HO-4-androstenedione were eluted after testosterone elution fraction. According to our experience, the only steroid which was eluted simultaneously with testosterone was the 17-OH progesterone. However, this steroid did not cross-react at all with the anti-testosterone-3-CMO-BSA, and did not interfere in our testosterone immunoassay. The use of a celite with very small median pore size (2.5 ␮m) allowed to reach such a

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good resolution. The chromatographic step before steroid immunoassays, today, is no more largely widespread, because it is considered to be cumbersome and time consuming. Thus, numerous steroids, including testosterone, are measured by immunoassays, RIA particularly, directly on serum or only after an extraction step. Consequently, it results in false high serum testosterone levels, particularly in women [18]. The use of polypropylene minicolumns filled with celite + ethylene glycol, and the migration of different solvents through celite columns using a slight depression in Visiprep has increased considerably the practicability of this chromatographic step compared to previous celite chromatographic procedures [11]. So 48 samples using 2 Visipreps can be easily processed simultaneously, and the testosterone containing fractions will be collected in less than 1 h and half. Moreover, the recovery of minute doses of tritiated testosterone added to each serum samples to calculate the testosterone level was high (82%) and homogenous. As a consequence of the good precision specificity and sensitivity, we could expect a good exactitude. Indeed, the standard tests exploring the exactitude quality, the dilution and recovery tests were satisfying. Moreover, we compared the testosterone levels in consecutive women consulting for hirsutism using this reported TR-FIA and RIA previously published [11]. We found a good correlation. At last, 36 sera were comparatively measured using our method and GC/MS. No significant difference and no systematic bias were found between the two assay methods and the correlation was correct (R2 = 0.98). This result is an additional argument for the good accuracy of our testosterone TR-FIA method. Considering several steroid hormones (including testosterone) measurements, similar analytical performances were also recently reported [19] between radioimmunoassays and GC/MS. In conclusion, this new non-isotopic testosterone TR-FIA has the advantage on RIA to use only very little quantities of radioactivity, the new minicolumns developed for the celite chromatographic step increase the practicability and this new TR-FIA present the analytical qualities required for a specific, sensitive reproducible and accurate measurements of testosterone in serum samples.

Acknowledgements We thank Dr Alain Belanger (Molecular Endocrinology Laboratory, CHUL Sainte Foy, Quebec, Canada) for comparison with GC/MS.

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