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The measurement of urinary estriol-116α-glucuronide by a solid-phase chemiluminescence immunoassay
0022-4731!(11,090941-OE~Z.~/O Copyright 8 1981 Pcrgnmon Press Ltd
Josmnl of Srrro~d Biro hmtiurr Vol. 14. pp. 941 lo 948. 1981 Printed m Great Britam All rights reserved
THE MEASUREMENT ESTRIOL-16mGLUCURONIDE CHEMILUMINESCENCE
OF URINARY BY A SOLID-PHASE IMMUNOASSAY
G. BARNARD*, W. P. COLLINS,* F. Kox-rENtand H. R. LINDNER~ *Department of Obstetrics and Gynaecology, King’s College Hospital Medical School, Denmark Hill, London SE5 8RX. England and tDepartment of Hormone Research, The Weizmann Institute of Science, Rehovot, Israel
(Received
7 January
1981)
SUMMARY A description is given of a simple, solid-phase chemiluminescence immunoassay for the measurement of estriol-16a-glucuronide in diluted urine. An IgG fraction of antiserum to estriol-16a-glucuronyl-6-bovine serum albumin is passively adsorbed to the walls of polystyrene tubes. The labelled antigen is estriol-l6a-glucuronyl-6-aminobutyl-ethyl-isoluminol. After the binding reaction, the solution is removed by aspiration. The antibody bound fraction is washed once with buffer, which removes interfering compounds from the biological sample. The light yield upon oxidation of the labelled conjugate with microperoxidase/hydrogen peroxide is inversely proportional to the concentration of estriol-16a-glucuronide in the standard or sample. A detailed evaluation of the method gave the following values: sensitivity 3.8 + 1.4 nmol/l (mean f SEM; n = 4) range for male urine 19.8 f 4.5 nmol/l (mean + SD; n = 6). The intra-assay precision was 7.4 (CVTJ”:,; 20 replicates; 240.5 nmol/l) and the inter-assay precision 11.4 @IV%;mean of three pools74.3. 139.2 and 256.7 nmol/l over 3 months). The mean bias was - 16.4% over the range 50 to 450 nmol/l and different aliquots (equivalent to 2.5, 5.0 and 10.0~1) of daily early morning urine throughout a complete menstrual cycle gave results that were in good agreement. The concentration (geometric mean
+90% limits) of E,-16a-G in early morning urine collected daily throughout four conception cycles was 35.2, 18.2 to 133nmol/l. In addition, the results obtained from the analysis of daily samples of early morning urine throughout three ovarian cycles were in good agreement with the values derived by radioimmunoassay (r = 0.97).
INTRODUCTION
The measurement of estrogen metabolites in urine is a routine procedure in many clinical laboratories. Initially, these compounds were measured by colorimetric [l], fluorimetric [2] or gas-liquid chromatographic methods [3] after the conjugates had been hydrolysed and the free steroids extracted with an organic solvent. Following the advent of radioimmunoassay for the measurement of plasma estrone, estradiol and estriol, the same approach was applied to the measurement of urinary estriol after the individual conjugates had been isolated on Sephadex LH20 and hydrolysed with glusulase [4]. More recently, specific antisera to various steroid glucuronides have been produced [S, 63 and this has lead to the development of rapid sensitive radioimmunoassays for the measurement of urinary estriol16a-glucuronide (E,-16~G) to monitor ovarian function [7-121 and assess fetal well-being [1319]. The increasing availability and usefulness of radioimmunoassays, however, has raised several serious problems which include: (i) health hazards associated with the use of radiolabelled ligands and the solvents necessary for liquid-scintillation counting; (ii) the increasing problem of waste disposal of radioactivity and organic solvents; and (iii) the high cost and maintenance 941
of equipment. Several approaches have been suggested to overcome the drawbacks of radioimmunoassay while retaining the specificity of the antigenantibody reaction [20-223. In particular, the use of chemiluminescent markers has been investigated since these compounds are commonly detectable at less than pmol amounts [23-251. Several homogeneous assays have been developed based upon the phenomenon of antibody-enhanced chemiluminescence [26-281. Recently, Kohen et al. [29] reported a method.for the determination of urinary Ea-16a-G which utilised this phenomenon and proved satisfactory with respect to the sensitivity, precision and accuracy. Furthermore, the results obtained by radioimmunoassay and chemiluminescence immunoassay were in good agreement. Nevertheless, one of the major difficulties with this and other homogeneous methods has been the presence of non-specific interference from samples of biological origin, thus making it necessary for the inclusion of time-consuming procedures for purifying the sample. To overcome this problem. Barnard et aL[30] investigated a heterogeneous chemihtminescence tmmunoassay for urinary pregnanediol-3aglucuronide utilising a solid-phase separation system; the specific IgG being adsorbed onto the surface of polystyrene
G.
942
BARNARD er al.
of the reaction mixture and subsequent washing with buffer removed all potentially interfering substances with the concomitant reduction of background chemiluminescence. The assay was tubes. The aspiration
Antibody-coated
The IgG fraction was suitably diluted in barbitol buffer (14.4g sodium barbitol in one litre of double distilled water; 0.07 M; pH 9.6). TwJo-hundred (200) pl of this solution was added to polystyrene assay tubes (LP3, Luckham Ltd, Labro Works, Victoria Gardens, Burgess Hill, Sussex). After an overnight incubation at 4°C the barbitol buffer was aspirated to waste and 200~1 immunoassay buffer was added to each tube. The tubes were stored at 4°C until required and the buffer removed immediately prior to use. The immunoassay buffer was 0.1 M phosphate in double distilled water containing O.l”,, BSA. 0.9”” sodium chloride. and O.l”,, sodium azide (pH 7.5).
comparable in specificity and sensitivity to an established radioimmunoassay and diluted urine could be assayed to monitor ovarian function and detect ovulation. In this report a description is given of the development of an heterogeneous chemiluminescence immunoassay for the measurement of urinary ES-l&G using antibody-coated polystyrene tubes. The method is assessed f&r specificity, sensitivity. accuracy and precision. In addition. a comparison is made between the values of urinary E,-16a-G as determined by chemi-
luminescence immunoassay and a conventional radioimmunoassay in samples collected throughout three menstrual cycles.
