Development of a chemiluminescence immunoassay for salivary progesterone using microtitre plates as a solid phase

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<ul><li><p>Analytrca Chimzca Acta, 227 (1989) 119-127 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands </p><p>119 </p><p>DEVELOPMENT OF A CHEMILUMINESCENCE IMMUNOASSAY FOR SALIVARY PROGESTERONE USING MICROTITRE PLATES AS A SOLID PHASE </p><p>J. DE BOEVER* and D. VANDEKERCKHOVE </p><p>Department of Obstetrics and Gynaecology, Universtty Hospital, De Pintelaan 185, B-9000 Gent (Belgzum) </p><p>F. KOHEN </p><p>Department of Hormone Research, Wettmann Instztute of Sctence, IL-7600 Rehovot (Israel) </p><p>(Received 24th May 1989) </p><p>SUMMARY </p><p>Progesterone is determined in whole saliva by a direct solid-phase chemiluminescence immu- noassay (CIA), which uses a monoclonal antibody raised against progesterone-llcu-hemisuccin- ate bovine serum albumin (BSA) and the homologous marker conjugate progesterone-lla-hem- isuccinate aminobutylethylisoluminol. Rabbit anti-mouse immunoglobulins (second antibody) are coated on to the wells of Nunc Maxisorb microtitre plates during an overnight incubation. Then monoclonal anti-estradiol antibody is bound overnight to the second antibody and residual binding places are blocked with BSA. Saliva is frozen, thawed and the clear supernate obtained after centrifugation is used. The calibration graph is linear over the range 3-200 pg of progesterone and the detection limit is 1.6 pg per well (0.096 nmol 1-r). The mean recovery of added proges- terone is 97%. The within-assay relative standard deviation (RSD) is 2.7-9.7% for 0.3-5.4 nmol 1-l progesterone and the between-assay RSD is 5.7-10.6% for 0.79-3.7 nmol 1-i progesterone. In pregnancy salivary progesterone rises from 1 nmol 1-l (6-15 weeks) to 4 nmol 1-l (36-40 weeks). </p><p>The determination of progesterone concentrations in blood is of great value for clinical endocrinological investigations in women. However, the assess- ment of ovarian function, ovulation induction and also threatening abortion require repeated determinations of progesterone in the peripheral circulation [l-3]. However, repeated blood sampling is time consuming and may be stressful. On several occasions it has been shown that some steroids, including progesterone, are present in saliva in concentrations that reflect those in the free fraction of serum or plasma [ 4-71. In addition, salivary steroid concen- trations are independent of flow-rate [ 6,8] and sampling is non-invasive and stress-free. Recently, it was found that the saliva estriol to progesterone ratio in late pregnancy changes considerably before the spontaneous onset of labour at term [ 7,9]. Because of the good correlations between the concentrations of </p><p>0003-2670/89/$03.50 0 1989 Elsevier Science Publishers B.V. </p></li><li><p>120 </p><p>estriol and progesterone in saliva with the concentrations of free estriol and progesterone in serum, this ratio in saliva reflects a similar ratio in blood. This latter could not be observed in an earlier study because it would have required frequent blood sampling over a long period of time [lo]. </p><p>The analysis of a large number of samples is tedious if performed by extrac- tion radioimmunoassay (RIA) with dextran-coated charcoal separation of an- tibody-bound and free fractions [ 71. It would be both practical and advanta- geous to have a non-extraction (direct) assay and to avoid the use of radioisotopes as labels and of dextran-coated charcoal for phase separation. In this paper, the development and evaluation of a simple direct solid-phase chemiluminescence immunoassay (CIA) for progesterone in saliva is described. </p><p>EXPERIMENTAL </p><p>Samples Unstimulated saliva was obtained between 0800 and 1200 h from pregnant </p><p>women (gestational age 