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Polarisation fluoroimmunoassay of serum and salivary cortisol
J. steroM Biochem. Vol. 19, No. 4. pp. 1475 1480, 1983
0022-4731/83 $3.00+0.00 Copyright .~'~ 1983 Pergamon Press Ltd
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POLARISATION F L U O R O I M M U N O A S S A Y OF SERUM A N D SALIVARY CORTISOL A. A. K. AL-ANSARI*, D. S. SMITH and J. LANDON Department of Chemical Pathology, St. Bartholomew's Hospital, London ECI, U.K. (Received 8 December 1982)
Summary Polarisation fluoroimmunoassays for serum and salivary cortisol were developed using the steroid labelled at the 3-position with fluorescein and antiserum raised in sheep. To avoid interfering factors present in the biological fluids, cortisol was extracted with dichloromethane prior to assay, which was then accomplished without the need for any further separation or blank-correction procedures. The methods were sufficiently sensitive to cover the nomml morning ranges for serum and salivary cortisoI and correlated acceptably with established fluorimetric and radioimmunoassay techniques. The salivary cortisol assay was more precise than the serum assay, reflecting the greater ease with which cortisol may be extracted from saliva. The potential usefulness of salivary cortisol assays was illustrated by a preliminary study showing that the circadian variation in three children was similar to that in an adult.
INTRODUCTION
Polarisation fluoroimmunoassay (PIA), introduced in 1973 by the groups of Dandliker[l] and Spencer[2], is one of the simplest immunoassay techniques. It is based on the increase in polarisation of fluorescence that occurs when a relatively small fluorescentlabelled antigen is b o u n d by a large antibody molecule [3] and hence is best suited to the assay of haptens such as drugs [4] and steroids[5, 6]. PIA requires no separation of free and bound fractions of the labelled antigen and employs stable and hazardfree reagents. PIA has not been widely applied in routine clinical practice for two main reasons. First, its sensitivity is limited by interfering factors that may be present intrinsically or abnormally in serum or plasma; these include fluorophores, haemoglobin and lightscattering materials that may interfere spectroscopically, and proteins (notably albumin) that may bind tracers non-specifically [7]. Secondly, suitable polarisation fluorimeters for precise and convenient end-point measurement have not been readily available. However, both manual[8,9] and fully automated [10] instrument systems have recently been described. Miyai, Watanabe and their colleagues have reported PIA methods for serum cortisol that employ steroid labelled at the 21-position with fluorescein and rabbit antibodies raised against a cortisol-21hemi-succinate-protein conjugate. In one system [5] interfering serum proteins are precipitated with methanol in a pre-treatment step, while in another [6] sodium dodecyl sulphate (SDS) is used as a blocking agent to prevent non-specific serum-protein binding *Present address: Endocrinology Unit, Sabah Hospital, P.O. Box 4078~ Safat, Kuwait. sl~ 19/4
(~
of the tracer and enable direct (non-extraction) assay. In both cases, correction for residual serum interferences (blank signals) is necessary and is achieved by making polarisation measurements both before and after the addition of antibody to each assay mixture. There are potential advantages in the measurement of steroids in saliva as opposed to serum or plasma[l 1]. First, salivary levels generally reflect those of the free (non-protein-bound) steroid fraction in the circulation, which is believed to be directly related to physiological effects. Secondly, saliva may be easily collected non-invasively and without stress and hence is suitable for studies on children and in diagnostic investigations requiring serial sampling. Thirdly, saliva contains relatively little protein, of which only traces are of plasma origin[12], thus protein matrix effects can be expected to be less of a problem in salivary immunoassays. Salivary cortisol levels average only about 50,~, of total serum levels and therefore relatively more sensitive assays are required. We have described an adaptation of a commercially-available radioimmunoassay (RIA) kit method for serum or plasma cortisol that permits direct assay of salivary specimens[13]. To study the PIA of serum and salivary cortisol, we raised antiserum against cortisol conjugated to carrier protein at the 3-position because this has generally been found to result in better specificity than when 21-1inked immunogens are used [14, 15]. Since antiserum consumption in PIA is high (relative to that in RIA) we used sheep as experimental animals rather than rabbits. MATERIALS
Cortisol and other steroids, N-hydroxysuccinimide, l-ethyl-3(3-dimethylaminopropyl) carbo-
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A . A . K . AL-ANSARIet al.