MATERIALS
AND
Synthesis of estriol-lfm-ylucuronide-ABEI
6-[N-(~aminobutyl~-N-ethylJ amino-2,3-dihydrothal~ine-l,~dione (ABEI) was prepared according to the method of Schroeder et al.[31]. The conjugation of ABE1 and E,-16a-G has been described previously [29]. The proposed structure of E,-16a-G-ABE1 is shown in Fig. 1.
METHODS
Random samples of early morning urine (EMU) were collected from six healthy men (aged 26 to 34 years). In addition, daily samples of EMU from four women who had conceived during the period of collection and from three patients attending an infertility clinic were recovered from storage at -2l”C, thawed and analysed for E,-16a-G. Prior to assay, 100pl of urine were diluted to one ml by the addition of 900 pi of assay buffer.
E,-16a-G(3.17~-dihydroxyestra-l.3,5(10)-triene-l6~yl-P-D-glucopyranosiduronic acid. estrone-3-glucuronide (El-3-G) and estradiol-17&glucuronide (E,-17/3G) were kindly donated by Dr W. F. Coulson of the
Courtauld Institute of Biochemistry, The Middlesex Hospital Medical School, London. Estriol-3-glucuronide (Ea-3-G), estriol-17~-glucuronide (E,-178-G) and estriol-3-sulphate (E,-3-S). microperoxidase (MP-1 I) and bovine serum albumin (BSA Fraction V) were purchased from Sigma London Chemical Co Ltd. Sepharose-Protein A was obtained from Pharmacia (Great Britain) Ltd. Antiserum to E,-16r-G-6-bovine serum albumin was kindly donated by Dr P. Samarajeewa of the Courtauld Institute of Biochemistry, The Middlesex Hospital Medical School, London. Tritiated E,-16cc-G (6,9-H3, 20--4OCi/mmol) was obtained from the Radiochemical Centre, Amersham, Bucks and all other reagents from Hopkin and Williams Ltd. Anti-estriol-I~-glucuronide IgG was prepared by chromatography on an affinity column of Protein A Sepharose CL-4B by a procedure already reported [29].
OH
&
1. The
reaction
One hundred (100) ~1 of diluted urine or 100 #I of standard (range 31.25 to 2~pg/l~~l of assay buffer) were added in duplicate to the antibodycoated tubes. Subsequently, 100 pl of E,-16a-G-ABE1 (1 ng/lOOpl of assay buffer) were added. The mixture was incubated for 1 h at 4’C and the contents of the tube were then removed by aspiration. Two hundred (2~)~l of wash buffer (0.1 M phosphate containing 0.9:,; sodium chloride, 0.19, sodium a&de, pH 8) were added to each tube and subsequently removed by aspiration.
51 f P C-ft-(CH2J4-N ’ Y
0
1’
OH
OH
OH
Estrid-I6ja-glucurcnide Fig.
Binding
__ ct -0 O
Ho= ’
tubes
- ABEI conjugate
proposed structure of E,-16x-G&ABE1
NH
I:
Chemiluminescence immunoassay Light emission and measurement
Microperoxidase was dissolved in 0.1 M phosphate buffer containing O.lQodium azide at pH 8 and the stock solution (1 mg/ml) was stored at 4°C. The working solution was prepared by dilution to lOpg/ml (1: 100. v/v). The oxidant solution was prepared by adding 100~1 of 307: hydrogen peroxide to 1Oml of double distilled water. The light emitted was measured with an LKB, Luminometer Model 1250 (kindly provided by LKB Instruments Ltd. Selsdon, Surrey), which was linked to a flat-bed potentiometric recorder (LKB Cat. No. 2210-032). The cuvette holder was fitted with an adaptor in order to accommodate the assay tube. An automatic system was used to inject the solution of hydrogen peroxide (Hook and Tucker Instruments Ltd, Croydon. Surrey). Two hundred (2OO)pi of the microperoxidase solution were added to the assay tube, which was then placed in the luminometer. The chemiluminescence reaction was initiated by the addition of 100~1 of diluted hydrogen peroxide. The light emitted was monitored by the recorder and the peak height (mV) was measured. Calculation of results and statistical analyses
The unknown values were derived from the calibration curve (light emitted, mV vs concentration of E,-16a-G, pg) and multiplied by 0.216 to obtain the results in nmol/l (MW 464). The concentrations from the analysis of male urine were best described by the arithmetic mean and standard deviation, whereas the overall distribution of amounts in daily samples of
W -D
: B 4 E p
943
EMU throughout four conception cycles was defined by the geometric mean and 90”/:,probability limits. RESULTS
Dilution of antiserum (IgG fraction)
The optimum dilution of the anti-Es-16a-G IgG fraction was determined by incubating tritiated E,-16a-G (5OOOc.p.m.)in a liquid phase system with serial dilutions of antibody in the presence or absence of 2ooOpg of the authentic compound. Dextrancoated charcoal was used to separate the antibody bound and free fractions. The results are shown in Fig. 2 and it was concluded that the optimum dilution was 1: 2000 (v/v). The diluted IgG (200 ~1) was incubated in the tubes overnight at 4°C and the barbitol buffer removed by aspiration. The titre of antibodies in the aspirate was found to be 1:6700 (v/v) and it was concluded that the procedure to coat the tubes was approximately 70% efficient. Reaction kinetics
Results showing the rate of binding of E,-16aG-ABE1 to the adsorbed IgG at 4°C and 37°C are shown in Fig. 3. The results of this experiment are consistent with those obtained for other steroid radioimmunoassays [32]. Calihrafion
Curve
Calibration curves were established using 1OOpg and 1 ng of E,-16a-G-ABE1 as labelled antigens and the results are shown in Fig. 4. Since adequate sensitivity was obtained with 1 ng of conjugate, this amount was used in all subsequent experiments. A recorder trace is shown in Fig. 5.
60o Initialbinding A DiiplOWmOnnt l Bindingin pmsaneo
7060so-
i g 40‘L 9
30-
F &
20-
& s
IO-
,
I
200400
830
I
1600
3200 I / dilution
1
6400
12800 25600
9200
Fig. 2. The effect of antibody (IgG) dilution on the initial binding of 6,9-H3-E3-16a-G and the inhibition by 2OMIpg of authentic compound.
