Analytica Chimica Acta 551 (2005) 92–97

Direct sub-ppt detection of the endocrine disruptor ethinylestradiol inwater with a chemiluminescence enzyme-linked immunosorbent assay

Christian Schneidera, Heinz F. Scholera, Rudolf J. Schneiderb,∗a Institute of Environmental Geochemistry, University of Heidelberg, Im Neuenheimer Feld 236, D-69120 Heidelberg, Germany

b Institute of Plant Nutrition, University of Bonn, Karlrobert-Kreiten-Str. 13, D-53115 Bonn, Germany

Received 6 May 2005; received in revised form 7 July 2005; accepted 13 July 2005Available online 22 August 2005

Abstract

A chemiluminescence ELISA for the direct detection of ethinylestradiol (EE2) in water at sub-ppt levels was developed and validated. Ata signal-to-noise ratio of three the detection limit is 0.2± 0.1 ng L−1, at a ratio of 10 the LOQ is found to be 1.4± 0.8 ng L−1. Based on aconservatively calculated precision profile the analytical working range is established from 0.8 to 100 ng L−1. The ELISA was tested in fourdifferent matrices, including surface water and effluent of sewage treatment plants. All measurements were validated using an LC–MS/MSm imet©

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anti-thodsh per-Cur-tec-

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ethod. Typical results were consistent in both methods below 1 ng L−1. Using this chemiluminescence ELISA facilitates for the first the direct detection of EE2 at ecotoxicologically relevant concentrations.

2005 Elsevier B.V. All rights reserved.

eywords: Chemiluminescence; ELISA; Ethinylestradiol; Sub-ppt concentrations; Direct measurement; Optimization

. Introduction

Endocrine disrupting effects observed in the aquaticcosystem have stimulated broad scientific and public inter-st. First studies already began in the 1970s when adverseffects of synthetic estrogens were discussed for the first

ime [1]. Research was intensified in the early 1990s withhe advent of reproductive problems in some freshwater fishopulations[2,3].

One of the most potent estrogenic hormones is the syn-hetic steroid ethinylestradiol (EE2). It has been shown thatE2 induces feminization in immature cyprinids at concen-

rations of 10 ng L−1 in water and in rainbow trout at levels of.1 ng L−1 [2]. In a thorough full life-cycle study using fat-

Abbreviations: PBS, phosphate buffered saline; TBS, tris bufferedaline; ELISA, enzyme-linked immunosorbent assay; CLEIA, chemilumi-escence enzyme immunoassay; CMO, carboxymethyloxime; EE2, 17�-thinylestradiol; POD, horseradish peroxidase; STP, sewage treatment plant;V, coefficient of variation; LOD, limit of detection∗ Corresponding author. Tel.: +49 228 732856; fax: +49 228 732489.

E-mail address: schneider@uni-bonn.de (R.J. Schneider).

head minnow a NOEC value of 1 ng L−1 was established foEE2[4]. Therefore there is an urgent need for measuremin the lower nanogram per litre range.

EE2 and other estrogenic hormones are usually qufied in aqueous matrices using standard instrumental mesuch as gas chromatography–mass spectrometry or higformance liquid chromatography–mass spectrometry.rent GC–MS/MS and LC–MS/MS methods achieve detion limits below 1 ng L−1 after sample enrichment[5–7].In addition to this enrichment that might cause problwith recovery, these chromatographic methods requirextensive sample clean-up due to ionization suppresEnrichment and clean-up steps are by themselvesprone and can lead to decreased precision and accurresults.

Alternatively immunoassays can be used for quantion of EE2 in the lower nanogram per liter range. Sevimmunoassays based on RIA and ELISA formats havedeveloped for measuring EE2 in body fluids[8–14]. In recenyears immunoassays for monitoring EE2 in environmesamples have been introduced, too[15–17]. Although mos

003-2670/$ – see front matter © 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.aca.2005.07.018

C. Schneider et al. / Analytica Chimica Acta 551 (2005) 92–97 93

of the latter assays exhibit improved sensitivity, none of themis capable of measuring in the ecotoxicologically relevantsub-ppt concentration range directly. In our work we presentfor the first time the development and optimization of anELISA for the direct measurement of EE2 in the sub-pptrange.

