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Clinical Chemistry 44: 1520-1528, 1998;
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(Clinical Chemistry. 1998;44:1520-1528.)
© 1998 American Association for Clinical Chemistry, Inc.

Endocrinology and Metabolism

Monitoring ovarian function by the simultaneous time-resolved fluorescence immunoassay of two urinary steroid metabolites

Geoff Barnard1,a, and Fortune Kohen2

1 Endocrine Unit, Department of Chemical Pathology, Southampton University Hospitals Trust, Tremona Road, Southampton SO16 6YD, UK.

2 Department of Biological Regulation, Weizmann Institute of Science, Rehovot 76100, Israel.
a Author for correspondence. Fax 44-1703-796898; e-mail GJRB{at}aol.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We report the development of a novel time-resolved fluorescence immunoassay utilizing two different assay strategies for the simultaneous measurement of estrone-3-glucuronide (EG) and pregnanediol-3{alpha}-glucuronide (PG) in samples of early morning urine (EMU). The method for the measurement of EG involves the use of a labeled anti-idiotype as a surrogate antigen, whereas the other method (for the measurement of PG) is a regular competitive immunoassay using a labeled antigen. In addition, the procedure uses different lanthanide chelates as labels to monitor ovarian function in women. After washing the streptavidin-coated plate, we added 10 µL of undiluted urine or mixed standard to the coated wells, followed by the addition of 100 µL of assay buffer containing the labeled reactants (i.e., europium-labeled PG and samarium-labeled anti-idiotype recognizing the binding site of the antibody to EG). Subsequently, we added 100 µL of assay buffer containing the two biotinylated specific monoclonal anti-steroid glucuronide antibodies. After incubation for 1 h on a shaker at room temperature, we washed the plate and added 200 µL of enhancement solution to each well. We measured europium and samarium fluorescence, using a gated plate fluorometer with appropriate emission filters. The method demonstrates appropriate sensitivity and precision (all CVs, 5–8%) across the relevant working ranges for each analyte. The technique has been applied to serial EMUs collected from women with normal and stimulated menstrual cycles.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Over the last two decades, clinical studies have indicated the utility of measuring estrone-3-glucuronide (EG)1 and pregnanediol-3{alpha}-glucuronide (PG) in samples of early morning urine (EMU) to monitor ovarian function in women (1)(2). Initially, RIA techniques for the measurement of these metabolites were developed (3)(4)(5) and extensively used in the delineation of the fertile period (6)(7)(8).

More recently, nonradioisotopic alternatives for the measurement of these analytes have been reported that have involved the use of (a) enzymes (9)(10)(11), (b) chemiluminophores (12)(13), and (c) fluorophores (14)(15). In particular, the application of time-resolved fluorometric assays using lanthanide chelates as labels has led to the development of methods with great sensitivity and precision over a very wide dynamic working range of analyte concentrations (16)(17)(18).

Moreover, it has been shown that the fluorescence emission wavelengths from alternative lanthanides are sufficiently different to be able to discriminate between them, using appropriate filters (19). This observation was successfully exploited in the development of a separation immunoassay for the simultaneous measurement of follitropin and lutropin (LH) in serum (20). Subsequently, dual kits for the simultaneous measurement of human chorionic gonadotropin (hCG) and alpha-fetoprotein in maternal serum to assist the screening of Down syndrome, and free and total serum prostate-specific antigen to facilitate the identification of prostatic cancer have become available commercially (Wallac Oy). To facilitate the development of simultaneous assays, a research fluorometer (Wallac 1234) that has the capability of measuring up to three different lanthanides (europium, samarium, and terbium) with the use of simple emission filters has been available for several years.

Accordingly, in this study, we describe the development of a simultaneous fluorescence immunoassay (FIA) for the measurement of EG and PG, using samarium and europium chelates to investigate ovarian function. In addition, the method for the measurement of EG utilizes an anti-idiotypic antibody as a surrogate antigen, whereas the method for the measurement of PG is a regular competitive immunoassay.

