Analytica Chcmica Acta, 205 (1988) 215-222 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

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CHEMILUMINESCENCE IMMUNOASSAY FOR ESTRIOL IN SALIVA

J. DE BOEVER*, W. ULRIX and D. VANDEKERCKHOVE

Department of Obstetrics and Gynaecology, University Hospital, De Pzntelaan 185, B-9000 Gent (Eelglum)

F. KOHEN

Department of Hormone Research, The Weizmann Instztute of Science, Rehouot 76100 (Israel)

(Received 12th May 1987)

SUMMARY

Estriol is determined in whole saliva of pregnant women by a direct solid-phase chemilumines- cence immunoassay. The assay uses a monoclonal antibody raised against estriol-6-carboxyme- thyloxime/bovine serum albumin and the homologous chemiluminescent marker conjugate estriol- 6-carboxymethyloxime/aminopentylethylisoluminol (E3/APEI). The anti-estriol antibody is bound to the wells of a microtitration plate via a second antibody directed against the monoclonal antibody; 50 ~1 of saliva and 12.5 pg of E3/APEI per well are used. The incubation time is 10 min at room temperature. The calibration graph covers 5-750 pg of estriol and the detection limit is 4.5 pg (0.31 nmol 1 -I). Mean recovery of added estriol is 98%. Within-assay coefficient of varia- tion is 11.8-5% for 0.21-6.5 nmol 1-l E3, and the between-assay value is 15.7-6.9% for 0.27-3.5 nmol 1-l E3. The correlation of E3 concentration in time-matched samples of saliva and sera from pregnant women was good (r = 0.934). Total assay time including calculation of results is 3 h for 40 saliva samples.

Fetoplacental function is often assessed by measuring estriol in urine and blood. However, considerable day-to-day and short-term fluctuations of estriol concentrations affect the reliability of such determinations [l-4]. In order to improve the diagnostic potential of estriol measurements, repeated sampling has been proposed [ 3,5,6]. This is, however, highly impractical and expensive. Saliva sampling is simple, easy and stress-free and might eventually be done by the patient at home. Salivary estriol reliably reflects the concentration of unconjugatedestriol levels in serum and of the biologically active “free” steroid fraction in serum [ 7-91. In addition, during the third trimester of pregnancy, the increase in salivary estriol parallels the rise of urinary estrogens and of unconjugated estriol in serum [ 7,9,10]. This makes monitoring of fetoplacen- tal function by measurement of estriol in saliva feasible and attractive.

Because repeat sampling of saliva may be a prerequisite for reliable follow-

0003-2670/88/$03.50 0 1988 Elsevier Science Publishers B.V.

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up [ 9,11,12], the clinical usefulness of estriol determinations greatly depends on the simplicity, speed and sample capacity of the assay method. Recently, a chemiluminescence immunoassay has been developed for salivary estriol that meets these requirements. The method and its characteristics are described below and its utility illustrated.

EXPERIMENTAL

Samples Unstimulated saliva was obtained between 08 00 and 12 00 h from pregnant

women (gestational age 7-38 weeks) attending the Womens’ Clinic at the Uni- versity Hospital. They received instructions for collecting whole mixed saliva: rinse the mouth several times with tap water and start collecting saliva in a plastic vial ca. 5 min later. Samples were frozen within 5 min of collection and were stored at - 20’ C. Before assay, the saliva was thawed and centrifuged (15 min at 3000 g) . Duplicate 50-~1 aliquots of the clear supernatant liquid were used for direct chemiluminescence immunoassay. Two pools of male sa- liva were obtained from the same person. Saliva samples (20-25 ml) were stored frozen, then thawed, pooled and centrifuged (15 min at 3000 g) . The clear supernatant liquid was stored frozen in portions. Part of one pool was treated with lyophilized dextran-coated charcoal (500 mg active charcoal and 50 mg of dextran per 100 ml of saliva), centrifuged twice for 15 min at 3000 g and stored frozen in portions.

Directly after saliva sampling, blood was collected by venepuncture and the serum was stored at -20°C. Duplicate 20-~1 aliquots were used in a direct radioimmunoassay (RIA) for unconjugated serum estriol.