G. BARNARDYI af.
944
16 -
L
t D
20
30
40
!50
I
I
60
120
Incubation time (min)
Fig. 3. The effect of time and temperature on the binding of E,-16~G-6-ABE1 to antibody coated assay tubes.
Specificity of antibodies
from four calibration curves. The value (mean + SEM) obtained over a period of three months was 3.8 f 1.4nmolJ. The concentration (mean k SD) of E3-16a-G in six random samples of male urine was 19.8 + 4.5 nmol/i.
culated
The percentage cross-reactivity of related compounds was determined by the method of Thorneycroft et aLC331. The results obtained with a liquid phase and solid phase radioimmun~say @IA) and the solid phase chem~lumines~n~e immunoassay (CIA) are shown in Table 1. Sensitivity
The minimum concentration of E,-16a-G that could be significantly distinguished from zero was cai-
Accuracy
Increasing amounts of authentic E3-16a-G were added to one ml of male urine. The samples were diluted and analysed by radioimmunoassay and chemiluminescence immunoassay. The results are
Edriol-lb&-~lucuronids Fig.
( pq /tube )
4. Calibration curves for Es-16a-G with either 1oOpg or 1 ng of E,-16a-G-6-ABE1 as labelled antigen.
945
Chemiluminescence immunoassay
Table 2. The measurement by radioimmunoassay and chemiluminescence immunoassay of Ea-l6a-G in male urine containing additive amounts of analyte
16 ;
1412-
e 0
IO-
f * %
RIA @g/ml)
CIA @g/ml)
25 50 100 200
16.43 33.45 64.98 113.98 198.10
9.22 21.83 48.10 103.92 232.17
- 11.57;
- 16.4”s;
Urine
E p
E,-l6a-G added Wmll
Mean bias
Precision
An estimate of the intra-assay variation was obtained by analysing a sample of EMU 20 times within a single assay. The corresponding value for inter-assay variation was obtained from the measurement of E3-16a-G in aliquots from three pools of urine used for internal quality control over a period of 3 months. The mean concentrations standard deviations and coefiicients of variation are shown in Table 3.
8642-
--_--_---_----_-_-.
-.---
.I
Estrbl -I6cglucuronide (pg/tuhe) Fig. 5. Th e recorder
tracing of a standard E,-16~G.
curve for
shown in Table 2. The extent of parallelism between serial dilutions of urine and a standard solution was checked. Daily samples of early morning urine throughout one complete menstrual cycle were analysed by taking the equivalent of 2.5,5.0 and 10.0 ~1 of sample. The application of Student’s paired r-test showed that there was no significant difference between the results at any dilution.
Table 1. The cross reactions of selected compounds, expressed as “/;,molar concentrations giving 507: inhibition
of labelled antigen bound to anti-E,-16a-G IgG, in 3 immunoassay systems
Compound
Liquid phase RIA
Solid phase RIA
Solid phase CIA
E3-l6a-G E,-3-G E3-17j?-G E,-3-G EI-17/%G ES-3-SO4 E,-W-G-ABE1 Estrone Estradioi Estriol
100.0 <0.5 11.7 <0.5 5.4 <0.5 20.9 17.7 21.8 25.3
100.0 <0.5 3.7 <0.5 3.2 <0.5 21.3 12.3 14.7 20.9
100.0 <0.5 22.3 co.5 19.7 <0.5 91.2 83.3 77.9
Normal range Daily samples of EMU from four conception cycles were analysed for E,-16a-G and LH. The concentration (geometric mean and 90% limits) of Er16a-G from Day LH peak -12 to Day LH peak +6 was 35.2, 18.2 to 133.0 nmol/l. Correlation with radioimmunoassay
The concentration of E3-16a-G in daily samples of EMU collected by three patients attending an infertility clinic was measured by chemiluminescence immunoassay (CIA) and by conventional radioimmunoassay (RIA). The results for each cycle are shown in Figs 6, 7 and 8. The overall correlation coefficient r between the two methods was 0.97 (y = 0.97x + 0.47) where y is the value obtained by RIA and x the value determined by CIA.
Table 3. Intra and inter-assay variation in the measurement of urinary E,-16a-G by CIA
Parameter
Mean cont. (nmol/l)
Intra-assay variation
240.5
17.8
74.3 139.2 257.2
12.2 15.2 15.4 Mean
Standard deviation
Coefficient of variation t:,, 7.4
Inter-assay variation, pool
: 3
16.4 10.9 6.0 11.1
G. BARNARD er al.
946 SUBJECT
I
360 320
z!
i 280-
o) 240e g 2 3 G
-
RIA
-
CIA
200-
‘$ T
160-
2
4
6
8
IO 12 14 16 18 20 22 24 26 28 Day of cycle
Fig. 6. The concentration
of E,-16a-G in samples of EMU collected throughout a complete menstrual cycle (subject 1).
) SUBJECT
.?j
50
z
40
2
30 20 (
Day of cycle Fig. 7. The concentration
of E,-16a-G in samples of EMU collected throughout a complete menstrual cycle (subject 2).
Chemiluminescence immunoassay
140
F
SUI~JECT 3
t
-
RIA CIA
Fig. 8. The concentration of E,-16a-G in samples of EMU collected throughout a complete menstrual cycle (subject 3).