The advantage in the measurement of urinary metabolites to the laboratory is a rapid method that can easily be automated and that does not suffer from some of the technical problems associated with the direct estimation of plasma steroids. Moreover, the advantage to the clinician is an overview of hormonal activity that cannot be obtained from the results obtained from serum samples taken at infrequent intervals.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
equipment
A time-resolved fluorometer (1234), plate washer (1296–024), plate shaker (1296–001), and plate dispenser (1296–041) were kindly provided by Wallac OY, Turku, Finland. NUNC Maxisorb microtitration plates (cat. no. 4–49824A) and sealing tape (Cat. No. 2–36269K) were obtained from Life Technologies Ltd. Amicon Microcon 30 Concentrators were purchased from Millipore (UK) Ltd.

basic reagents
Steroids, biotinamidocaproate-N-hydroxysuccinimide ester, Tris, bovine serum albumin (BSA, Fraction V), Tween 20, diethylene triamine pentaacetic acid, bovine {gamma}-globulin, incomplete and complete Freund's adjuvant, 1-ethyl-3(3-dimethyl aminopropyl) carbodiimide (water-soluble carbodiimide), ovalbumin, and other buffer ingredients were purchased from Sigma Chemical Co. Affinity-purified rabbit immunoglobulins were obtained from Dakopatts. Rabbit anti-rat IgG was purchased from Jackson. Sepharose-Protein A and Sephadex G-25 were products of Pharmacia. Anti-mouse immunoglobulins (used for Ouchterlony immunodiffusion) were from Serotec.

Streptavidin-coated plates (cat. no. C122–105), assay buffer (cat. no. 1244–106), wash concentrate (cat. no. B117–100) and enhancement solution (cat. no. 1244–104) were kindly provided by E.G.&G. Wallac UK Ltd., Milton Keynes, UK. The assay buffer consisted of a ready-for-use Tris-HCl-buffered (pH 7.8) salt solution with BSA, bovine {gamma}-globulin, Tween 40, an inert red dye, and <1 g/L NaN3 as a preservative. The wash concentrate consisted of a 25-fold concentration of Tris-HCl-buffered (pH 7.8) salt solution with Tween 20 and preservative. We prepared the wash solution by diluting the wash concentrate 25-fold (i.e., 40 mL of concentrate diluted to 1 liter) with distilled water. The enhancement solution was a ready-for-use solution containing the appropriate concentrations of Triton X-100, acetic acid, and chelators. This was stored at room temperature, avoiding direct sunlight.

The labeling reagent N-1(p-isothiocyanatophenyl)-diethylene-triamine N,N,N1 -tetraacetate chelated with europium (cat. no. 1244–302) and samarium (cat. no. 1244–303) were kindly provided by E.G. & G. Wallac UK Ltd., Milton Keynes, UK. Europium-labeled PG was synthesized by Dr. Heikki Mikola, Wallac Oy, Turku, Finland, by a procedure described previously (16). Affinity-purified monoclonal antibodies to PG-BSA (Clone 2555.6) were kindly provided by M. Gani, Unilever Research, Colworth Laboratory, Sharnbrook, Bedford, UK. The specificity of these antibodies has been reported elsewhere (16). The immunogen, EG conjugated to BSA, was a gift from the late Dr. P. Samarajeewa from University College, London, UK.

preparation of monoclonal anti-estrone-3-glucuronide antibodies
We used EG-BSA conjugate (50 µg/animal) in complete Freund's adjuvant to immunize female Wistar-derived rats (age, 2 months). Subsequently, we gave the rats two booster injections using the same conjugate in Freund's incomplete adjuvant. After 2 months of immunization, we checked the antibody titer, using rabbit anti-rat IgG-coated plates and europium-labeled EG with time-resolved fluorescence detection. Three months after the initial immunization, we fused the rat spleen cells showing the highest titer of antibodies with a mouse myeloma cell line (NSO, kindly provided by Prof. C. Milstein, Medical Research Council, Cambridge, UK) (21). We screened the culture supernatants of growing hybridomas for antibody activity, using rabbit anti-rat IgG-coated plates and europium-labeled EG by a procedure described previously (22). We selected four hybridomas that secreted antibodies against EG. Of these, we propagated one clone (8A3) that exhibited the highest affinity and specificity as ascites in pristane-primed irradiated CD2 male mice. Clone 8A3 belonged to the IgG2a class, and we purified the ascites derived from this clone by affinity chromatography on Sepharose-Protein A as described previously (23). We labeled 1 mg of the IgG fraction with europium chelate as described previously (24) and stored the rest at -20 °C until use. For screening purposes, we labeled another irrelevant rat-mouse hybridoma (clone 2F9) of the same isotype as clone 8A3 with europium.