2. Materials and methods

2.1. Reagents and materials

All reagents were of analytical or biochemical grade andwere used as received. Horseradish peroxidase (EIA grade)was obtained from Roche (Mannheim, Germany), 1,3,5(10)-estratrien-17�-ethinyl-3,17-diol-6-one-6-carboxymethyl-oxime (EE2-6-CMO) was from Steraloids (London, UK),GuardianTM and SuperSignalTM femto was from Perbio(Bonn, Germany). 3,3′,5,5′-Tetramethylbenzidine (TMB)and TweenTM 20 were purchased from Serva (Heidel-berg, Germany), sodium azide was from VWR (Darmstadt,Germany), ultrapure water was obtained by running deminer-alized water through a Milli-Q water purification system fromMillipore (Eschborn, Germany).N,N-Dimethylformamide,N-methylmorpholine, isobutylchloroformiate, ethinylestra-diol, estradiol, tris(hydroxymethyl)aminoethane (Tris) anda en,G diolw eref mica vel-o n oft ds ty-l sd onc teb fferc acidp eachr t 96fla h-i sher( ncew witha r-m 30 nmu lo-g romM ndS 6,16-D fromC andC ny),r

2.2. Preparation of enzyme conjugate

Horseradish peroxidase (POD) was coupled to the 6-CMO derivative of EE2 using modifications of a methoddescribed for coupling POD and derivatives of progesterone[19]. The oxime (0.958 mg EE2-6-CMO, 2.5�mol) dissolvedin 50�L N,N-dimethylformamide in the presence of 0.4�LN-methylmorpholine was activated with 0.4�L isobutylchlo-roformiate under inert gas at−15◦C. After stirring for threeminutes this solution was slowly added to 2.5 mg POD (ca.5.7× 10−2 �mol with M = ca. 44,000 g mol−1) dissolved ina mixture of 25�L water and 15�L DMF cooled to the sametemperature. The reddish solution was stirred for 1 h andallowed to reach 0◦C, using an ice-bath. Stirring continuedfor another 2 h at 0◦C, after which the solution was directlypoured on a Sephadex G-25 column previously conditionedwith TBS (pH 7.5, 0.1 M Tris, 0.15 M NaCl). The same bufferwas used for elution of the enzyme conjugate. Fractions werecollected in a microtiter plate and their absorbance measuredphotometrically at 405 nm. Fractions of highest absorbancevalues were pooled, mixed with an equal amount of PODstabilizer (GuardianTM), aliquoted and stored at 4◦C. Using−15◦C as the initial temperature and 0◦C for the terminationof the reaction enables kinetic control in the activation of thesteroid and coupling to lysine residues of POD. Of the totalsix lysine amino groups present in POD due to steric restric-t ions.C ounto tingi ture[ atei

2

ndt reb gated singsE titerp tion1 mc withP ba-t BS1 5%T on-s wasp urew cleww Thisw ilu-t 6,c oom

ll buffer salts were supplied by Sigma–Aldrich (Taufkirchermany). All metabolites of estradiol and ethinylestraere from Steraloids (London, UK). Sephadex columns w

rom Amersham Biosciences (Freiburg, Germany). Hucid was from Carl Roth (Karlsruhe, Germany). The depment of the polyclonal antibody and the preparatio

he buffers have been described before[15], the TMB-baseubstrate (TMB-solution: 41 mM TMB, 8 mM tetrabu

ammoniumborohydride inN,N-dimethylacetamide) waeveloped by Frey et al.[18]. The actual substrate solutionsists of 540�L TMB-solution in 21.5 ml substrauffer (200 mM citric acid monopotassium salt buontaining 3 mM hydrogen peroxide and 0.01% sorbicotassium salt, pH 3.8) and was freshly prepared forun. Microtiter plates used were white and transparenat-bottom wells with high binding capacity (MaxisorpTM

nd FluoroNuncTM) from Nunc (Roskilde, Denmark). Wasng steps were carried out using an automatic plate waFlexiwash, Asys Hitech, Eugendorf, Austria). Absorbaas measured at 450 nm and referenced to 650 nmplate reader (Vmax®, Molecular Devices, Munich, Geany). Luminescence was detected between 380 and 6sing a MicroLumat Plus LB 96 V (Berthold Technoies, Bad Wildbad, Germany). Filters were obtained facherey–Nagel (Duren, Germany) and Schleicher achuell (Dassel, Germany). Deuterated Estradiol (2,4,14) and deuterated estrone (2,4,16,16-D4) wereambridge Isotope Laboratories (Andover, MA, USA)DN Isotopes (via Dr. Ehrenstorfer, Augsburg, Germa

espectively.