DISCUSSION
In recent years there has been much discussion and several publications with regard to the development of alternative labels for immunoassay. Several different approaches have been taken with varying degrees of success. The labels that have been used include enzymes [20], organic free radicals [34], organometallit complexes [35], cofactors [21] and fluorescent molecules [36]. Of the many different assay systems that have been developed, however, only very few have attained the high degree of sensitivity, comparable to the equivalent radioimmunoassay, essentially because the signal (generated by the label bound to antigen or antibody) to noise (non-specific background) ratio has been too low to enable the detection of very low levels of individual analytes. The use of chemiluminescence to monitor specific protein-binding reactions was first proposed by Schroeder et a/.[23]. Since that time, several homogeneous chemiluminescent immunoassays for steroids have been published [26-291. The steroid-isoluminol conjugates have been shown to be very stable; the assays are sensitive and specific; the detection systems relatively inexpensive and reliable. Nevertheless, it has been shown that homogeneous assays in general, are affected by interference from samples of biological origin which limit the sensitivity of the procedures. To
947
overcome this fundamental problem, several techniques have been proposed. These include solvent extraction [26] and ion-exchange chromatography [29]. An alternative approach has been described, which involves the development of a simple heterogeneous chemiluminescent immunoassay using a solid-phase separation system based on the passive adsorption of specific IgG onto the walls of the polystyrene assay tubes. This method successfully combines the advantages of a stable, sensitive non-isotopic label, simple instrumentation and an immunoassay that is not significantly affected by background interference, and thus can be applied to diluted urine without prior purification. The measurement of urinary E,-16a-G has become a useful parameter for monitoring ovarian function and assessing fetal well-being. The results of the present study have enabled the development of a nonisotopic immunoassay which is essentially similar in terms of sensitivity and reliability to an equivalent radioimmunoassay. The results from the experiments on cross reactivity, however, indicate a change in the specificity of the antibodies, in particular with E,-17/7-G, E,-17/?-G, estrone, estradiol and estriol. This finding would indicate a reduced avidity of the antibodies towards E,-16a-G-ABEL Nevertheless, in practice there is not a significant difference in the values as measured by CIA and RIA, which implies that the concentrations of the cross reacting compounds are relatively low in early morning urine from non pregnant women. The chemiluminescence immunoassay has the advantage of being more rapid, it does not involve a centrifugation step and the method can be completed in 2 h. Furthermore, the instrument used in these experiments is inexpensive and easily maintained. We believe that the potential to develop other chemiluminescence immunoassays is very great. With the synthesis of novel luminescent markers, the produo tion of simple immunoassay kits, and luminescence photometers it is envisaged, that this technique will provide a useful alternative method to radioimmunoassay. A~~no~ledyements-This work has been supported by grants to WPC from the Medical Research Council and to HRL from WHO, Ford Foundation and Rockefeller. HRL is an International Fogarty Fellow at the National Institute of Health. We thank LKB-Wallac for the use of the luminometer and Drs W. Coulson and P. Samarajeewa. Department of Steroid Biochemistry. The Courtauld Institute of Biochemistry, The Middlesex Hospital Medical School, London, for the generous donation of reagents and for helpful discussions. REFERENCES
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948 3. Knorr
D. W. R., Kirschner M. A. and Taylor J. P.: Estimation of estrone and estradiol in low level urines using electron-capture gas-liquid chromatography. J. c/in,-Endocr. 31 (i970) 409-416. 4. Youne B. K.. Jirku H.. Kadner S. and Levitz M: Renal clearances of estriol conjugates in normal human pregnancy at term. Am. J. Obsrer. Gynecol. 126 (1976) 3842. 5. Kellie A. E., Samuel V. K., Riley W. J. and Robertson D. M.: Steroid glucuronoside-BSA complexes as antigens: The radioimmunoassay of steroid conjugates. J. steroid Biochem. 3 (1972) 275-288. 6. Kellie A. E.: The radioimmunoassay of steroid conjugates. J. sreroid biochem. 6 (1975) 277 -28 1. 7. Wright K., Collins D. C.. Musey P. I. and Preedy J. R. K.: Direct radioimmunoassay of specific estrogen glucosiduronates in normal men -and non-pregnant women. Steroids 31 (1978) 407426. 8. Wright K., Collins 6. C. and Preedy J. R. K.: Urinary excretion of estrone glucosiduronate, 17P-estradiol-17glucosiduronate, and estriol-16a-glucosiduronate. Significance of proportionate differences during the menstrual cycle. Steroids. 34 (1979) 445457. T. S.. Jennison K. M. and Kellie A. E.: 9. Baker The direct radioimmunoassay of oestrogen glucuronides in human female urine. Biochem. J. 177 (1979) 729-738. 10 Stanczyk F. Z., Miyakawa I., Soares J. R. and Goebelsmann U.: Radioimmunoassay of estriol-16a-glucuronide using tritiated and radioiodinated radioligands: Direct radioimmunoassay of urinary estriol-lba-glucuronide during the menstrual cycle. J. steroid Biochem. 10 (1979) 443448. I. and Goebelsmann U.: 11 Stanczyk F. Z., Miyakawa Direct radioimmunoassay of urinary estrogen and pregnanediol glucuronides during the menstrual cycle. Am. J. Obstet. Gynecol. 137 (1980) 443450. ._ 12. Adlercreutz H., Lehtinen T. and Kairento A-L.: Prediction OF ovulation by urinary estrogen assays. J. steroid Biochem. 12 (1980) 395-401. 13. Davis S. E. and Loriaux D. L.: A simple specific assay for estriol in maternal urine. J. c/in. Eidoci. Metab. 40 (1975) 895-899. 14. Di pietro D. L.: Assay of estriol-16c@-D-glucuronide) in pregnancy urine with a specific antiserum. Am. J. Obstet. Gynecol. 125 (1976) 841-845. 15. Hanning R. V., Satin K. P., Lynskey M. T., Levin R. M. and Speroff L.: A direct radioimmunoassay for estriol-16a-glucuronide in urine for monitoring pregnancy and induction of ovulation. Am. J. Obster. Gynecol. 128 (1976) 793-801. 16. Adlercreutz H., Lehtinen T. and Tikkanen M.: Preliminary studies on the determination of estriol-16a-glucuronide in pregnancy urine by direct radioimmunoassay without hydrolysis. J. steroid Biochem. 7 (1976) 105~107. 17. Soares J. R.. Zimmerman E. and Gross S. J.: Direct radioimmuneassay of 16-glucosiduronate metabolites of estriol in human plasma urine. FEBS Lett. 61 (1976) 263-266. T. and Adlercreutz H.: Solid-phase radio18. Lehtinen immunoassays of estriol-16a-glucuronide in urine and pregnancy plasma. J. steroid Biochem. 8 (1977) 99-104. 19. Sugar J.. Alexander S.. Dessy C. and Schwers J.: Radioimmunoassay of estriol-16a-glucuronide J. c/in. Endocr.