preparation of monoclonal anti-idiotypic antibodies against anti-eg
We immunized female CD2 mice (age, 2 months) with anti-EG IgG (clone 8A3, 50 µg/mouse) in Freund's complete adjuvant. Subsequently, we gave the mice one booster injection, using EG IgG in Freund's incomplete adjuvant. Ten days afterwards, we checked the anti-idiotypic response as described previously (24). Ten weeks after the initial immunization, we fused the spleen cells of the CD2 mouse showing the highest serum titer of antibodies recognizing europium-labeled anti-EG IgG with mouse myeloma cell line NSO (as above). Subsequently, we screened the culture supernatants of growing hybridomas for antibody activity, using the following screening procedures.

screening assay 1: identification of IgG-secreting hybridomas
In this screening assay, we added hybridoma culture supernatants (50 µL/well) to anti-mouse IgG-coated microstrips containing assay buffer (150 µL/well). We incubated the plates for 1 h and then washed them three times. We blocked the plates by adding 10 µL of mouse serum, followed by 5 min of rapid shaking. Subsequently, we added 200 µL of europium-labeled anti-EG IgG (clone 8A3, ~10 cps/µL) to each well. We incubated the plates for 1 h and then washed and processed them for time-resolved fluorescence detection by procedures already described (24).

screening assay 2: identification of the epitope specificity (1)
In the second screening procedure, we added, in duplicate, the hybridomas (50 µL/well) that gave positive results in the first screening assay to anti-mouse IgG-coated strips containing 150 µL of assay buffer. We incubated the plates and then washed and blocked them as described in the first screening procedure (see above). Subsequently, we added 200 µL of assay buffer containing europium-labeled clone 2F9 (10 cps/µL; same isotype as anti-EG clone 8A3) to each well. The plates were processed as described in the first screening procedure. We classified the immobilized hybridoma culture supernatants that failed to bind europium-labeled clone 2F9 as anti-idiotypic antibodies (i.e., IgG binding to epitopes specific to 8A3) and those that gave positive results as xenotypic antibodies (i.e., IgG recognizing common epitopes expressed by both 2F9 and 8A3).

screening assay 3: identification of epitope specificity (2)
In this screening procedure, we added, in quadruplicate, the nine hybridomas (50 µL/well) classified as anti-idiotypes in the second screening assay to anti-mouse IgG-coated microtiter wells containing 150 µL of assay buffer. We incubated the plates for 2 h and then washed and blocked them as described for the first screening procedure. Subsequently, we added 200 µL of assay buffer containing europium-labeled anti-EG (clone 8A3; 10 cps/µL) to one set of duplicates and 200 µL of assay buffer containing EG (500 µg/L) and europium-labeled anti-EG (clone 8A3; 10 cps/µL) to the other set of duplicates. After 1 h of incubation, the plates were processed as described in the first screening procedure. We classified the immobilized hybridoma culture supernatants that failed to bind europium-labeled anti-EG in the presence of EG as betatypes (i.e., anti-idiotypic IgG that binds at the binding site of 8A3). On the other hand, we classified the IgG in the wells that gave positive results as alphatypes (i.e., anti-idiotypic IgG that binds near but not at the binding site of 8A3). For the purposes of the dual assay, we propagated one strong betatype (clone 11C7) against anti-EG (clone 8A3) in vivo as ascites in the peritoneum of pristane-primed male mice.

We prepared an IgG fraction of this betatype by Protein A–Sepharose affinity chromatography and labeled this purified fraction with samarium by a similar procedure to that described for the preparation of the europium-labeled conjugates.

biotinylation of primary monoclonal antibodies
We added 500-µL aliquots of the primary antibodies (anti-EG, 8A3; and anti-PG, 2555.6) to separate Microcon-30 devices in Eppendorf tubes. We capped the tubes and centrifuged them for 15 min in a Microfuge (6700g; MSE Micro Centaur). Subsequently, we removed the eluate from the tube and diluted the retentate by adding 400 µL of carbonate biotinylation buffer (50 mmol/L, pH 9.3). After centrifugation for an additional 15 min (6700g), we removed the eluate and added another 400 µL of carbonate biotinylation buffer. This procedure was repeated twice more. We diluted the retentates by the addition of 300 µL of biotinylation buffer to each tube and transferred them to separate glass bottles. We carefully washed the Microcon-30 devices by the addition of another 400 µL of bioinylation buffer, with removal of solution to the appropriate glass bottles. We determined the protein concentrations at 280 nm. We added additional biotinylation buffer to give final concentrations of the monoclonal antibodies of ~1 g/L.

