Analytica Chimica Acta 444 (2001) 79–86

Simple dip strip ELISA for airborne estrogenic steroids

Stewart Armstrong a, Z.-F. Miao a, Frederick J. Rowell a,∗, Zulfiqur Ali b

a North East Biotechnology Centre, Institute of Pharmacy and Chemistry,School of Sciences, University of Sunderland, Sunderland SR1 3SD, UK

b Biosensors Group, School of Science and Technology, University of Teesside, Middlesborough, UK

Received 4 May 2000; received in revised form 10 October 2000; accepted 10 November 2000

Abstract

An ELISA for the potent estrogenic steroids, ethinyl estradiol (ETED), 17-�-estradiol (ED) and estrone (ES) has beendeveloped and used to determine the recovery of ED and ETED following spiking from seven filters commonly used insamplers for ascertaining personal exposure of workers to airborne biochemicals. Best results were obtained with cellulosenitrate (CN) and polytetrafluoroethylene (PTFE) filters. The assay reagents have also been used to develop a simple dip stripassay that can be used to determine the presence of steroids on the filters. Steroids captured from the air on the surface of filterswithin a conventional personal exposure sampler are extracted with specific antiserum. A spot of hapten-protein conjugate isimmobilised on a small square of CN filter attached to a plastic strip. This is immersed in the sampler solution where unboundantibodies bind to the hapten on the spot. The strips are then transferred to a 96 well filter plate located within a filter manifold.Following washing, strips are incubated with alkaline phosphatase-labelled second antibody and the spots were developedby addition of substrate. The intensity of the developed blue spot is inversely proportional to the amount of steroid originallycaptured on the filter. A batch of over 50 samplers can be screened within 1 h. Spots on strips from filters on which 10 ng ofED, ES or ETED are present can be visually discerned from strips from filters where no steroid is present. © 2001 ElsevierScience B.V. All rights reserved.

Keywords: Airborne estrogenic steroids; Health and safety; Recovery from filters; Dip strip ELISA

1. Introduction

Ethinyl estradiol (ETED), 17-�-estradiol (ED) andestrone (ES), a metabolite of ED, are highly potentestrogenic steroids. Recent reports describe their pres-ence in environmental water samples [1,2] althoughthe locations of highest concentrations of ETED andED are likely to be chemical and pharmaceuticalmanufacturing sites since the former is often used in

∗ Corresponding author. Tel.: +44-191-515-2504;fax: +44-191-515-2625.E-mail address: frederick.rowell@sunderland.ac.uk (F.J. Rowell).

female contraceptive formulations and the latter inhormone replacement therapies. Due to their potencyit is necessary to monitor airborne levels within thefactories in order to demonstrate that workers havenot inhaled levels that could be detrimental to theirhealth, and also to show that these compounds are notbeing released from the factories.

Although there are no legislative occupational en-vironmental standards for ETED and ED in-houseexposure levels have been set by manufacturers. Thusthe occupational exposure limit (OEL) for ETED iscurrently 100 ng m−3 although levels one-tenth of

0003-2670/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved.PII: S0003 -2670 (01 )01165 -5

80 S. Armstrong et al. / Analytica Chimica Acta 444 (2001) 79–86

this or less are expected in practice [3]. If a samplingdevice is used for 8 h at a flow rate of 2 l per min,then sampling air containing the drug at its OELwould deposit 96 ng of it on the filter or 9.6 ng if theconcentration was one-tenth of the OEL.

This paper describes development of a simple im-munoassay screen for the presence or absence ofETED or ED captured on filters within IOM-typeor cassette samplers following air sampling. Thesesamplers are chosen since they are commonly usedin industry, they provide a simple and portable mon-itoring system capable of capturing particles of res-pirable sizes, and the analysis of captured steroidsmay be amenable to simple immunoassay followingsampling. Such analysis could obviate the need forcomplex extraction coupled with expensive physic-ochemical methods of analysis such as HPLC [4]and GC [5] which are performed at dedicated lab-oratories often located far from the sampling site.This new approach would rapidly provide results onsite rather than waiting for days or weeks as is thecurrent case.