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Josmnl of Srrro~d Biro hmtiurr Vol. 14. pp. 941 lo 948. 1981 Printed m Great Britam All rights reserved
THE MEASUREMENT ESTRIOL-16mGLUCURONIDE CHEMILUMINESCENCE
OF URINARY BY A SOLID-PHASE IMMUNOASSAY
G. BARNARD*, W. P. COLLINS,* F. Kox-rENtand H. R. LINDNER~ *Department of Obstetrics and Gynaecology, King’s College Hospital Medical School, Denmark Hill, London SE5 8RX. England and tDepartment of Hormone Research, The Weizmann Institute of Science, Rehovot, Israel
(Received
7 January
1981)
SUMMARY A description is given of a simple, solid-phase chemiluminescence immunoassay for the measurement of estriol-16a-glucuronide in diluted urine. An IgG fraction of antiserum to estriol-16a-glucuronyl-6-bovine serum albumin is passively adsorbed to the walls of polystyrene tubes. The labelled antigen is estriol-l6a-glucuronyl-6-aminobutyl-ethyl-isoluminol. After the binding reaction, the solution is removed by aspiration. The antibody bound fraction is washed once with buffer, which removes interfering compounds from the biological sample. The light yield upon oxidation of the labelled conjugate with microperoxidase/hydrogen peroxide is inversely proportional to the concentration of estriol-16a-glucuronide in the standard or sample. A detailed evaluation of the method gave the following values: sensitivity 3.8 + 1.4 nmol/l (mean f SEM; n = 4) range for male urine 19.8 f 4.5 nmol/l (mean + SD; n = 6). The intra-assay precision was 7.4 (CVTJ”:,; 20 replicates; 240.5 nmol/l) and the inter-assay precision 11.4 @IV%;mean of three pools74.3. 139.2 and 256.7 nmol/l over 3 months). The mean bias was - 16.4% over the range 50 to 450 nmol/l and different aliquots (equivalent to 2.5, 5.0 and 10.0~1) of daily early morning urine throughout a complete menstrual cycle gave results that were in good agreement. The concentration (geometric mean
+90% limits) of E,-16a-G in early morning urine collected daily throughout four conception cycles was 35.2, 18.2 to 133nmol/l. In addition, the results obtained from the analysis of daily samples of early morning urine throughout three ovarian cycles were in good agreement with the values derived by radioimmunoassay (r = 0.97).
INTRODUCTION
The measurement of estrogen metabolites in urine is a routine procedure in many clinical laboratories. Initially, these compounds were measured by colorimetric [l], fluorimetric [2] or gas-liquid chromatographic methods [3] after the conjugates had been hydrolysed and the free steroids extracted with an organic solvent. Following the advent of radioimmunoassay for the measurement of plasma estrone, estradiol and estriol, the same approach was applied to the measurement of urinary estriol after the individual conjugates had been isolated on Sephadex LH20 and hydrolysed with glusulase [4]. More recently, specific antisera to various steroid glucuronides have been produced [S, 63 and this has lead to the development of rapid sensitive radioimmunoassays for the measurement of urinary estriol16a-glucuronide (E,-16~G) to monitor ovarian function [7-121 and assess fetal well-being [1319]. The increasing availability and usefulness of radioimmunoassays, however, has raised several serious problems which include: (i) health hazards associated with the use of radiolabelled ligands and the solvents necessary for liquid-scintillation counting; (ii) the increasing problem of waste disposal of radioactivity and organic solvents; and (iii) the high cost and maintenance 941
of equipment. Several approaches have been suggested to overcome the drawbacks of radioimmunoassay while retaining the specificity of the antigenantibody reaction [20-223. In particular, the use of chemiluminescent markers has been investigated since these compounds are commonly detectable at less than pmol amounts [23-251. Several homogeneous assays have been developed based upon the phenomenon of antibody-enhanced chemiluminescence [26-281. Recently, Kohen et al. [29] reported a method.for the determination of urinary Ea-16a-G which utilised this phenomenon and proved satisfactory with respect to the sensitivity, precision and accuracy. Furthermore, the results obtained by radioimmunoassay and chemiluminescence immunoassay were in good agreement. Nevertheless, one of the major difficulties with this and other homogeneous methods has been the presence of non-specific interference from samples of biological origin, thus making it necessary for the inclusion of time-consuming procedures for purifying the sample. To overcome this problem. Barnard et aL[30] investigated a heterogeneous chemihtminescence tmmunoassay for urinary pregnanediol-3aglucuronide utilising a solid-phase separation system; the specific IgG being adsorbed onto the surface of polystyrene
G.
942
BARNARD er al.
of the reaction mixture and subsequent washing with buffer removed all potentially interfering substances with the concomitant reduction of background chemiluminescence. The assay was tubes. The aspiration
Antibody-coated
The IgG fraction was suitably diluted in barbitol buffer (14.4g sodium barbitol in one litre of double distilled water; 0.07 M; pH 9.6). TwJo-hundred (200) pl of this solution was added to polystyrene assay tubes (LP3, Luckham Ltd, Labro Works, Victoria Gardens, Burgess Hill, Sussex). After an overnight incubation at 4°C the barbitol buffer was aspirated to waste and 200~1 immunoassay buffer was added to each tube. The tubes were stored at 4°C until required and the buffer removed immediately prior to use. The immunoassay buffer was 0.1 M phosphate in double distilled water containing O.l”,, BSA. 0.9”” sodium chloride. and O.l”,, sodium azide (pH 7.5).
comparable in specificity and sensitivity to an established radioimmunoassay and diluted urine could be assayed to monitor ovarian function and detect ovulation. In this report a description is given of the development of an heterogeneous chemiluminescence immunoassay for the measurement of urinary ES-l&G using antibody-coated polystyrene tubes. The method is assessed f&r specificity, sensitivity. accuracy and precision. In addition. a comparison is made between the values of urinary E,-16a-G as determined by chemi-
luminescence immunoassay and a conventional radioimmunoassay in samples collected throughout three menstrual cycles.