ions only three of them are accessible for coupling reactonditions described above, using a 44-fold excess amf oxime lead to a quantitative coupling to POD, resul

n a steroid to enzyme ratio of 3:1, as described in litera20–22]. Thus the initial concentration of enzyme conjugs estimated to be 6× 10−2 mmol L−1.

.3. Immunoassay procedures

Both immunoassays, the EE2 ELISA utilizing TMB ahe EE2 CLEIA using SuperSignalTM femto as substrate, aased on the polyclonal antibody and the enzyme conjuescribed above. The direct competitive ELISA format uequential saturation was adapted for both assays[23]. TheE2 ELISA was performed as follows. Transparent microlates were coated with the polyclonal antibody (dilu:200,000, 200�L per well) in coating buffer (50 mM sodiuarbonate buffer, pH 9.6). The plates were coveredarafilm® to prevent evaporation. After overnight incu

ion at 20◦C, the plates were washed three times with P(10 mM PBS pH 7.6, containing 150 mM NaCl and 2.8weenTM 20) using the automatic plate washer. For ctruction of the calibration curve an EE2 stock solutionrepared in methanol and then further diluted with ultrapater to obtain calibration solutions. After the three-cyashing step the plates had EE2 standards added (100�L perell) and were shaken at room temperature for 30 min.as followed by the addition of the enzyme conjugate (d

ion 1:50,000, 100�L per well) in PBS 2 (8 mM PBS pH 7.ontaining 145 mM NaCl); the plates were shaken at r

94 C. Schneider et al. / Analytica Chimica Acta 551 (2005) 92–97

temperature for 10 min, followed by a second three-cyclewashing step. Finally, substrate solution was added (200�Lper well) and incubated for 30 min. The enzyme reaction wasstopped by addition of sulfuric acid (1 M, 50�L per well).

The EE2 CLEIA was performed as follows. Whitemicrotiter plates (FluoroNuncTM) were coated according tothe ELISA procedure using a serum dilution of 1:200,000.EE2 standards (100�L per well) were incubated for 30 min,followed by a 10 min incubation together with the enzymeconjugate (dilution 1:50,000; 100�L per well). After incuba-tion plates were washed and placed in the luminometer. Twohundred microlitres of substrate were used per well, measure-ments were performed before addition of substrate (blank),immediately after addition and after a 3 min incubation time.Calibration curves were calculated using the difference of thethird and the first measurement.

All determinations were at least made in triplicate on dif-ferent plates. The mean values were fitted to a four-parametriclogistic equation (4PL)[24], optical densities and relativelight units were normalized according to Eq.(1) and plottedagainst the logarithmic concentration.

YN = Y − D

A − D× 100 (1)

YN is the normalized optical density (OD) or the normal-ized relative light unit (RLU), respectively,Y is OD (RLU,r p-t elyl Ld na-l entf andm

2

thers ne bya ross-r ionsao per-c

C

wi t thei hec

2

ven-t eres er-

cial humic acid as interfering agent. Concentrations of humicacid covered ranged from 0.2 to 5 mg L−1.

2.6. Water samples

Water samples were collected in brown glass bottles,stored at 4◦C and analyzed within 24 h. Two spiked sampleswere prepared using ultrapure water and tap water (of origin1/3 purified surface water and 2/3 groundwater). Eight sur-face water samples were taken at the river Rhine and two ofits tributaries in Rhineland-Palatinate (Ahr) and North Rhine-Westphalia (Agger). Four effluent samples were taken fromdifferent municipal sewage treatment plants (STPs) in thesurroundings of Bonn and Cologne.