Chemicals and solutions Steroids were purchased from Steraloids (Wilton, NH 03086) and from

Sigma Chemical Co. Microperoxidase (MP-11; EC 1.11.1.7), bovine serum albumin (Cohn Fraction V) , and Tween-20 (polyoxyethylene (20) sorbitan monolaureate) were from Sigma. Ethanol, hydrogen peroxide (300 g 1-l)) so- dium hydroxide pellets and activated charcoal were from Merck and Dextran T70 was from Pharmacia. Microtitre plates (“Immunoplate 1 with certifi- cate”), and “sealing tape”, i.e., adhesive-backed plastic film to cover and seal off the plates, were obtained from Nunc AS, (Kampstrup, Denmark). Second antibody, purified anti-mouse IgG, code no. 2259, was from Dakopatts (Glos- trup, Denmark). “Lumacuvettes” (12 x 50 mm polystyrene tubes) were from Lumac Systems (Basel, Switzerland). For the determination of unconjugated estriol in serum, commercial ‘251-labeled estriol RIA kits, code no. IM. 2041 were obtained from Amersham International.

The assay buffer was sodium phosphate (50 mmol l-l, pH 8.0) containing 9 g of sodium chloride, 100 mg of bovine serum albumin, and 1 g of sodium

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azide per litre. The coating buffer was a sodium carbonate (50 mmol l-l, pH 9.6) solution containing 0.1 g 1-l thimerosal. The wash solution, microperox- idase, oxidant solution (0.1 ml of 300 g 1-l hydrogen peroxide mixed with 15 ml of distilled water), dextran-coated charcoal and steroid stock solutions were all as previously described [ 131.

Procedures Preparation of isoluminoLE3 conjugate and antibody to estriol. Estriol-6-car-

boxymethyloxime/aminopentylethylisoluminol was synthesized as described previously [ 14,151. A stock solution in ethanol (122 ,ug ml-l) stored at 4°C was stable for two years. A substock solution (100 ng ml-‘) in assay buffer, was prepared every two months.

Monoclonal anti-estriol antibodies were obtained from the rat-mouse hy- bridoma line 9B4. This cell line was derived by fusion of rat spleen cells, im- munized with estriol-6-carboxymethyloxime/bovine serum albumin, with mouse myeloma cell line NSO, kindly donated by Dr. C. Milstein (Cambridge, England). This line was propagated as ascitic fluid in irradiated CD2 female mice [ 161. Monoclonal antibody 9B4 belongs to the IgGl class, and it was used in the assay as diluted ascitic fluid, without further purification. A stock so- lution prepared by 1:lO dilution in assay buffer was stored at 4°C.

Coating of microtitration plates. This procedure was described in detail for estradiol antibody [ 171. It included overnight incubation at 4” C of 200 ~1 of diluted second antibody per well, followed by thorough washing. Then 200 ~1 of diluted monoclonal antibody per well was incubated overnight at 4°C. For the present assay, a 7500-fold dilution of antibody 9B4 was used. Next the plate was washed four times and stored at 4°C for up to 8 days.

Optimization of assay conditions. During experiments designed to optimize the assay performance. incubations were tested at different temperatures for varying periods of time, i.e., overnight at 4’ C, 1 min to 2 h at room temperature and 15 to 30 min at 37’ C. The best results in terms of limit of detection, stan- dard deviation for duplicate standards and slope of the calibration graphs were obtained with brief incubations at room temperature. The equilibrium between antibody-bound and free steroid was reached within 3 min. Because identical results were obtained for between 3 and 20 min of incubation (Fig. 1) , it was decided to incubate for 10 min at room temperature.

Chemiluminescence immunoassay. A 1040 nmol 1-l solution of estriol was prepared by direct mixing of 60 ~1 of an ethanolic, 34.7 pm01 1-l solution with 1.94 ml of assay buffer. From this solution a first standard of 52 nmol 1-l E3 was prepared, and serial dilutions were used to obtain other standards between 17.75 and 0.35 nmol 1-l. To each well were added 50 ,~l of saliva, 50 ~1 of stan-

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100- 90.. 80-e 70-m

60. 50.

iO.m . .\,

40. 30.

20.