We added 1 mL of dry dimethyl formamide to the bottle containing 10 mg of biotinamidocaproate N-hydroxysuccinimide ester (NHS-LC-biotin) and mixed the solution for ~20 s until the solid material was dissolved. We added 20 µL (for each milligram of IgG) of this NHS-LC-biotin solution to each bottle containing the diluted monoclonal antibodies. The bottles were capped, mixed, and left at 4 °C overnight. The following day, we transferred the solutions to clean Microcon 30 microconcentrators and centrifuged them at 6700g for 20 min as described previously. Subsequently, the eluates were discarded and ~400 µL of Tris-buffered saline (TBS) was added to the retentates. The centrifugation was repeated three times, with the samples being diluted with 400 µL of TBS each time. We prepared the 50 mmol/L solution of TBS using the pre-set TRIZMA® crystals (pH 7.8), sodium chloride (9 g/L), and sodium azide (1 g/L).

Finally, we diluted the retentates with 400 µL of TBS, and the solutions were transferred to appropriate glass bottles. As previously, each Microcon-30 device was carefully washed by the repeated addition of 400 µL of TBS. We obtained the protein concentration of the purified biotinylated anti-idiotype solutions after the measurement of absorbance at 280 nm. We stabilized the solutions by adding purified BSA (Wallac) to give a final concentration of ~1 g/L.

urine samples
Daily samples of EMU were collected from three healthy volunteers throughout their complete menstrual cycles. In addition, daily samples of EMU were collected from one patient being treated with gonadotropin to stimulate multiple follicular development before oocyte donation. All samples were stored at -20 °C until analysis.

immunoassay
We washed the required number of streptavidin-coated plates once using the plate washer. After tapping the plates dry on absorbent paper, we added 10 µL of sample (undiluted EMU) or mixed standard solution containing EG (range, 0–4480 nmol/L) and PG (range, 0–200 µmol/L). This was followed by the addition of 100 µL of assay buffer containing europium-labeled PG (1:5000, by volume) and samarium-labeled anti-idiotype (11C7; 1:200, by volume) to each microtiter well. Subsequently, we added 100 µL of assay buffer containing biotinylated anti-PG (2555.6; 1:1000, by volume) and biotinylated anti-EG (8A3; 1:500, by volume) to each microtiter well. The plates were incubated for 1 h at room temperature on the plate shaker and then washed six times using the plate washer.

We added 200 µL of enhancement solution to each microtiter well, using a designated dispenser, and incubated the plates for 5 min at room temperature on the plate shaker. The plates were transferred to the time-resolved fluorometer to measure europium and samarium fluorescence using the appropriate emission filters. Filter selection and data reduction was performed by the iterative computer program MULTICALC (Wallac Oy).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
characterization of monoclonal antibodies to estrone-3-glucuronide
The fusion experiment using the spleen of the rat immunized with EG-BSA yielded eight hybridomas that secreted antibodies to EG with varying specificity. Of these, two hybdridomas recognized estradiol-3ß-glucuronide as well as EG. From the remaining six hybridomas, two hybridomas recognized EG but had no cross-reaction with estradiol-3-glucuronide (25). Consequently, we propagated one of the more specific hybridomas (clone 8A3) as ascites. Subsequently, this was used for the preparation of anti-idiotypic antibodies. Clone 8A3 belonged to the IgG2a class, and the affinity to EG was in the order of 10-9 mol/L.

characterization of monoclonal anti-idiotypic antibodies against anti-eg
The fusion experiment yielded 13 hybridomas that secreted various types of anti-idiotypic antibodies against anti-EG. Of these, four demonstrated strong betatypic activity. From the remaining nine hybridomas, six demonstrated alphatypic activity, and the rest exhibited xenotypic activity. We chose a strong betatype (clone 11C7) for the development of the dual assay. Clone 11C7 belonged to the IgG1 class and competed strongly with EG for the binding sites of the primary anti-EG antibody, clone 8A3.

calibration curves
Calibration curves and within-batch precision profiles (26) for EG (range, 0–4480 nmol/L) and PG (range, 0–200 µmol/L) obtained with the simultaneous FIA are shown in Fig. 1



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  		Figure 1. Calibration curves and precision profiles (within batch) for the measurement of EG and PG, using samarium (Sm), and europium (Eu) labels, respectively.