7-42 weeks) attending the Womens Clinic at the Uni- versity Hospital, Ghent. They received instructions to rinse their mouths sev- eral times with tap water and to start collecting 2-3 ml of 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. Pools (20-25 ml) of male saliva were obtained from the same person. They were stored frozen, then thawed and centrifuged. The clear supernate was pooled and mixed to homogeneity, then divided into small portions and stored frozen at - 20 C. </p><p>Chemicals and solutions Steroids were purchased from Steraloids (Wilton, NH) and Sigma. Micro- </p><p>peroxidase (MP-11; E.C. 1.11.1.7), bovine serum albumin (Cohn Fraction V) and Tween-20 [ polyoxyethylene (20) sorb&amp;an monolaureate] were from Sigma. Ethanol, hydrogen peroxide (300 g 1-l) and sodium hydroxide pellets were from Merck. Microtitre plates (Maxisorb, with certificate) and adhesive- backed plastic film to cover and seal off the plates (sealing tape) were ob- tained from Nunc (Kampstrup, Denmark). Second antibody, purified anti- mouse IgG, code no. 2259, was from Dakopatts (Glostrup, Denmark). Luma- cuvettes (12 x 50 mm polystyrene tubes ) were from Lumac Systems (Basle ). </p><p>The assay buffer was sodium phosphate (50 mmol l-l, pH 8.0) containing 9 g 1-l sodium chloride, 100 mg 1-l bovine serum albumin (BSA) and 1 g 1-l sodium azide. The coating buffer was sodium carbonate solution (50 mmol l- , pH 9.6) containing 0.1 g 1-l thimerosal. The wash solution, microperoxidase, oxidant solution (0.1 ml of 300 g 1-l hydrogen peroxide mixed with 15 ml of </p></li><li><p>121 </p><p>distilled water) and steroid stock solutions were all as described previously </p><p>[ill. </p><p>Procedures Preparation of chemiluminescent marker conjugate and antibody toprogester- </p><p>one. Progesterone-lla-hemisuccinate aminobutylethyl isoluminol (Pll- ABEI) was synthesized as described [ 121. A stock solution in ethanol (5 pg ml-) was stored at 4C. A sub-stock solution in assay buffer (100 ng ml-) was prepared every 2 months. Working solutions (62.5-4000 pg ml-l) were prepared daily in assay buffer. Monoclonal anti-progesterone antibody was obtained from the rat-mouse hybridoma line 1E 11. This cell line was derived by fusion of rat spleen cells, immunized with progesterone-lla-hemisuccin- ate/BSA, with mouse myeloma cell line NSO, kindly donated by Dr. C. Mil- stein (Cambridge). This line was propagated as ascitic fluid in irradiated CO, female mice [ 131. Monoclonal antibody 1E 11 belongs to the IgG, class, and was used in the assay as diluted ascitic fluid, without further purification. A stock solution prepared by 1: 100 dilution in assay buffer was stored at 4 C. </p><p>Coating of microtitre plutes. The procedure has been described in detail for estradiol antibody [ 141. It included overnight incubation at 4 C of 200 ~1 di- luted second antibody per well, followed by washing. Then 200 ,~l of diluted monoclonal antibody per well were incubated overnight at 4 o C. For the present assay, a 200 OOO-fold dilution of antibody 1E 11 in assay buffer was used. Next, the plate was washed four times and used either directly or after storage at 4 C for up to 8 days. </p><p>Optimization of assay conditions. The steepness of the calibration graph, the standard deviation (SD) of duplicates of the different points on the graph and the relative standard deviation (RSD) for each point on consecutive graphs were studied under varying assay conditions. This included different buffers for incubation, different incubation times and either direct or delayed addition of the labelled conjugate. </p><p>The pH 8 buffer with BSA (assay buffer) was better than buffers with less or no BSA or at pH other than 8. An increase in binding of the Pll-ABE1 conjugate was observed with increasing incubation from 15 min to 3.5 h. How- ever, the relative