MATERIALS
AND
Synthesis of estriol-lfm-ylucuronide-ABEI
6-[N-(~aminobutyl~-N-ethylJ amino-2,3-dihydrothal~ine-l,~dione (ABEI) was prepared according to the method of Schroeder et al.[31]. The conjugation of ABE1 and E,-16a-G has been described previously [29]. The proposed structure of E,-16a-G-ABE1 is shown in Fig. 1.
METHODS
Random samples of early morning urine (EMU) were collected from six healthy men (aged 26 to 34 years). In addition, daily samples of EMU from four women who had conceived during the period of collection and from three patients attending an infertility clinic were recovered from storage at -2l”C, thawed and analysed for E,-16a-G. Prior to assay, 100pl of urine were diluted to one ml by the addition of 900 pi of assay buffer.
E,-16a-G(3.17~-dihydroxyestra-l.3,5(10)-triene-l6~yl-P-D-glucopyranosiduronic acid. estrone-3-glucuronide (El-3-G) and estradiol-17&glucuronide (E,-17/3G) were kindly donated by Dr W. F. Coulson of the
Courtauld Institute of Biochemistry, The Middlesex Hospital Medical School, London. Estriol-3-glucuronide (Ea-3-G), estriol-17~-glucuronide (E,-178-G) and estriol-3-sulphate (E,-3-S). microperoxidase (MP-1 I) and bovine serum albumin (BSA Fraction V) were purchased from Sigma London Chemical Co Ltd. Sepharose-Protein A was obtained from Pharmacia (Great Britain) Ltd. Antiserum to E,-16r-G-6-bovine serum albumin was kindly donated by Dr P. Samarajeewa of the Courtauld Institute of Biochemistry, The Middlesex Hospital Medical School, London. Tritiated E,-16cc-G (6,9-H3, 20--4OCi/mmol) was obtained from the Radiochemical Centre, Amersham, Bucks and all other reagents from Hopkin and Williams Ltd. Anti-estriol-I~-glucuronide IgG was prepared by chromatography on an affinity column of Protein A Sepharose CL-4B by a procedure already reported [29].
OH
&
1. The
reaction
One hundred (100) ~1 of diluted urine or 100 #I of standard (range 31.25 to 2~pg/l~~l of assay buffer) were added in duplicate to the antibodycoated tubes. Subsequently, 100 pl of E,-16a-G-ABE1 (1 ng/lOOpl of assay buffer) were added. The mixture was incubated for 1 h at 4’C and the contents of the tube were then removed by aspiration. Two hundred (2~)~l of wash buffer (0.1 M phosphate containing 0.9:,; sodium chloride, 0.19, sodium a&de, pH 8) were added to each tube and subsequently removed by aspiration.
51 f P C-ft-(CH2J4-N ’ Y
0
1’
OH
OH
OH
Estrid-I6ja-glucurcnide Fig.
Binding
__ ct -0 O
Ho= ’
tubes
- ABEI conjugate
proposed structure of E,-16x-G&ABE1
NH
I:
Chemiluminescence immunoassay Light emission and measurement
Microperoxidase was dissolved in 0.1 M phosphate buffer containing O.lQodium azide at pH 8 and the stock solution (1 mg/ml) was stored at 4°C. The working solution was prepared by dilution to lOpg/ml (1: 100. v/v). The oxidant solution was prepared by adding 100~1 of 307: hydrogen peroxide to 1Oml of double distilled water. The light emitted was measured with an LKB, Luminometer Model 1250 (kindly provided by LKB Instruments Ltd. Selsdon, Surrey), which was linked to a flat-bed potentiometric recorder (LKB Cat. No. 2210-032). The cuvette holder was fitted with an adaptor in order to accommodate the assay tube. An automatic system was used to inject the solution of hydrogen peroxide (Hook and Tucker Instruments Ltd, Croydon. Surrey). Two hundred (2OO)pi of the microperoxidase solution were added to the assay tube, which was then placed in the luminometer. The chemiluminescence reaction was initiated by the addition of 100~1 of diluted hydrogen peroxide. The light emitted was monitored by the recorder and the peak height (mV) was measured. Calculation of results and statistical analyses
The unknown values were derived from the calibration curve (light emitted, mV vs concentration of E,-16a-G, pg) and multiplied by 0.216 to obtain the results in nmol/l (MW 464). The concentrations from the analysis of male urine were best described by the arithmetic mean and standard deviation, whereas the overall distribution of amounts in daily samples of
W -D
: B 4 E p
943
EMU throughout four conception cycles was defined by the geometric mean and 90”/:,probability limits. RESULTS
Dilution of antiserum (IgG fraction)
The optimum dilution of the anti-Es-16a-G IgG fraction was determined by incubating tritiated E,-16a-G (5OOOc.p.m.)in a liquid phase system with serial dilutions of antibody in the presence or absence of 2ooOpg of the authentic compound. Dextrancoated charcoal was used to separate the antibody bound and free fractions. The results are shown in Fig. 2 and it was concluded that the optimum dilution was 1: 2000 (v/v). The diluted IgG (200 ~1) was incubated in the tubes overnight at 4°C and the barbitol buffer removed by aspiration. The titre of antibodies in the aspirate was found to be 1:6700 (v/v) and it was concluded that the procedure to coat the tubes was approximately 70% efficient. Reaction kinetics
Results showing the rate of binding of E,-16aG-ABE1 to the adsorbed IgG at 4°C and 37°C are shown in Fig. 3. The results of this experiment are consistent with those obtained for other steroid radioimmunoassays [32]. Calihrafion
Curve
Calibration curves were established using 1OOpg and 1 ng of E,-16a-G-ABE1 as labelled antigens and the results are shown in Fig. 4. Since adequate sensitivity was obtained with 1 ng of conjugate, this amount was used in all subsequent experiments. A recorder trace is shown in Fig. 5.
60o Initialbinding A DiiplOWmOnnt l Bindingin pmsaneo
7060so-
i g 40‘L 9
30-
F &
20-
& s
IO-
,
I
200400
830
I
1600
3200 I / dilution
1
6400
12800 25600
9200
Fig. 2. The effect of antibody (IgG) dilution on the initial binding of 6,9-H3-E3-16a-G and the inhibition by 2OMIpg of authentic compound.
