Enzyme immunoassay of progesterone in saliva

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ENZYME IMMUNOASSAY OF PROGESTERONE IN SALIVA D. F. Tallon and E. M. O'Dwyer Department of Obstetrics and Gynaecology, Regional Hospital and University College, Galway, and P. F. Fottrell Department o! Biochemistry, University College, Galway. Summary A VERY sensitive enzyme immunoassay for direct measurement of proges- terone has been developed. It is a solid phase assay on microtitre plates and there are no extraction, separation or centrifugation steps. The assay is suit- able for frequent monitoring of proges- terone levels in saliva during the men- strual cycle. Expensive equipment or special facilities are not required. The assay is very suitable for hospital labor- atories that currently lack facilities for measuring progesterone by radioim- munoassay. Introduction Measurement of plasma progesterone, a product of follicular luteinization after the mid-cycle surge of luteinizing horm- one is used as a biochemical indicator of ovulation. It is superior to determina- tions of basal body temperatures which may respond to relatively small altera- tions in progesterone (Shepard and Sen- turia, 1977). However, there is consider- able variation in plasma progesterone values between individuals at similar times in the menstrual cycles and even in separate cycles of a given individual (Moghissi and Wallach, 1983). Frequent plasma samples during the menstrual cycle are therefore recommended in pre- ference to single samples (Moghissi and Wallach, 1983). Although frequent measurements of plasma progesterone during the men- strual cycle provide a more accurate assessment of corpus luteum function it Correspondence: P. F. Fottrell. is not practical because of expense, in- convenience and possible stress on the patient. An alternative approach is the measurement of progesterone in saliva samples taken in the patient's home and sent to the laboratory for analysis. Recent reports have demonstrated the efficacy of saliva for monitoring the levels of several important steroid hor- mones including progesterone and have shown excellent correlation between steroid levels in saliva and serum (Riad- Fahmy et al, 1982). Salivary progester- one measurements have been done to date by radioimmunoassay following extraction with organic solvent (Riad- Fahmy et al, 1982). This has restricted the assays to laboratories with the rel- evant facilities and equipment. We de- scribe an enzyme immunoassay for pro- gesterone in saliva that has the requisite sensitivity and specificity to allow direct measurement of progesterone without extraction and centrifugation steps. The suitability of the assay for frequent mon- itoring of progesterone during the men- strual cycle is demonstrated in this report. Materials and Methods Reagents The basic procedures and the reag- ents used in the current method are similar to those outlined in extensive reviews on enzyme immunoassay (Schuurs and Van Weeman, 1977; Eng- vail, 1980). Antiserum against 11o~- hydroxyprogesterone-bovine serum albu- min conjugate was raised in rabbits and horseradish peroxidase was coupled to the 11(x-hydroxyhemisuccinate of proges- 195 196 Tallon and O'Dwyer I.J,M.S. June, 1984 terone (see Engvall, 1980). The chromo- gen used to detect peroxidase activity was o-phenylendiamine. Assay Saliva samples (50 FI), enzyme-lab- elled progesterone, progesterone stand- ards and controls areadded to individual wells of microlitre plates previously pre- coated with progesterone antibody. The plates are incubated at 37~ for 60 min- utes and then washed to remove unbound reactants. The substrate, hydrogen per- oxide (5.3 mM) in potassium acetate buffer and 8 mM o-phenylenediamine are added to the wells and the plates are incubated at room temperature in a dark environment for 30 minutes. The enzyme reaction is stopped by adding 50 /~1 of 8N sulphuric acid and the optical density at 492 nm is measured with a Biotek EIA microtitre plate reader (Noctech, Dublin). Progesterone values are calculated on a B.B.C. microcomputer connected to the microtitre plate reader. One person may process 200 saliva samples per day. Saliva samples Non-pregnant females between the. ages 18-40 who were not taking oral steroid contraceptives and who reported having regular menstrual cycles (28 days -+ 2) over the previous six months gave daily samples of saliva. Each volunteer collected about 5 ml of saliva between 7 a.m. - 10 a.m. each day following an overnight fast and preliminary rinsing of the mouth with water. Samples were collected in 20 ml universal containers and placed in the deep freeze as soon as possible. Deep freezing greatly re- duced viscosity