.

precision
Estimates of within-batch precision for the simultaneous FIA were obtained by measuring EG and PG in replicate samples of EMU (n = 24) from three urine pools within a single assay. To estimate between-batch precision, we measured the two metabolites in samples taken from the same urine pools in separate assays (n = 6). The results are summarized in Table 1 .


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  		Table 1. Estimates of assay precision for the measurement of EG and PG by simultaneous FIA.

correlation with individual analyses
Concentrations of EG and PG were measured in 150 random samples of EMU by the simultaneous assay and by sequential (separate) analysis. The results of the linear regression analyses are shown in Table 2 .


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  		Table 2. Linear regression analysis of results obtained from the measurement of EG and PG in 150 samples of EMU by simultaneous and sequential assays.

normal menstrual cycles
Daily samples of EMU were obtained from a healthy female volunteer throughout her complete menstrual cycle. EG and PG were determined simultaneously (i.e., by dual assay) and sequentially (i.e., by separate analysis). The results are shown in Fig. 2 . Fig. 3 shows the EG/PG ratio together with the concentration of urinary LH determined by a time-resolved fluorescence immunoassay (Wallac 1244–006).



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  		Figure 2. The measurement of EG and PG throughout a normal menstrual cycle by simultaneous (dual) assay and sequential analysis.



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  		Figure 3. EG (nmol/L)/PG (µmol/L) ratio and LH throughout a normal menstrual cycle.

To convert ratios to molar ratios, divide by 1000.

stimulated cycle
Daily samples of EMU were collected from one patient being treated with buserilin (days 1 to 3) and then with gonadotropin to stimulate multiple follicular development before oocyte donation. EG and PG were determined simultaneously (i.e., by dual assay) and sequentially (i.e., by separate analysis). The results are shown in Fig. 4 . Fig. 5 shows the EG/PG ratio together with the concentration of LH (actually hCG used to initiate luteinization before egg collection) determined by a time-resolved fluorescence immunoassay (Wallac 1244–006).



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  		Figure 4. The measurement of EG and PG throughout a stimulated cycle by simultaneous (dual) assay and sequential analysis.



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  		Figure 5. EG (nmol/L)/PG (µmol/L) ratio and hCG throughout a stimulated cycle.

To convert to molar ratios, divide by 1000.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Immunoassays are still being refined for the measurement of analytes that are indicative of reproductive endocrine status. In particular, there is an on-going requirement for laboratory tests to assess ovarian function in women who are being monitored or treated to help achieve or avoid conception, or who are undergoing hormone replacement therapy administered to alleviate symptoms associated with menopause and premenstrual syndrome. In addition, the technical problems associated with the measurement of gonadal steroids in serum together with the difficulties associated with serial sampling have led to an investigation of alternative noninvasive hormonal components in other biological fluids, such as saliva or urine.

Accordingly, over the last 25 years, clinical studies have indicated the utility of measuring urinary EG and PG to monitor ovarian function in women (1)(2). Furthermore, other reports have suggested that the ratio of EG to PG in EMU may constitute a useful guideline in the delineation of the fertile period and the biochemical assessment of ovarian status (27)(28)(29)(30). Consequently, attempts were made to develop an assay that would assess this ratio directly (31). This has proved a difficult task.

The introduction of separation immunoassays involving the use of time-resolved fluorescence has been highly successful and has enabled the development and routine use of a wide range of assays for the measurement of haptens and large molecular weight analytes with appropriate sensitivity, specificity, precision, and robustness (32). In particular, several FIAs using europium chelates as labels have been reported for the measurement of EG and PG in samples of EMU (14)(15)(16)(17)(18). These procedures have been used to monitor spontaneous and stimulated ovarian function, and also to predict the periods of potential fertility.

Moreover, it has been demonstrated that the fluorescence emission spectra from alternative lanthanides may be distinguished using appropriate filters in the fluorometer. This feature has led to the development of several simultaneous immunoassays for the measurement of two (20)(33)(34), three (35), or even four (36) analytes. Accordingly, the application of reactants labeled with europium and samarium has enabled the development of a dual assay for the measurement of EG and PG and has facilitated a direct assessment of the ratio of EG to PG.

To achieve this objective, it was necessary to produce a high specific activity tracer for the samarium assay. The reason for this is that samarium can be measured with only ~5% of the efficiency of europium, using the same enhancement solution (32). Consequently, it has not been possible to synthesize a samarium-labeled steroid (e.g., EG) that would give sufficient signal to enable the development of a sensitive assay. On the other hand, we have demonstrated the utility of anti-idiotypic antibodies, which we have termed betatypes, in recognizing the binding sites of their corresponding primary antibodies (17)(22)(24). The anti-idiotype may be labeled with samarium to high specific activity and used as a surrogate antigen to facilitate the development of sensitive procedures.