diimide hydrochloride (EDC), tetrazolium blue and bovine albumin (type A4503) were obtained from Sigma, Poole, Dorset, U.K.: organic solvents (all Analar grade and used without further purification), gelatin, sodium salicylate, SDS and Triton X-100 from BDH, Poole, Dorset, U.K.: silica-gel thin-layer chromatography (TLC) sheets (DC-Alufotien Kieselgel 60 F254, Art. 5554) from Merck, Darmstadt, F.R.G.: keyhole limpet hemocyanin (KLH), 133 g/l in 50!}i;glycerol, from Calbiochem, San Diego, California, U.S.A.: complete and incomplete Freund's adjuvant from Difco, West Molesey, Surrey, U.K.; activated charcoal from Hopkin and Williams, Chadwell Heath, Essex, U.K.; pooled normal human serum from ILS, Newbury Street, London ECI, U.K.; and [1,2,6,7-3H] cortisol from Amersham International, Amersham, Buckinghamshire, U.K. Glass centrifuge tubes ( l l 0 x 17ram) were obtained from Coming, Stone, Staffordshire, U.K.; glass test tubes (76 × 16mm) from Hoslab, llford, Essex, U.K.: polystyrene test tubes type LP3 (63.5 x 9.5 mm) from Luckham, Burgess Hill, Sussex, U.K.; polystyrene Universal containers (90 x 24 mm) from Sterilin, Richmond, Surrey, U.K.; and polystyrene fluorimeter cuvettes No. 67.754 (10 x 10 ram) from Sarstedt, Leicester, Leicestershire, U.K. Cortisol solid-phase ~2SI-RIA (Immophase ~M) kits were provided by Coming Medical Halstead, Essex, U.K, METHODS
Cortisol immunogen
Anti-eortiso[ serum
Six mature female Border-Leicester cross sheep were each immunised with 2.5 mg of the cortisolKLH conjugate in 8 ml of an emulsion of water and complete Freund's adjuvant (1:3 parts by volume). The immunogen was divided between several intramuscular and subcutaneous sites. The animals were given booster injections 20 days later in the same way, except that half the amount of immunogen was used and the adjuvant was incomplete Freund's. Subsequently the sheep were boosted every 20 days and bleeds taken 10 days after alternate boosts. Antiserum obtained from the fourth bleed of one sheep was used in the studies reported in detail below. Fluoreseein-labelled eortisol
Fluoresceinthiocarbamyl ethylenediamine, prepared by reaction of fluorescein isothiocyanate with ethytenediamine, was conjugated with cortisol-3-(Ocarboxymethyl) oxime by an active-ester procedure and the product purified by TLC on silica gel as described previously [16]. Labelled cortisol, eluted from silica gel with methanol, was stored at - 2 0 C . Its concentration was estimated spectrophotometrically [16]. Cortisol-l?ee human serum
Pooled normal human serum was mixed with activated charcoal (100 g/l) by gentle rotation for 18 h at 37 C, followed by centrifugation for 45 min at 4 C (10,000 rpm). Charcoal fines were removed from the supernatant by pressure filtration through membranes with a series of pore sizes (2, 1, 0.8, 0.45 and 0.22/~m) using equipment from Sartorius, Belmont, Surrey, U.K. When a trace amount of tritiated cortisol was equilibrated with pooled normal human serum for 4 h at room temperature prior to treatment, it was found that 99.6'},] of the cortisol was removed by the charcoal. When tested by the RIA kit method, no cortisol was detected in the charcoal-treated serum.