To this end, we have reported simple on-filterDOT–ELISA methods for �-lactam antibiotics [6]and some estrogenic steroids [7] following theircapture on filters within IOM-type samplers. Theformat adopted for those assays had several lim-itations. Firstly, prolonged air sampling depositedgrey dust from the air over the surface of the filterand unless the sampler was modified, the dust inter-fered with the endpoint of the assay. Secondly, thesample of analyte solution recovered from the filterwas lost during the assay, hence, if analyte were de-tected this could not be confirmed by a conventionalphysico-chemical assay. Finally, if a cassette samplerwas used its narrow air sampling orifice made additionand removal of reagents into and from the samplerdifficult.

This study aims to overcome these problems by re-locating the spot of immobilised antigen/hapten fromthe surface of the filter within the sampler onto thetip of a strip that can be inserted into the solution ofanalyte above the surface of the filter within the airsampler. The study will use 17-�-estradiol and relatedestrogenic steroids as the target analytes. The influ-ence of filter type on the efficiency of recovery andkinetics of dissolution of these steroids from the filterswas examined. The influence of dust captured during

air sampling on the recovery of captured analyte wasalso studied.

2. Experimental

2.1. Reagents

Cellulose nitrate (CN) membrane filters, diameter25 mm, pore size 1.0 �m, were from Whatman Inter-national Ltd. (Maidstone, Kent, UK), all other filterswere from Schleicher & Schuell (London, UK). Im-ages of spots were captured by a Microtek Phantom336CX scanner with ScanWizard for Windows soft-ware, Microtek Lab Inc. (Redondo, CA, USA). Theintensities of grey shades for spots on the dip stripswere measured by a Band Leader Application V 2.01shareware software, produced by Maayan Techknowl-edge (Tel-Aviv, Israel). IOM personal inhalable dustsamplers for the membrane filters were purchasedfrom SKC Ltd. (Blandford, Dorset, UK). Acetateplate sealers (8.3 cm × 13.3 cm) were from DynexTechnologies Inc. (Chantilly, VA, USA). The flat bedshaker was model HS 250 from Janke and Kunkel andsupplied by S.H. Scientific (Blyth, Northumberland,UK). The microtitre filter plate was a Uni-filter®

350, category No. 7700-3307, from Whatman Inc.(Clifton, NJ, USA) and the filtration manifold wasfrom Polytronics®, Whatman Inc., at the same ad-dress. The portable air pumps were from Gil-Air Per-sonal Air Samplers, Pine Environmental Services Inc.(Cranbury, NJ, USA).

All chemical reagents were purchased from SigmaChemical Co. (Poole, Dorset, UK) unless otherwisestated. The buffer salts were of analytical-reagentgrade (BDH Chemicals, Poole, Dorset, UK). Phos-phate buffered saline with Tween 20, pH 7.4(PBST), and carbonate buffer, pH 9.6 were preparedas previously described [7]. BCIP stock solutionwas 50 mg ml−1 bromochloroindolylphosphate(BCIP) in 100% dimethyl formamide (DMF).Nitrobluetetrazolium (NBT) stock solution was75 mg ml−1 NBT in 70% DMF. Substrate solu-tion for the dip strip assay was prepared from amixture of BCIP stock, NBT stock and carbonatebuffer with volume ratio of 1:1:100. Chemicals usedin the cross reactivity experiment were obtainedfrom the following sources; nafarelin acetate and

S. Armstrong et al. / Analytica Chimica Acta 444 (2001) 79–86 81

17-�-ethinylestradiol-2-methyl ester, Monsanto (St.Louis, MS, USA); ethynodiol diacetate, 17-�-estradioland nonyl phenol, Sigma; and ethyl, butyl, octyl anddecyl phthalate esters, CSL Food Science Laboratory(Norwich, Norfolk, UK).

2.2. Production of polyclonal antibodiesto estradiol

Antiserum was raised in rabbits to an immunogen of17-�-estradiol–hemisuccinate–bovine serum albumin(ED–HS–BSA) following a reported protocol [7].