G. BARNARDYI af.
944
16 -
L
t D
20
30
40
!50
I
I
60
120
Incubation time (min)
Fig. 3. The effect of time and temperature on the binding of E,-16~G-6-ABE1 to antibody coated assay tubes.
Specificity of antibodies
from four calibration curves. The value (mean + SEM) obtained over a period of three months was 3.8 f 1.4nmolJ. The concentration (mean k SD) of E3-16a-G in six random samples of male urine was 19.8 + 4.5 nmol/i.
culated
The percentage cross-reactivity of related compounds was determined by the method of Thorneycroft et aLC331. The results obtained with a liquid phase and solid phase radioimmun~say @IA) and the solid phase chem~lumines~n~e immunoassay (CIA) are shown in Table 1. Sensitivity
The minimum concentration of E,-16a-G that could be significantly distinguished from zero was cai-
Accuracy
Increasing amounts of authentic E3-16a-G were added to one ml of male urine. The samples were diluted and analysed by radioimmunoassay and chemiluminescence immunoassay. The results are
Edriol-lb&-~lucuronids Fig.
( pq /tube )
4. Calibration curves for Es-16a-G with either 1oOpg or 1 ng of E,-16a-G-6-ABE1 as labelled antigen.
945
Chemiluminescence immunoassay
Table 2. The measurement by radioimmunoassay and chemiluminescence immunoassay of Ea-l6a-G in male urine containing additive amounts of analyte
16 ;
1412-
e 0
IO-
f * %
RIA @g/ml)
CIA @g/ml)
25 50 100 200
16.43 33.45 64.98 113.98 198.10
9.22 21.83 48.10 103.92 232.17
- 11.57;
- 16.4”s;
Urine
E p
E,-l6a-G added Wmll
Mean bias
Precision
An estimate of the intra-assay variation was obtained by analysing a sample of EMU 20 times within a single assay. The corresponding value for inter-assay variation was obtained from the measurement of E3-16a-G in aliquots from three pools of urine used for internal quality control over a period of 3 months. The mean concentrations standard deviations and coefiicients of variation are shown in Table 3.
8642-
--_--_---_----_-_-.
-.---
.I
Estrbl -I6cglucuronide (pg/tuhe) Fig. 5. Th e recorder
tracing of a standard E,-16~G.
curve for
shown in Table 2. The extent of parallelism between serial dilutions of urine and a standard solution was checked. Daily samples of early morning urine throughout one complete menstrual cycle were analysed by taking the equivalent of 2.5,5.0 and 10.0 ~1 of sample. The application of Student’s paired r-test showed that there was no significant difference between the results at any dilution.
Table 1. The cross reactions of selected compounds, expressed as “/;,molar concentrations giving 507: inhibition
of labelled antigen bound to anti-E,-16a-G IgG, in 3 immunoassay systems
Compound
Liquid phase RIA
Solid phase RIA
Solid phase CIA
E3-l6a-G E,-3-G E3-17j?-G E,-3-G EI-17/%G ES-3-SO4 E,-W-G-ABE1 Estrone Estradioi Estriol
100.0 <0.5 11.7 <0.5 5.4 <0.5 20.9 17.7 21.8 25.3
100.0 <0.5 3.7 <0.5 3.2 <0.5 21.3 12.3 14.7 20.9
100.0 <0.5 22.3 co.5 19.7 <0.5 91.2 83.3 77.9
Normal range Daily samples of EMU from four conception cycles were analysed for E,-16a-G and LH. The concentration (geometric mean and 90% limits) of Er16a-G from Day LH peak -12 to Day LH peak +6 was 35.2, 18.2 to 133.0 nmol/l. Correlation with radioimmunoassay
The concentration of E3-16a-G in daily samples of EMU collected by three patients attending an infertility clinic was measured by chemiluminescence immunoassay (CIA) and by conventional radioimmunoassay (RIA). The results for each cycle are shown in Figs 6, 7 and 8. The overall correlation coefficient r between the two methods was 0.97 (y = 0.97x + 0.47) where y is the value obtained by RIA and x the value determined by CIA.
Table 3. Intra and inter-assay variation in the measurement of urinary E,-16a-G by CIA
Parameter
Mean cont. (nmol/l)
Intra-assay variation
240.5
17.8
74.3 139.2 257.2
12.2 15.2 15.4 Mean
Standard deviation
Coefficient of variation t:,, 7.4
Inter-assay variation, pool
: 3
16.4 10.9 6.0 11.1
G. BARNARD er al.
946 SUBJECT
I
360 320
z!
i 280-
o) 240e g 2 3 G
-
RIA
-
CIA
200-
‘$ T
160-
2
4
6
8
IO 12 14 16 18 20 22 24 26 28 Day of cycle
Fig. 6. The concentration
of E,-16a-G in samples of EMU collected throughout a complete menstrual cycle (subject 1).
) SUBJECT
.?j
50
z
40
2
30 20 (
Day of cycle Fig. 7. The concentration
of E,-16a-G in samples of EMU collected throughout a complete menstrual cycle (subject 2).
Chemiluminescence immunoassay
140
F
SUI~JECT 3
t
-
RIA CIA
Fig. 8. The concentration of E,-16a-G in samples of EMU collected throughout a complete menstrual cycle (subject 3).