binding, B/B0 (where B = number of counts in the presence of progesterone and B ,-, = number of counts in the absence of progesterone ) was stable for all points on the graph. There was also no change in the reproduci- bility of duplicates. A 1: 200 000 dilution of monoclonal antibody gave a steeper calibration graph than either higher or lower dilutions on all occasions, i.e., 30 min up to 3.5 h of incubation. Therefore, it was decided to use a 1: 200 000 dilution of monoclonal antibody 1E 11 and incubation for 3.5 h. </p><p>Chemihminescence immunoassay (CIA). Standards were prepared by dilut- ing an ethanolic 1 E ml- solution of progesterone in assay buffer so that 50 ~1 of each dilution contained between 3.1 and 200 pg. The sub-stock solution </p></li><li><p>122 </p><p>of 100 ng ml- Pll-ABE1 in assay buffer was diluted 240-fold to give 83 pg of conjugate in 100 ~1. To the wells were added 50 ~1 of standard or buffer, 50 ,~l of saliva and 100 ~1 of conjugate. The plate was incubated for 3.5 h at room temperature on a horizontal shaker at 170 cycles min-. Subsequently, the wells were emptied by inversion and washed three times with wash solution. A 200-~1 portion of 2 M sodium hydroxide was added to all wells and the plate was heated for 30 min in a water bath at 60 C. The plate was cooled to ambient temperature and 150 ~1 of the contents of each well were transferred into a Lumacuvette. Diluted microperoxidase and hydrogen peroxide were added and the light emitted for 10 s was measured in a luminometer (Lumac Biocounter M2000 or M2010) [ 141. The total assay time for one plate (i.e., 40 saliva sam- ples) was 6 h. Calculation of results. Non-specific blank values were subtracted from the </p><p>luminometer readings and B/B0 vs. log (dose of standards) was plotted as a calibration graph using the RIA-CALC program from Pharmacia-LKB. </p><p>0 2 5 10 20 5Ll 100 200 </p><p>Progesterone, pg I well </p><p>Fig. 1. Dose-response curve of direct CIA for progesterone in saliva. Each point is the mean % B/B, f SD of 10 consecutive determinations. </p><p>TABLE 1 </p><p>Cross-reactivity (% ) of anti-progesterone antibody clone 1E 11 in two immunoassay systems </p><p>Steroid RIA CIAb Steroid RIA CIAb </p><p>Progesterone 100 100 3p-Hydroxypregn-5-en-20-one </p></li><li><p>123 </p><p>RESULTS </p><p>The well-to-well variation of coated microtitre plates was assessed by mea- suring zero standards in all wells. The RSDs observed with plates 1-8 days after coating were between 1.9 and 3.2%. Thereafter, the RSD increased to &gt; 4% and edge effects were observed. Plates were therefore used within 8 days of coating. </p><p>A slight matrix effect of added saliva was observed. Therefore, 50 ~1 of pooled male saliva were added to all standards, including the zero binding (B,) . The limit of detection, defined as the least amount of progesterone that could be distinguished from zero (mean - 2 standard deviations), was calculated from 10 replicate measurements of the assay response at zero dose (Fig. 1) and was 1.5 pg per well. Cross-reactivities of the monoclonal antibody 1E 11, as mea- sured by RIA [ 151 and by the present method, are given in Table 1. </p><p>In order to assess the analytical recovery, different amounts of progesterone (5-75 pg per 50 fl, 0.312-4.69 nmol 1-l) were added to male saliva and 50-~1 portions of each were assayed in five duplicates. Between 90.8 and 102% of added progesterone was recovered (mean 96.8%; +5 pg, 93.5%; + 10 pg, 90.8%; + 25 pg, 101.6%; and+ 75 pg, 101.4% ). </p><p>The within-assay precision was determined from replicate measurements in one assay and the between-assay precision from repeated analysis in consec- utive CIAs (Table 2). </p><p>Progesterone concentrations in saliva during normal and pathological preg- nancy are presented in Fig. 2. The increase in progesterone from 1 