In terms of precision and specificity, the characteristics of this simultaneous FIA are excellent. This is illustrated by the excellent performance of the dual assay in terms of within- and between-batch variation (Table 1Up ) and the fact that the values obtained from the measurement of the analytes by the simultaneous FIA correlate well with the equivalent results obtained from sequential analysis (Table 2Up ). The simultaneous FIA, however, has many advantages over the sequential methods in terms of speed and potential clinical utility. Furthermore, the rapid counting time and data-handling facilities of the time-resolved fluorometer make the assay extremely simple and convenient to perform.

The technique has been applied to serial EMUs collected from women with normal (Fig. 2Up ) and stimulated (Fig. 4Up ) cycles. The data clearly demonstrate that the values obtained by the simultaneous assay are not significantly different from the equivalent values obtained from sequential analysis. The analytes may be measured in daily samples of EMU, or in serial pooled collections over 24-h periods. Alternatively, the individual analyte values may be corrected for creatinine concentration (16)(37). Nevertheless, extensive studies have indicated that this is generally unnecessary (1)(5)(6)(29). The measurement of the EG/PG ratio, however, avoids this problem because it is a value independent of urine volume (Figs. 3Up and 5Up ).

We envisage that defined changes in the urinary EG/PG ratio could be used to indicate the appropriate time for insemination with donor semen. In addition, the technique may be used in conjunction with pelvic ultrasonography to monitor multiple follicular development before the administration of hCG to initiate the resumption of meiosis before oocyte collection or to effect multiple ovulation. The prospective application of the dual urinary metabolite assay may assist the clinician in the administration of the appropriate dose of gonadotropin to avoid the potential danger of hyperstimulation. In addition, the measurement of PG may indicate the occurrence of premature luteinization of the follicle(s), which may lead to an unsuccessful outcome.

Very recently, the measurement of urinary EG and/or PG has been used to investigate other aspects of ovarian function, which include: (a) the hormonal dynamics in perimenopause (38); (b) ammenorhea (39); (c) anovulation and transient luteal function (40); (d) monitoring hormone replacement therapy (41); and (e) precocious puberty (42). Moreover, the measurement of EG has formed the basis for the development of home tests to facilitate pregnancy avoidance (43)(44). The concept of measuring urinary steroid metabolites rather than the primary steroids in serum is becoming more acceptable in routine reproductive endocrinology.

It has been our experience that the serial collection of EMU (0.5 mL) by volunteers and patients has been well tolerated and can be performed at home. We recommend that the samples are stored at -20 °C after collection and before their arrival at the laboratory. For prolonged storage, it is suggested that preservatives such as glycerol are added to the urine (45)(46). The advantage in the measurement of urinary metabolites to the laboratory is a rapid method that can easily be automated (AutoDelfia(TM), Wallac Oy, Turku, Finland). In addition, the measurement of serum steroids (e.g., estradiol) poses serious technical problems (47)(48) that are not applicable to the analysis of urinary metabolites. Moreover, the dual analysis of these analytes in daily samples of urine present the clinician with a comprehensive overview of ovarian function.


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  		Table 0.


   Acknowledgments
 
We are grateful to Wallac Oy, Turku, Finland, and E.G. & G. Wallac UK Ltd., Milton Keynes, UK for the very generous provision of equipment and reagents. In addition, we thank our colleagues B. Gayer, S. Lichter, and Y. Amir-Zaltsman for excellent technical assistance. In particular, we acknowledge with gratitude the contribution made to this work by the late Dr. P. Samarajeewa (University College, London) and we dedicate this paper to his memory.


   Footnotes
 
1 Nonstandard abbreviations: EG, estrone-3-glucuronide; PG, pregnanediol-3{alpha}-glucuronide; EMU, early morning urine; LH, lutropin; hCG, human chorionic gonadotropin; FIA, fluorescence immunoassay; BSA, bovine serum albumin; and TBS, Tris-buffered saline.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. . for the Determination of the Fertile Period. World Health Organization Task Force on Methods. The measurement of urinary steroid glucuronides as indices of the fertile period in women. J Steroid Biochem 1982;17:695-702. [Web of Science][Medline] [Order article via Infotrieve]
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