2.7. Sample preparation

For CLEIA measurements surface water and effluent sam-ples were passed subsequently through folded filter papers(Macherey–Nagel, ref. no. 619) and by vacuum through glassfibre filter GF 8 (Schleicher and Schuell, ref. no. 370120).For LC–MS/MS measurements all samples were enrichedusing solid phase extraction following a method describedby Zuhlke et al.[25].

2.8. LC–MS/MS procedure

utef heH gi-l IM x,A diente ents.M ing anA ny)e iza-t ribedi alce threea

3

3

sen-s edt wasv -t (seeT cal-i angeo e

espectively),A is a parameter of the 4PL (“upper asymote” representing the OD (RLU, respectively) at infinitow analyte concentration,D is the parameter of the 4Pescribing the OD (RLU, respectively) at infinitely high a

yte concentration, the “lower asymptote”, which is differrom zero due to intrinsic optical density of substrateicrotiter plate and unspecific binding of the tracer).

.4. Cross-reactivity determination

The relative sensitivity of the immunoassay towards oteroid hormones was determined for estradiol and estrossaying a dilution series of each substance in water. Ceactivity is calculated as the ratio of molar concentratt the inflection points (midpoints, parameterC in the 4PL)f the corresponding calibration curves and expressed inentage relative to the midpoint for EE2 (see Eq.(2)).

R = Cstandard

Ctest× 100 (2)

here CR describes the cross-reactivity in percent,Cstandards a parameter of the 4PL giving the EE2 concentration anflection point andCtest refers to the concentration of tross-reacting compound at its inflection point.

.5. Matrix interference

Matrix constituents can be part of real samples and eually affect antibody or enzyme performance. Effects wtudied using serial dilutions of EE2 in solutions of comm

LC–MS/MS determinations were run by the Institor Hygiene and Public Health, University of Bonn. TPLC system consisted of an Agilent 1100 system (A

ent, Boblingen, Germany) with a Phenomenex SYNERGTM

ax-RP C18, 4.0�m, 2.0 mm× 150 mm (Phenomeneschaffenburg, Germany) as the stationary phase. Gralution was applied using acetonitrile and water as solvass spectrometric measurements were performed usPI 2000TM (Applied Biosystems, Darmstadt, Germaquipped with APCI source operated in the negative ion

ion mode. The method is an adaption of a method descn literature[25]. The detection limits for EE2 after internorrection for the surrogate standards d4-estrone and d4-stradiol was established at a signal-to-noise ratio oft 0.3 ng L−1.

. Results and discussion

.1. Assay optimization

Both immunoassays were optimized with regard toitivity. The initial serum dilution of 1:50,000 was increaso 1:200,000. Incubation time of the enzyme conjugatearied from 10 to 180 min (seeTable 1), the serial diluion of the conjugate in PBS 2 extended to 1:500,000able 2). Based on the optimized conditions, the EE2

bration curve was constructed in the concentration rf 0.001–1000 ng L−1. A typical calibration curve and th

C. Schneider et al. / Analytica Chimica Acta 551 (2005) 92–97 95

Table 1Assay optimization: photometric ELISA

Incubation time of enzyme conjugate (min)

10 30 60 180

ParameterAa (OD) 0.06± 0.001 0.32± 0.01 0.38± 0.01 0.51± 0.003ParameterCa (�g L−1) 0.10± 0.01 0.51± 0.05 0.79± 0.11 14.9± 5.0LOD (�g L−1) 0.02± 0.02 0.04± 0.004 0.14± 0.01 2.3± 2.8

Influence of the tracer incubation time on signal intensity and test sensitivity using an antiserum dilution of 1:200,000 and a tracer dilution of 1:50,000.a cf. Eqs.(1) and(2).

Table 2Assay optimization: chemiluminescence enzyme immunoassay (CLEIA)

Tracer dilution

1:500,000 1:250,000 1:50,000

ParameterAa (RLU 103) 38.0± 9.3 114± 24 436± 136ParameterCa (ng L−1) 7.39± 4.18 20.2± 4.5 102± 13LOD (ng L−1) 0.17± 0.10 0.77± 1.1 9.4± 4.6

Influence of the tracer dilution on signal intensity and test sensitivity usingan antiserum dilution of 1:200,000 and a tracer incubation time of 10 min.

a cf. Eqs.(1) and(2).

corresponding precision profile[24] is compared to a cali-bration curve under suboptimal conditions inFig. 1. Resultsof the optimization process can be clearly identified inthe calibration curve shifted to lower analyte concentra-tions. The LOD of the optimized test at a signal-to-noiseratio (S/N) of 3 is found to be 0.2± 0.1 ng L−1, the LOQ(S/N of 10) is 1.4± 0.8 ng L−1 calculated from eight indi-vidual calibration curves. The analytical working rangeof the optimized assay extends with a reasonable preci-sion (CV < 20%) from 0.8 to 100 ng L−1 making the assayusable over more than two orders of magnitude in analyteconcentration.