2.3. Microtitre plate ELISA development

An indirect competitive ELISA for ED was de-veloped using the immunogen conjugate adsorbedonto 96 well microtitre plates, antiserum diluted inPBST containing 0.1% BSA and 10% ethanol byvolume (PBST–BSA–EtOH) (Ab1), and an alkalinephosphatase-labelled monoclonal mouse anti-rabbitIgG whole molecule second antibody (Ab2). Thesubstrate used was p-nitrophenyl phosphate (PNPP,1 mg ml−1) in substrate buffer and the end point of theassay was determined from the absorbance reading at405 nm. A range of concentrations of each reagent wasused as was a range of incubation times for the individ-ual steps within the assay. Reagent concentrations andassay conditions were chosen to produce a standardcurve over the range 10−9 to 10−6 M for ED, ETEDand ES. The specificity of the antiserum in this for-mat was tested using a standard cross-reactivity studywith ED, ES, ETED, 17-�-estradiol, nafarelin acetate,ethynodiol diacetate, 17-�-ethinylestradiol-3-methylester, nonyl phenol and four alkyl phthalate esters.

Table 1Recovery of 17-�-estradiol from selected filters

Filter type Pore size (�m) Recovery of17-�-estradiol (%)

Non-specific adsorp-tion of antibody (%)

Time toplateau (min)

Cellulose acetate 2.00 22 10 40Cellulose nitrate 1.00 150 50 45Regenerated cellulose 1.00 37 0 45Mixed ester 2.00 55 17 45Durapore (DVPP) 0.65 64 6 35Nylon 0.20 63 0 35PTFE 1.00 83.3 0 30

2.4. Recovery of steroids from filters

Standards of the three steroids of 10−5 M concen-tration were prepared in PBST–BSA–EtOH. Aliquotsof 10 �l were placed at the centre of each filter, whichwere then allowed to air dry. This process depositedabout 30 ng of the steroid on to each filter. As a con-trol 10 �l of the PBST buffer was added to matchedfilters. Addition of this solution wetted the surface forall the filter types apart from the polytetrafluoroethy-lene (PTFE) filters. Initially, the ED standard was usedwith the full range of seven filters, but only PTFE andCN filters were used with ETED and ES. Details ofthe filters are shown in Table 1.

Following air drying for 30 min the filters wereplaced in IOM samplers and the samplers securedon a flat bed shaker. To each sampler was thenadded 1 ml of Ab1 diluted to a final dilution of1:10 K with PBST–BSA–ethanol. The samplers werethen shaken for up to 2 h and 10 �l aliquots ofthe solution above the filters were periodically re-moved to a 96 well microtitre plate pre-coated withED–HS–BSA conjugate for ELISA analysis. The as-say was performed by adding 90 �l of Ab1 (1:10 Kdilution in PBST–BSA–EtOH) from the IOM sam-plers to the wells designated for samples followedby 10 �l of PBST–BSA–EtOH buffer per well.Standards of steroid (10−9 to 10−6 M) in 10 �l ofPBST–BSA–EtOH were added to other wells fol-lowed by 90 �l of Ab1 as above. The plate was left for30 min, and then the wells were emptied by gravityand then washed under the tap with dH2O. The wellswere tapped dry and 100 �l of Ab2 (diluted 1:2 K inPBST) added. After incubation for 30 min the wellswere emptied, washed and dried as before. Substratesolution (100 �l) was then added to each well and the

82 S. Armstrong et al. / Analytica Chimica Acta 444 (2001) 79–86

plate incubated at RT for about 30 min when the ab-sorbance (optical density, OD) reading at 405 nm forthe zero standards was about 1.5 U. The concentrationof steroid in the samples was then interpolated fromthe resulting standard curve. This information wasused to plot the percent recovery of steroid from thefilter against time. As a second control, experimentswere also run in which steroid was deposited on thefilter as described, but 1 ml of PBST–BSA–EtOHreplaced Ab1. Hence, it was possible to compare theefficiency of extraction of the steroid from the filterwith and without antibody present.