nmol l-l at 6-15 weeks of pregnancy to 4 nmol 1-l at 36-40 weeks of pregnancy parallels the rise in salivary estriol up to 33-35 weeks. Thereafter the progesterone con- centrations stabilize whereas the estriol levels increase further [ 161. As a re- sult, the estriol to progesterone ratio is 0.6-l between 6 and 34 weeks and increases to 1.5 from 33-35 weeks on (5 weeks before delivery). </p><p>TABLE 2 </p><p>Precision of the direct CIA for progesterone (P4) in saliva </p><p>Within-assay Between-assay </p><p>No. of P4 concentration RSD No. of P4 concentration RSD determi- (nmol 1-l) (W) determi- (nmoll-) (%) nations nations </p><p>Mean SD Mean SD </p><p>7 0.320 0.023 7.4 10 0.796 0.057 7.2 9 1.368 0.133 9.7 10 0.806 0.042 5.7 9 1.975 0.070 3.5 10 2.403 0.188 7.8 9 2.501 0.188 7.5 10 2.587 0.274 10.6 5 3.336 0.174 5.2 10 3.696 0.367 9.9 5 5.426 0.145 2.7 </p></li><li><p>124 </p><p>12 - </p><p>Y- </p><p>+ </p><p>+ t + + </p><p>3- </p><p>+ Y - </p><p>+ </p><p>WEEKS OF PREGNANCY </p><p>Fig. 2. Progesterone concentrations in saliva during normal and pathological pregnancy. (A) 0, Normal; + , twin pregnancy. (B) 0, Intra-uterine growth retardation; + , pre-term delivery; * , toxaemia. </p></li><li><p>125 </p><p>DISCUSSION </p><p>This direct chemiluminescence immunoassay for salivary progesterone is convenient and simple. The main advantages are that no radioisotopes, no extraction of the samples and no centrifugation steps are needed. Most of the assays described so far for the determination of progesterone in saliva are RIA with extraction of the samples and dextran-coated charcoal separation of bound and free fractions. Of the direct RIAs, two are with overnight incubation. </p><p>A direct enzyme immunoassay, using microtitre plates as a solid phase, has been described with an excellent detection limit (0.012 nmol 1-l) and an assay time of 4 h [ 171. The present CIA has a sensitivity of 0.096 nmol 1-l and an assay time of 6 h. It meets the requirements of sensitivity, accuracy and spec- ificity. Both 5cy- and 5P-pregnanedione cross-react considerably. Binding of Pll-ABE1 to the antibody is inhibited 50% by 135 pg (5cw-) and 150 pg (5/&amp; ) of pregnanedione (compared with 24 pg of progesterone). The concen- tration of these cross-reacting steroids in saliva is unknown but is probably very low. Indeed, their concentrations in the circulation are very low [ 181 and if their concentrations in saliva were ca. 2% of the concentrations in the pe- ripheral circulation, as is observed for progesterone [ 71, the salivary levels would be negligible. Consequently, pregnanediols would not interfere in the direct measurement of progesterone by the present method. 17a-Hydroxypro- gesterone has not been determined in saliva throughout pregnancy. In preg- nancy plasma, however, its concentration is much lower than that of proges- terone [ 191. </p><p>The results presented here closely parallel those obtained using RIA with extraction of samples [ 7,9] in terms of progesterone concentrations and of the changing ratio of estriol to progesterone shortly before labour. Other workers have found conflicting results [ 201. </p><p>The determination of steroid hormones in saliva offers an attractive alter- native to the use of serum or plasma. Saliva is easily obtained and, if needed, serial samples can conveniently be collected. The concentrations of most ster- oid hormones have been found to parallel the concentrations of free steroids, i.e., the biologically active fraction, in the peripheral circulation [ 4-71. The use of saliva, however, requires either large samples to be extracted or highly sensitive methods for direct assay and if only small samples for extraction are available. In addition, saliva sampling must be reliable: saliva must not con...</p></li></ul>

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