F ndingp unders indi-c lightu

3.2. Specificity of the antiserum

Specificity of the antiserum was tested in the EE2 CLEIAby determination of the molar cross-reactivities for estradioland estrone, steroids frequently occuring in STP effluents.Cross-reactivity of an antiserum is characterized by intrin-sic properties of the antibodies and binding kinetics appliedduring incubation of sample, enzyme conjugate and anti-serum. Sequential saturation methods working under non-equilibrium conditions are to be distinguished from equilib-rium type assays[26]. This assay and a Bio-LC EE2 ELISAdescribed before[15] are both non-equilibrium type assaysusing the same antiserum. The values for estradiol were 0.2%in both the CLEIA and the Bio-LC-ELISA and for estronewere 0.1% (CLEIA) and <0.1% (Bio-LC-ELISA), respec-tively [15]. The antiserum proved highly specific for EE2,exhibiting only negligible reactivities for structurally simi-lar compounds. Discrimination of these compounds can beexplained through superior steric requirements of the IgGpopulations and the respective binding pockets involved inrecognition and binding of the analyte.

3.3. Matrix interference

Humic acid was used as a model substance for the determi-nation of matrix effects. The assay exhibited a rather robustb simi-l tionc s ofh toh sen-su mics ferenta vail-a

3

m-p ande runa sedd r useo d ass re

ig. 1. Optimized calibration curve (solid line, squares) and corresporecision profile (dotted line, stars) compared to a calibration curveuboptimal conditions (dashed line, circles). Standard deviations areated as error bars. Signal intensity is shown in normalized relativenits and normalized optical density, respectively.

ehaviour in the presence of humic acids. In contrast toar studies in literature the sigmoidal shape of the calibraurves could be maintained at elevated concentrationumic acids[27]. However, curves were slightly shiftedigher concentrations of EE2 resulting in some loss ofitivity. Effects are shown in detail inFig. 2. The humic acidsed in this study is produced from pulverized lignite, huubstances present in water samples might have a difnd more complex composition than this commercially able one, though.

.4. Water samples

The optimized CLEIA was tested with four different sale matrices: ultrapure water, tap water, surface waterffluent of sewage treatment plants. All samples weret least in quadruplicate. Adsorption of EE2 to filters uuring sample preparation was not observed, moreovef these filters is a common procedure frequently applieample clean-up[6,28]. Relative standard deviations we

96 C. Schneider et al. / Analytica Chimica Acta 551 (2005) 92–97

Fig. 2. Influence of commercial humic acid on EE2 calibration curves.Curves are obtained using spiked calibrators: (squares) ultrapure water, (cir-cles) 0.2 mg L−1, (open circles) 0.5 mg L−1, (triangles) 1 mg L−1 and (opentriangles) 5 mg L−1 humic acid. Inset shows the influence on the correspond-ing midpoints (as a measure of sensitivity).

below 10%. EE2 was below the detection limit in three of theeight surface water samples. Two samples collected from therivers Rhine and Agger showed values above the LOD. Alleffluent samples taken from local STPs showed values abovethe LOD from 0.4 to 0.7 ng L−1 (Table 3). Results obtainedin reference measurements using LC–MS/MS were generallyin good agreement with the CLEIA results, the immunoassayshowing false-positive results with some surface and waste-water samples.