0 5 23 47 94 265 750

ESTRI OL (PG ‘WELL)

Fig. 1. Bate of binding of E3/APEI conjugate to monoclonal antibody 9B4 at room temperature. Antibody is bound to the wells of microtitration plates. Each well contains 50 ~1 of pooled male saliva, 50 ~1 of assay buffer and 100 ,~l (12.5 pg) of ES/APEI.

Fig. 2. Dose/response curve of the direct chemiluminescence assay for estriol. Each point is the mean % B/B,, f SD of 13 consecutive determinations.

dard or assay buffer and 100 ,ul of 0.9 nmol l-l E3/APEI in assay buffer. The plate was incubated for 10 min at room temperature on a horizontal shaker at 170 cycles min-‘. Subsequently, the wells were emptied by inversion and washed three times for 30 s with wash solution. A 200~~1 portion of 2M sodium hy- droxide was added to all wells and the plate was heated in a waterbath at 60’ C.

The plate was cooled to ambient temperature and 150 ~1 of the contents of each well was transferred to a lumacuvette. Diluted microperoxidase and hy- drogen peroxide were added and the light emitted for 10 s was measured in a luminometer (Biocounter M2000, Lumac) [ 171. The total assay time for one plate (i.e., 40 saliva samples) was 3 h.

Direct radioimmunoassay of unconjugated serum estriol. The procedure of the commercial kit was briefly as follows. To each plastic assay tube were added 20 ~1 of standard or unknown serum sample, 100 ~1 of ‘251-labelled estriol and 500 ~1 of solid-phase antibody. The mixture was incubated at room tempe- rature for 30 min and centrifuged for 15 min at 2000 g, and the supernatant liquid was decanted. The pellets were allowed to drain on absorbent tissue for 10 min, and the antibody-bound radioactivity in the pellets was counted (model 1270 Rackgamma- counter, LKB Wallac) .

Calculation of results. Nonspecific blank values were subtracted from the luminometer readings and B/B0 vs. log dose of the standards (I? = no. of counts in presence of estriol, B0 = no. of counts in absence of estriol) was plotted as a calibration graph. For RIA, a logit-log calibration was used for on-line calcu- lation of estriol concentration. Best-fit linear regression lines were calculated according to Davies [ 181.

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TABLE 1

Cross reactivity ( % ) of anti-estriol antibody clone 9B4 in two immunoassay systems

Steroid RIA” Chemiluminescence Steroid immunoassayb

RIA” Chemiluminescence immunoassayb

Estriol 100 100 Estrone 0.6 0.04 16/Y, 17/I-Estriol rid’ 3.2 16cr OH-Estrone nd” 0.04 16q 17ar-Estriol 0.1 0.05 Cortisol 0.1 0.05 Estriol-3-glucuronide nd’ 77 Progesterone 0.1 0.01 Estradrol 13 2 Testosterone 0.1 0.01

“RIA, antibody in solution; phase separation by dextran-coated charcoal [ 191. bPresent method; solid-phase antibody, direct assay; phase separation by washings, ‘Not determined.

RESULTS

The well-to-well variation of coated microtitration plates was investigated by assaying zero standards in all wells. With freshly coated plates, coefficients of variation between 1.9 and 3.4% were observed. Plates assayed more than eight days after coating had coefficients of variation > 4% and edge effects were observed. Plates were normally used within four days of coating.

No matrix effect of added saliva was observed. Graphs constructed with as- say buffer or with 25-100 ~1 of untreated male saliva per well overlapped com- pletely. Saliva treated with dextran-coated charcoal caused a lateral shift and could therefore not be used. Thus 50 ~1 of pooled male saliva was added to the standards, including zero binding.

The limit of detection, defined as the least amount of estriol that could be distinguished from zero (mean - 2 standard deviations), was evaluated from 13 replicate measurements of the assay response at zero dose (Fig. 2 ) . It was 4.5 pg per well.

Cross-reactivities of the monoclonal antibody 9B4, as measured by RIA [ 191 and by the present method, are given in Table 1.