DISCUSSION
In recent years there has been much discussion and several publications with regard to the development of alternative labels for immunoassay. Several different approaches have been taken with varying degrees of success. The labels that have been used include enzymes [20], organic free radicals [34], organometallit complexes [35], cofactors [21] and fluorescent molecules [36]. Of the many different assay systems that have been developed, however, only very few have attained the high degree of sensitivity, comparable to the equivalent radioimmunoassay, essentially because the signal (generated by the label bound to antigen or antibody) to noise (non-specific background) ratio has been too low to enable the detection of very low levels of individual analytes. The use of chemiluminescence to monitor specific protein-binding reactions was first proposed by Schroeder et a/.[23]. Since that time, several homogeneous chemiluminescent immunoassays for steroids have been published [26-291. The steroid-isoluminol conjugates have been shown to be very stable; the assays are sensitive and specific; the detection systems relatively inexpensive and reliable. Nevertheless, it has been shown that homogeneous assays in general, are affected by interference from samples of biological origin which limit the sensitivity of the procedures. To
947
overcome this fundamental problem, several techniques have been proposed. These include solvent extraction [26] and ion-exchange chromatography [29]. An alternative approach has been described, which involves the development of a simple heterogeneous chemiluminescent immunoassay using a solid-phase separation system based on the passive adsorption of specific IgG onto the walls of the polystyrene assay tubes. This method successfully combines the advantages of a stable, sensitive non-isotopic label, simple instrumentation and an immunoassay that is not significantly affected by background interference, and thus can be applied to diluted urine without prior purification. The measurement of urinary E,-16a-G has become a useful parameter for monitoring ovarian function and assessing fetal well-being. The results of the present study have enabled the development of a nonisotopic immunoassay which is essentially similar in terms of sensitivity and reliability to an equivalent radioimmunoassay. The results from the experiments on cross reactivity, however, indicate a change in the specificity of the antibodies, in particular with E,-17/7-G, E,-17/?-G, estrone, estradiol and estriol. This finding would indicate a reduced avidity of the antibodies towards E,-16a-G-ABEL Nevertheless, in practice there is not a significant difference in the values as measured by CIA and RIA, which implies that the concentrations of the cross reacting compounds are relatively low in early morning urine from non pregnant women. The chemiluminescence immunoassay has the advantage of being more rapid, it does not involve a centrifugation step and the method can be completed in 2 h. Furthermore, the instrument used in these experiments is inexpensive and easily maintained. We believe that the potential to develop other chemiluminescence immunoassays is very great. With the synthesis of novel luminescent markers, the produo tion of simple immunoassay kits, and luminescence photometers it is envisaged, that this technique will provide a useful alternative method to radioimmunoassay. A~~no~ledyements-This work has been supported by grants to WPC from the Medical Research Council and to HRL from WHO, Ford Foundation and Rockefeller. HRL is an International Fogarty Fellow at the National Institute of Health. We thank LKB-Wallac for the use of the luminometer and Drs W. Coulson and P. Samarajeewa. Department of Steroid Biochemistry. The Courtauld Institute of Biochemistry, The Middlesex Hospital Medical School, London, for the generous donation of reagents and for helpful discussions. REFERENCES
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G. BARNARD er ul.
948 3. Knorr
D. W. R., Kirschner M. A. and Taylor J. P.: Estimation of estrone and estradiol in low level urines using electron-capture gas-liquid chromatography. J. c/in,-Endocr. 31 (i970) 409-416. 4. Youne B. K.. Jirku H.. Kadner S. and Levitz M: Renal clearances of estriol conjugates in normal human pregnancy at term. Am. J. Obsrer. Gynecol. 126 (1976) 3842. 5. Kellie A. E., Samuel V. K., Riley W. J. and Robertson D. M.: Steroid glucuronoside-BSA complexes as antigens: The radioimmunoassay of steroid conjugates. J. steroid Biochem. 3 (1972) 275-288. 6. Kellie A. E.: The radioimmunoassay of steroid conjugates. J. sreroid biochem. 6 (1975) 277 -28 1. 7. Wright K., Collins D. C.. Musey P. I. and Preedy J. R. K.: Direct radioimmunoassay of specific estrogen glucosiduronates in normal men -and non-pregnant women. Steroids 31 (1978) 407426. 8. Wright K., Collins 6. C. and Preedy J. R. K.: Urinary excretion of estrone glucosiduronate, 17P-estradiol-17glucosiduronate, and estriol-16a-glucosiduronate. Significance of proportionate differences during the menstrual cycle. Steroids. 34 (1979) 445457. T. S.. Jennison K. M. and Kellie A. E.: 9. Baker The direct radioimmunoassay of oestrogen glucuronides in human female urine. Biochem. J. 177 (1979) 729-738. 10 Stanczyk F. Z., Miyakawa I., Soares J. R. and Goebelsmann U.: Radioimmunoassay of estriol-16a-glucuronide using tritiated and radioiodinated radioligands: Direct radioimmunoassay of urinary estriol-lba-glucuronide during the menstrual cycle. J. steroid Biochem. 10 (1979) 443448. I. and Goebelsmann U.: 11 Stanczyk F. Z., Miyakawa Direct radioimmunoassay of urinary estrogen and pregnanediol glucuronides during the menstrual cycle. Am. J. Obstet. Gynecol. 137 (1980) 443450. ._ 12. Adlercreutz H., Lehtinen T. and Kairento A-L.: Prediction OF ovulation by urinary estrogen assays. J. steroid Biochem. 12 (1980) 395-401. 13. Davis S. E. and Loriaux D. L.: A simple specific assay for estriol in maternal urine. J. c/in. Eidoci. Metab. 40 (1975) 895-899. 14. Di pietro D. L.: Assay of estriol-16c@-D-glucuronide) in pregnancy urine with a specific antiserum. Am. J. Obstet. Gynecol. 125 (1976) 841-845. 15. Hanning R. V., Satin K. P., Lynskey M. T., Levin R. M. and Speroff L.: A direct radioimmunoassay for estriol-16a-glucuronide in urine for monitoring pregnancy and induction of ovulation. Am. J. Obster. Gynecol. 128 (1976) 793-801. 16. Adlercreutz H., Lehtinen T. and Tikkanen M.: Preliminary studies on the determination of estriol-16a-glucuronide in pregnancy urine by direct radioimmunoassay without hydrolysis. J. steroid Biochem. 7 (1976) 105~107. 17. Soares J. R.. Zimmerman E. and Gross S. J.: Direct radioimmuneassay of 16-glucosiduronate metabolites of estriol in human plasma urine. FEBS Lett. 61 (1976) 263-266. T. and Adlercreutz H.: Solid-phase radio18. Lehtinen immunoassays of estriol-16a-glucuronide in urine and pregnancy plasma. J. steroid Biochem. 8 (1977) 99-104. 19. Sugar J.. Alexander S.. Dessy C. and Schwers J.: Radioimmunoassay of estriol-16a-glucuronide J. c/in. Endocr.
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