Table 3Determination of EE2 in spiked water, surface water and wastewater samples

Sample CLEIA (ng L−1) LC–MS/MS (ng L−1)

Spiked samplesa

Ultrapure water 1.4 2.1Tap water 1.5 1.3

Surface water (river, location)Ahr, spring <0.2 <0.3Ahr, springb <0.2 <0.3Ahr, Dumpelfeld <0.2 <0.3Ahr, Dumpelfeldb <0.2 <0.3Agger, Lohmar <0.2 <0.3Agger, Lohmarb 0.7 <0.3Rhine, Bonn 0.6 <0.3Rhine, Bonnb <0.2 <0.3

W

4. Conclusion

Extensive discussions of factors affecting immunoassaysensitivity have been published[23,29,30]. The underlyingconcept of sensitivity in such reagent-limited assays is basedon the antibody affinity. In contrast to immunometric assaysthe “detectability” of the label is of minor importance. How-ever, if in reagent limited assays the prerequisite of optimumdilutions cannot be achieved due to sensitivity restrictions ofthe label, then the detectability becomes a significant factorimpeding further improvements. Under identical conditionsboth labels, photometric and luminescent, yield identicalcalibration curves of the same sensitivity, yet the photomet-ric assay is slow in developing a rather low absolute ODsignal. Exemplary parameters of calibration curves obtainedusing both labels are as follows:Aluminescent 100± 6.2,Aphotometric 100± 7.6; Bluminescent 0.65± 0.1, Bphotometric

0.76± 0.2; Cluminescent 42.7± 14.0 ng L−1, Cphotometric

31.9± 10.4 ng L−1; Dluminescent0± 3.6,Dphotometric0± 2.4.Although the photometric method based on the detectionof peroxidase by the TMB/H2O2 substrate system witha detection limit of 2× 10−15 mol L−1 POD is alreadyvery sensitive[31], the switch to a luminol based substrateenabled us to achieve better detection limits due to itsenhanced detectability. Our assessment of assay charac-teristics is based on conservative conventions: LOD andL and1 cedu tha ion( ofel nge,s her-m ionsb forr

nt arei /MSm mplesm Them notu ncesp ver-e sultsd diesh rdt rva-t ptr xcel-l tomB ateso eforef re notp

astewaterSTP I 0.6 <0.3STP II 0.4 0.8STP III 0.4 0.3STP IV 0.7 0.6

a Samples were spiked with 2.5 ng L−1 EE2.b Second sample collection, some weeks later.

OQ are calculated using signal-to-noise ratios of 30, respectively, the analytical working range is dedusing Ekins’ description of the “precision profile” wi

comparatively low admissible coefficient of variatCV) of 20% [32]. It should be mentioned that any usempirical rules such as the Horwitz equation[33] would

ead to arbitrary assumptions regarding the working raimulating unsustainably low assay sensitivities. Furtore application of the Horwitz equation to concentratelow 100�g L−1 yields unacceptably high valueseproducibility CVs[34].

EE2 values measured in surface water and STP effluen good accordance with the results obtained in LC–MS

easurements. Deviations observed in wastewater saight be due to matrix effects regarding the antibody.olecular mechanism of this potential interference isnderstood, recognition of moieties from humic substaresent in the sample might be an explanation for ostimated EE2 concentrations. Such false positive reue to matrix components interfering with the antiboave been reported before[35]. As expected, with rega

o human excretion, dilution factor and more recent obseions by other researchers[5–7], all values are in the sub-pange. The antiserum employed in this study shows eent specificity for EE2 with negligible cross-reactivities

etabolites and conjugates[36]. Additionally, according toaronti[37] deconjugation of estrogens excreted as sulphr glucuronides takes place already in the sewers. Ther

alse-positive results due to cross-reacting compounds arobable to occur.

C. Schneider et al. / Analytica Chimica Acta 551 (2005) 92–97 97

As shown in this study antibody affinity and labeldetectability are of major importance for assay optimization.Under optimized conditions the reduction of experimentalerror and elimination of potential sources of imprecisionbecomes relevant. Although we were able to establish excel-lent detection limits in this manually performed assay, furtheradvances are expected from implementing this assay on anautomated system for water analysis[38].

Acknowledgement

C. Schneider thanks the German Federal EnvironmentalFoundation (DBU) for a Ph.D. scholarship. We express ourappreciation to Perbio and Nunc for excellent support and toD. Skutlarek and Dr. H. Farber of the Institute for Hygieneand Public Health, University of Bonn, for reference mea-surements.

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