2.5. Development of the dip strip ELISA

Strips were prepared by sticking squares of CN fil-ters (1 cm × 5 cm) to sticky backed plastic microtitreplate sealer. Strips of about 6 mm width and 5 cmlength were cut from the plastic sheet. These retainedthe plastic cover adjacent to the attached CN mem-brane with a gap of about 2 mm between the top ofthe membrane and the bottom of the covering sheet.

A spot of ED–HS–BSA (1 �l of a 0.5–20 �g ml−1

solution in coating buffer) was dispensed on to thecentre of the membrane and was allowed to dry in theair for 5 min. It was then transferred to an oven whereit was incubated at 37◦C for 30 min. Strips were thenstored sealed in aluminium foil in a desiccator at roomtemperature until used.

The assay was performed by spiking PTFE or CNfilters with 3 or 10 �l of PBST or 10−5 M solutionsof ED, ETED or ES (equivalent to about 10 or 30 ngof the steroid) as described above. The dried filterswere placed in IOM-type samplers and the side armsealed with rubber teats. Ab1 (1 ml of 1:10–100 K di-luted in PBST–BSA–EtOH) was added to cover eachfilter. The samplers were then shaken for 15 min atroom temperature with 150 motions per min shaking.The dip strips were then inserted into the antibodysolution. It was necessary to slightly tip each sampleron the shaker to achieve adequate coverage of themembrane by the solution. After shaking for a further15 min the strips were removed and inserted into thewells of a 96 well microtitre filter plate. The platewas located within a filter manifold that was attachedto a vacuum pump. Each strip was washed withdistilled water with the vacuum on. The tap on themanifold was then closed and Ab2 (100 �l, diluted

2 K in PBST) added to the wells. After incubating for15 min the strips were washed with water as beforeand substrate solution added to each well (100 �l ofBCIP/NBT). After about 2–5 min blue spots werevisible on the zero (PBST-spiked) strips. The sub-strate solution was removed by filtration and washedwith water. The intensities of the resulting spots werethen recorded following visual inspection or using anoptical scanner after the strips had dried.

2.6. Analysis of ambient air forestrogenic steroids

Six CN filters were placed within IOM samplers.Two were attached to portable pumps which were setat a flow rate of 2 l min−1. Ambient air from the labo-ratory was sucked through these samplers at this ratefor 10 h. An aliquot (10 �l) of a 10−5 M standard ofED was then added to the filter surface of two of theunused samplers. These were allowed to dry in air for30 min after which Ab1 was added to all six samplersand the dip strip assay performed.

2.7. Recovery from filters exposed to air andspiked with 17-β-estradiol

The above experiment was repeated with two sam-plers. Following air sampling as described, the palegrey surface of each CN filter was spiked with 10 �l ofstandard containing 30 ng of ETED. Fresh, unexposedfilters within two other samplers were also spiked inthis way. Finally, a further two samplers containingfresh unexposed filters were also used. These were notspiked with the drug. After about 30 min antiserumwas added to each sampler and the dip strip assay per-formed as described above. The intensities of the spotsthat developed on the six strips were measured usingan optical scanner. The intensities of the spots on thestrips were also visually compared.

3. Results

3.1. Antiserum production

The antiserum used in this study was from the thirdbleed of rabbit 3 [7].

S. Armstrong et al. / Analytica Chimica Acta 444 (2001) 79–86 83

3.2. ELISA in the 96 well microtitre plate format

The conditions chosen (volumes per well) were:coating conjugate, 100 �l of a 1 �g ml−1 solutioncoated overnight at 4◦C; Ab1, 90 �l diluted 1:10 K inPBST–BSA–EtOH buffer; standard or sample, 10 �ldissolved in PBST–BSA–EtOH; Ab2, 100 �l diluted1:2 K in PBST; substrate, 100 �l per well; incubationtimes, first and second, both 30 min, and final 30 min.This gave a linear standard curve over the range 10−9

to 10−6 M ED (typically r2 = 0.977, n = 4) andreasonable precision with typical R.S.D. values of4–12% for standards. The assay was shown to bespecific for estrogenic steroids having an unsubsti-tuted A-ring as only ED (100%), ES (33%), ETED(29%) and 17-�-estradiol (33%) exhibited significantdisplacement. None of the other compounds testedexhibited significant cross reactivity in the ELISA.