Cortisol-3-(O-carboxymethyl)oxime, prepared by reaction of cortisol with carboxymethoxylamine hemihydrochloride as described previously [16], was added (35 mg) to a solution of N-hydroxysuccinimide ( l l . 5 m g ) and EDC (15.3rag) in 3.5ml of dimethylformamide. After 45 min at room temperature, TLC of a small aliquot of the reaction mixture on silica gel developed with ethyl acetate methanol-glacial acetic acid (90:8:2 by vol) indicated the disappearance of the oxime starting material (Rt 0.35 ) and its conversion to the active ester (R~- Cortisol serum standards 0.75). Steroid-containing spots were visualized on the Cortisol dissolved in methanol (1 mmol/l) was adTLC plate by spraying with tetrazolium blue reagent [17] to give the blue colour characteristic of ded to cortisol-free human serum. The standards the dihydroxyacetone group. The reaction mixture were stored in aliquots at - 2 0 C. was diluted with dimethylformamide (I.5 ml) and Specimen collection distilled water (5 ml), then immediately added to a Normal sera were obtained from volunteers in the well-stirred solution of KLH (133 mg diluted to 6 ml with distilled water). The mixture was stirred for 24 h Departments of Chemical Pathology and Reat room temperature, dialysed against four changes productive Physiology, St. Bartholomew's Hospital, of 21 of distilled water over 48 h at 4 C , and the and sera from subjects undergoing adrenal stimulation tests from the Department of Endocrinology, product freeze-dried A degree of incorporation of 300 steroid residues St. Bartholomew's Hospital and the Endocrinology per protein molecule (assuming a mol. wt of Unit, Sabah Hospital, Kuwait. Whole, mixed saliva was collected. Subjects were 3,000,000 for KLH) was estimated for the cortisol immunogen by absorption measurements made in instructed to rinse their mouths with tap water and, 15 min later, to salivate into a polystyrene Universal 2 mol/l aqueous potassium hydroxide.
Polarisation fluoroimmunoassay of cortisol container. Salivary specimens were stored frozen (-20c~C) until required, when they were thawed, centrifuged for 10 rain (2,000 rpm) to sediment any debris or aggregated material, and the supernatants taken for assay. Specimens were obtained between 09.00 and 10.30 from normal volunteers in the Departments of Chemical Pathology and Reproductive Physiology, St. Bartholomew's Hospital. Saliva was also collected at various times during the course of one day from one normal adult and three children (ages 3, 4 and 6 years).
Polarisation fluorimeter A Model 4000 polarisation fluorimeter (SLM Instruments, Urbana, Illinois, U.S.A.), as described in detail elsewhere [18], was used. All measurements of fluorescence polarisation were made at ambient room temperature after transfer of sample solutions (l.5ml) from test tubes into a glass fluorimeter cuvette (10 × 10 ram) from Hellma, Westcliff-on-Sea, Essex, U.K. A single measurement based on total fluorescence emission was made in each case, and no corrections for background or blank signals were applied.
Assay diluent buffer Sodium phosphate buffer (100retool/I, pH 8.0) containing gelatin (1 g/l) and sodium azide (1 g/l) was used to prepare all working assay reagents.
Polarisation fluoroimmunoassay of serum cortisol Serum specimens (or standards) were pipetted (500pl) into glass centrifuge tubes and thoroughly vortexed with 4.5 ml of dichloromethane for 2 rain. The tubes were stood for l h then centrifuged for 10rain (2,000rpm). Aliquots (3 ml) of the organic (lower) layer were transferred to glass test tubes, the solvent evaporated under vacuum (Searle Vortex Evaporator, Buchler Instruments, Fort Lee, New Jersey, U.S.A.), and the residues taken up in 300/~1 of diluent buffer. Aliquots (100#1) of the reconstituted serum extracts were pipetted in duplicate into polystyrene test tubes, followed by 100 pl of fluorescein-labelled cortisol (40nmol/l), then 100/~1 of anti-cortisol serum (diluted 1: 600). After incubation for 30 rain at room temperature, 1.2 ml of diluent buffer was added and fluorescence polarisation measured.