In order to assess the analytical recovery, different amounts of estriol (12.5-600 pg/50 ,ul, 0.83-41.6 nmol l-l) were added to male saliva and 50-~1 portions of each were assayed in quadruplicate. Between 89 and 105% of added E3 was accounted for (mean 98%, coefficient of variation 6.2%; i.e., + 12.5 pg, 94%; + 25 pg, 89%; + 50 pg, 92.5%; 100 pg, 99%; + 200 pg, 102%; + 400 pg, 104.6% and + 600 pg, 105% ) .

Within-assay precision was assessed from replicate measurements in one chemiluminescence immunoassay and between-assay precision from repeated analysis in consecutive assays (Table 2).

Comparison of results from serum and saliva Time-matched samples of saliva and serum were collected between 08 00

and 12 00 h from 26 pregnant women attending the clinic. The estriol concen-

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TABLE 2

Precision of the direct chemiluminescence immunoassay for estriol in saliva

Within-assay Between-assay

No. of E3 cont.’ (nmol 1-l) CV No. of E3 conc.a (nmol 1-l) determina- Mean determina- Mean tions tions

10 0.21 (0.025) 11.8 7 0.27 (0.042) 10 0.81 (0.073) 8.9 8 0.39 (0.037) 8 1.39 (0.082) 5.9 10 0.55 (0.044) 8 1.82 (0.133) 7.3 8 1.64 (0.168) 8 2.66 (0.125) 4.7 7 2.70 (0.221)

10 3.27 (0.165) 5.0 7 3.47 (0.240) 10 6.49 (0.405) 6.2

“Mean with standard deviation in parentheses and coefficient of variation (CV, % ) .

CV

15.7 9.3 7.8

10.2 8.2 6.9

tration in saliva (SAL, nmol 1-l) correlated well with the serum estriol con- centration (SER, nmol 1-l ) with a correlation coefficient of 0.934. Regression parameters were SAL = 0.071 x SER + 0.16 nmol l-l. Salivary E3 concen- trations gradually rose from 0.186 nmol l-l at 7 to 10 weeks of gestation to 5 nmol l-l at 38 weeks; they represented a mean proportion of 9.45% of uncon- jugated estriol in time-matched serum samples of the same patients.

DISCUSSION

The direct chemiluminescence immunoassay for salivary estriol described in this paper meets the requirements of sensitivity, accuracy and precision. In addition it is simple and fast, with an assay capacity of 40 duplicate samples in 3 h. The concentration range of the assay, from 0.31 to over 20 nmol 1-l E3, amply covers a complete pregnancy. The good correlation observed between estriol concentrations of time-matched samples of saliva and serum is consis- tent with the concept that salivary estriol accurately reflects the serum uncon- jugated estriol levels [ 7,9,10]. In addition, from individual matched samples, it was calculated that the salivary E3 concentration is ca. 9.5% (range 4.4-24% ) that of the corresponding serum E3 concentrations. This confirms earlier re- sults obtained with other methods, such as direct RIA [ 7,11,20] and enzyme immunoassay [ 211 or assay after extraction of the steroid from both fluids [ 9,10,22].

Although the monoclonal antibody clone 9B4 cross-reacts to an extent of 77% with estriol-3-glucuronide, this poses no problems in the assay of salivary estriol because conjugates of estriol are shown not to be present in saliva in significant concentrations [ 6,11,23]. In general, high selectiv$y of antibodies

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with respect to estriol conjugates is not necessary. This obviates the need of time-consuming and error-prone extractions and it also decreases the required volume of saliva delivered by the patient (e.g., to 250 ~1 for the direct chemi- luminescence immunoassay). After freezing and centrifuging, 100 ~1 of clear supernate for duplicate 50-~1 specimens can easily be pipetted. This may be of value in normal pregnancy and even more in high-risk pregnancy or in emer- gency cases where multiple sampling would be required.

This study was supported in part by a grant from the National Fund for Scientific Research (NFWO Grant No. 3.0030.86 to J.d.B.), and a grant to F.K. from the Binational Science Foundation (Jerusalem). F.K. is the Fanny and Samuel Kay Research Fellow at the Weizmann Institute. The authors are grateful to Dr. R.D. Mackintosh and Mr J. Vossen of Lumac B.V., Landgraaf, The Netherlands, for the loan of a Biocounter M2000 luminometer, and thank D. Leyseele, R. De Kimpe, S. Hoste and J. Plum for their help.

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