3.3. Recovery of steroids from filters

Different recovery versus time profiles were ob-served for ED added to the six filters. Good and mostrapid recovery was seen with the PTFE filters. Al-though good and rapid recovery was also seen with CN

Fig. 1. Recovery–time profiles for 17-�-estradiol from the seven filters tested.

filters, it was observed that after 30 min non-specificabsorption of antibodies occurred resulting in apparentrecoveries of 150% for the spiked filters and 50% forthe unspiked ones. However, negligible non-specificbinding was seen with the PTFE filters. These resultsare summarised in Table 1, and the recovery versustime profiles are shown in Fig. 1. There were no sig-nificant differences in recovery profiles for the spikedfilters between Ab1 and the PBST–BSA–EtOH buffer.Due to these results, CN and PTFE filters were singledout for further study for recovery with ETED and ES,and for the dip strip ELISA. The recovery values forETED and ES were not significantly different fromthose observed with ED.

3.4. Performance of the dip strip ELISA

The initial incubation time of 15 min was chosenfrom the results obtained in the recovery experi-ments. The second incubation step was terminatedafter 15 min as use of CN membrane for longer pe-riods was found to increase background colorationon substrate addition presumably due to non-specificabsorption of the second antibody. Good visual spotintensity was observed with a final incubation step

84 S. Armstrong et al. / Analytica Chimica Acta 444 (2001) 79–86

Fig. 2. Spots from dip strip assays using PTFE filters. Strips 4, 5, and 11 were from unspiked filters; strip 6 from a filter spiked with 1 ngof ED; strip 7 from a filter spiked with 10 ng of ED; strip 8 from a filter spiked with 1 ng of ES; strip 9 from a filter spiked with 10 ngof ES; and strip 12 from a filter spiked with 10 ng of ETED.

of 2 min with a 1:60 K dilution of first antibody and5 min with a 1:100 K dilution. The 1:100 K dilutionwas used since this produced an intense spot for thezero standard and a pale spot for the 10 ng standardsof ED, ES or ETED. Examples of the end points ob-tained with these steroids extracted from PTFE filtersare shown in Fig. 2.

Fig. 3. Effect of airborne dust on the dip strip assay. Strip 1 (no dot at top) was from a fresh unspiked CN filter; strip 2 (single dot attop) from a fresh CN filter spiked with 30 ng of ED; and strip 3 (double dot at top) from a CN filter exposed to 1200 l of air over 10 hand then spiked with 30 ng of ED.

3.5. Analysis of ambient air forestrogenic steroids

Following air sampling for 10 h when 1200 l of airhad passed through each filter, a grey deposit was seenon the surface of each filter due to captured particles.These samplers and the four unexposed samplers were

S. Armstrong et al. / Analytica Chimica Acta 444 (2001) 79–86 85

subjected to the antibody extraction and the dip stripassay. Filters in two of the unexposed samplers hadbeen spiked with 30 ng of ED and the other two servedas fresh zero controls. There was no significant dif-ference in the intensities of spots obtained from stripsadded to the exposed samplers and those from thefresh zero controls. The spots on the two strips fromthe spiked filters were significantly paler to the eyethan these four spots. Hence, there was no detectablesteroid present within the laboratory air.

3.6. Recovery from filters exposed to air andspiked with 17-β-estradiol

The intensities of the spots on strips from the spikedfilters were observed to be less intense than those fromthe unspiked filter. There were no visible differencesin the intensities from the two sets of spiked filtersalthough the intensities of the spots from the filtersexposed to 1200 l of air over 10 h were marginallyless intense than the corresponding spots from thefresh unexposed filter also spiked with 30 ng of ED(Fig. 3). The mean values were 210 and 192, re-spectively, compared with the blank value of 130 U(the intensity is inversely related to the signal fromthe scanner).