of saliva and after freez- ing and subsequent thawing salivary progesterone values remained stable for several days at 4~ or 18 ~ After freezing samples may be transported to the lab- oratory for analyses. Results Precision Intra and inter-assay variance were determined using low (120 pmol/L) medium (190 umol/L) and high (440 pmol/L) progesterone concentrations. The coefficients of variation for intra- assay are 6.5% - 8.6% and for inter- assay variation 5.9% - 8.5%. Sensitivity The sensitivity defined as the least amount distinguishable from zero at two standard deviations is 0.2 picograms progesterone per well of microtitre plate; this is equivalent to 12.7 picomoles per litre. Specificity The antiserum displayed a high degree of specificity for progesterone. The cross reactions of the antiserum were calcu- lated from the formula Picograms progesterone at B/Bo of 50% Picograms test steroid at B/Bo of 50% The percentage cross reaction with 5o~- pregnane- 3- 20- dione, 17~- hydroxy- progesterone and 11-deoxycorticoster- one is 5.7%, 4.6% and 2.0% respectiv- ely. The following steroids gave values of about 1% or lower, corticosterone, testosterone, oestradiol-17-/3 and preg- nenolone. Recovery of progesterone There is excellent correlation between expected and detected values when dif- ferent concentrations of progesterone were added to control male saliva (Fig. 1). Progesterone levels in saliva Daily saliva samples were collected from a control group and stored in the deep-freeze. Samples from one menstrual cycle were assayed simul- taneously. Progesterone levels during one menstrual cycle in two different females are shown in Figure 2. The val- ues display the typical cyclic variation reporter for both plasma and salivary progesterone as determined by radioim- munoassay (Riad-Fahmy et al, 1982). Discussion A rapid enzyme immunoassay for measuring progesterone in human saliva Vo lume 153 Number 6 Enzyme immunoassay 197 I~C07~Y C~" I :~(~I~CI~ AC~ED 'J~ NO~,AL MA~ S.ALIYA. r = 0 .999 ( I r = I = I 25 50 150 250 Progesterone Expected pg /ml Fig. 1--Recovery of progesterone added to normal male saliva. without the need for prior extraction and separation has been described. The precision and specificity of the assay are very good and the sensitivity is superior to published radioimmunoassays for pro- gesterone in saliva. A recent radioim- muncassay for salivary progesterone (Sorgo, Manella and Zachmann, 1983) reported a sensitivity of 9 picograms progesterone per test tube compared with 0.2 picograms obtained with the present assay. Although cycles were not dated in this study by determining luteinizing horm- one in matched samples of plasma it is nevertheless interesting that the proges- terone levels in the controls in Fig. 2 compare favourably with reported values (Riad-Fahmy et al, 1982). The latter re. ported in a more extensive study using radioimmunoassay that salivary proges- terone levels do not exceed 150 pico- moles per litre during the follicular phase but increase to 300-750 picomoles per litre around day 21 of the cycle. Expensive equipment is not necessary to measure progesterone by this present SALIVARY PROGESTERONE LEVELS THROUGHOUT TEE MENSTRUAL CYCLE OF TWO NORMAL FEMALES. r , , ~ . . . . ~1 ' , a , ,~ . . . . ~o I I ' '2's . . . . 30 Day of Cycle f . . . . . . . . . . , . . . . . . . . . . . . 5 10 15 20 24 1 2 3 Day of Cycle Fig. 2--Progesterone levels in daily saliva samples from 2 control females. procedure and unlike radioimmunoassay special facilities are not required and there are no problems with disposal of isotopes or solvents. The method is therefore of particular relevance to lab- oratories interested in measuring pro- gesterone but lacking the necessary equipment and facilities for radioim- m u noassay. We are grateful to W. F. Cleere and; J. Gosling for advice and to Noctech for generous gifts of reagents. Request for Reprints : P. F. Fottrell. References Engvall, E. 1980. Enzyme immunoassay ELISA and EMIT. Methods in Enzymol. 70, 419. Moghissi, K. S. and~ Wallach, E. E. 1983. Unex- plained infertility. Fertil. Steril. 39, 5. Riad~Fahmy, D., Read, G. F., Walker, R. F. and~ Griffiths, K. 1982. Steroids in saliva for assess- ing endocrine function. Endocrine Revs. 3, 367. Schuurs, A. H. W. M!. and~ van Weeman, B. K. 1977. Enzyme immunoassay. Clin. Chim. Acta, 81, 1. Shepherd, M. K. and Senturia, Y. D. 1977. Com- parison of serum progesterone an~ endometrial biopsy for confirmation of ovula,tior~ and evalu, ation of luteal function. Fertil. Steril. 28, 541. Sorgo, W., Manella, B. and Zachmann, M. 1983. Radioimmunoassay of progesterone in saliva. Hormone Res. 17, 153.

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