4. Discussion

The object of the study was to develop a robust butrelatively simple and cheap semi-quantitative assayfor detecting estrogenic steroids on filters followingair sampling. The assay is to be used within a factoryenvironment as part of a screen to assess exposure ofworkers to airborne ED or ETED. The assay wouldneed to be completed within 60 min and as describedabove to detect 10 ng of these steroids captured on thesurface of the filters.

The assay assumes reasonable recovery of steroidcaptured on the filter during the initial incubationwith specific antibody added to the filter within thesampler. Hence, it was first necessary to identify thefilter composition which afforded the most rapid,most consistent and highest recovery of steroid. Thiswas achieved by using the indirect ELISA for ED andadding Ab1 from the filter for analysis in this assay. Itwas therefore possible to determine the amount of ED

bound to the antiserum taken from above the filtersover the time course of the experiment. As seen fromTable 1 and Fig. 1, most rapid recovery was seen withPTFE filters although CN filters also had acceptableproperties. However, significant non-specific antibodybinding to the filters was seen with the CN filters after30 min. These two filters were therefore chosen foruse in the assay with an extraction time of 15 min andcontact with the dip strip for a further 15 min. Thus,total contact of the filter with Ab1 was 30 min whichwas designed to produce adequate recovery for bothfilter types (>70%) without undue non-specific bind-ing with the CN filters. It was deemed unnecessaryto obtain 100% recovery since identical filters spikedwith standards were to be processed at the same timeas actual samples.

A variety of parameters were studied in the develop-ment of the dip strip assay. Good visual discriminationbetween unspiked and those spiked with 10 ng of ED,ETED or ES (Fig. 2) was found using the conditionsdescribed. The overall assay time was less than thetarget of 60 min and use of the microtitre filter plateand filter manifold enabled facile incubation with Ab2and substrate and easily washing steps. This formatalso allowed processing of a batch of up to 96 strips.

The specific antiserum was shown in the ELISA tocross react only with estrogenic steroids which pos-sessed underivatised phenolic A-rings. Presence ofsubstituents at the 17-position in the D-rind had onlyminor effects on the binding characteristics. Othernon-estrogenic steroids or estrogenic steroids deriva-tised in the A-ring or their pro-drugs did not exhibitsignificant cross-reaction. This was also observed fornafarelin, a synthetic peptide that is used therapeuti-cally as an inhibitor of gonadotropin release [8], nonylphenol and the four alkyl phthalates studied. Hence,presence of progestrogens that are also used in somefemale contraceptive pills [8] would not interfere inthe assay for ETED.

No estrogenic steroids were detected in ambient lab-oratory air using the dip strip assay. The effect of dustco-captured on the filter with ED on recovery of thedrug during the dip strip assay was also determinedby spiking with ED filters coated with dust particlesfollowing air sampling. No reduction in the recoverywas observed in this experiment indicating that pres-ence of dust on the filters is unlikely to interfere withthe assay for the drug. The coated dip strips are also

86 S. Armstrong et al. / Analytica Chimica Acta 444 (2001) 79–86

stable for extended periods (over 4 weeks) when storedas described (data not presented). Good stability wasalso observed for the spots of the same coating con-jugate located on CN filters [7].

The precision of the dip strip assay was adequate fora semi-quantitative assay. A relative standard deviationof 18% was found for the intensity of spots from abatch of six untreated filters (data not shown).

The dip strips used in the assay are narrow enoughto be easily inserted through the air inlet orifice of cas-sette type air samplers and the format described cantherefore be used with both IOM and cassette sam-plers. Insertion of the dip strip only results in adsorp-tion of unbound antibody onto the spot of immobilisedhapten leaving the analyte in the solution within thesampler. Hence, if the screen results in detection of thesteroid then quantitation of the amount of steroid isthen required. The solution containing the steroid canbe removed and the steroid extracted and analysed byconventional physico-chemical methods. Since only asmall fraction of samples are likely to result in positivedip strip tests, the approach described herein shouldprove to be highly cost effective.

Acknowledgements

We gratefully acknowledge part funding of thiswork by the Monsanto Corporation, MO, USA.

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