Polarisation .fluoroimmunoassay of salivary cortisol Salivary specimens (or standards prepared in diluent buffer) were pipetted (1 ml) into glass centrifuge tubes and extracted with 5 ml of dichloromethane as described above. Aliquots (4.2 ml) of the organic layer after centrifugation were evaporated as above and taken up in 700/~1 of diluent buffer. Aliquots (300 kel) of the reconstituted salivary or buffer extracts were pipetted in duplicate into polystyrene test tubes, followed by 100~1 of fluorescein-labelled cortisol (40 nmol/l), then 100 ~tl
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of anti-cortisol serum (diluted 1 : 1,000). After incubation for 30min at room temperature, l ml of diluent buffer was added and fluorescence polarisation measured.
Established assay methods Serum cortisol assay by the Mattingly fluorimetric method[19] was performed in the Department of Chemical Pathology, St. Bartholomew's Hospital. Using the ~:SI-RIA kit, direct assay of serum cortisol was performed according to the manufacturer's instructions and direct assay of salivary cortisol by a modified procedure [13].
RESULTS
Choice of diluent b~4ffer Fluorescein-labelled cortisol at 2.67nmol/l (the final assay concentration) in sodium phosphate buffer containing only sodium azide gave a fluorescence polarisation signal of 0.060. In the presence of gelatin (1 g/l), bovine albumin (1 g/l), Triton X-100 detergent (1 ml/l), SDS (1 g/l) or a mixture of Triton X-100 and SDS (1 ml/I and 1 g/l) signals of 0.069, 0.105, 0.133, 0.199 and 0.103, respectively, were recorded. Diluent buffer containing gelatin was chosen for assay use.
Polarisation fluorimetrv The intensity of the fluorescence emission from 2.67 nmol/l labelled cortisol in diluent buffer, measured in the Model 4000 instrument[18], was 14.2 times that of the background from diluent buffer alone. Replicate immunoassay mixtures that gave an average polarisation signal of 0.138 were used in assessments of the precision of end-point determination in PIA. Twenty measurements made on one mixture in the glass cuvette gave a within-sample coefficient of variation (CV) of 0 8°4, while measurements made on 10 different mixtures gave a between-sample CV of 1.2'~. When similar assay mixtures were measured in 10 different polystyrene fluorimeter cuvettes, a between-sample CV of 16.5% was found.
Effects o[ serum and saliva The presence of 100#1 of cortisol-free human serum in a mixture (1.5 ml total volume) of labelled cortisol (final concentration 2.67 nmol/1) and anticortisol serum (final dilution 1:9,000) was sufficient to raise the measured polarisation from 0.143 to 0.270. Similarly, the polarisation of labelled cortisol in diluent buffer (1.5 ml total volume) increased from 0.069 to 0.090 when 300/~1 of normal saliva was present. Neither SDS[6] nor sodium salicylate [16, 20] at concentrations up to 40g/1 in diluent buffer significantly blocked the effects of serum or saliva in the PIA system. Correction for blank signals was also ineffective in eliminating the interference.
A . A . K . AL-ANSAR]et al.
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0.20
.~_ o o~ E 0.15
u-
0,10
'H*¢ I 0 30
I 150
I 600
I 3000
Serum cortisol (nmol/I)
Fig. 1. Standard curve for polarisation fluoroimmunoassay of serum cortisol.
Extraction of cort&ol fi'om serum and saliva Trace amounts of tritiated cortisol were added to normal human sera or salivary specimens, or to diluent buffer, and allowed to equilibrate for at least 4 h at room temperature. Recovery after extraction with dichloromethane ranged from 86 to 99% (mean 92%) for serum and from 92 to 105% (mean 99%) for saliva or buffer. The serum cortisol PIA was accordingly calibrated with standards prepared in cortisolfree human serum and the salivary assay with standards prepared in diluent buffer.
Polarisation fluoroimmunoassay o f serum cortisol Figure 1 shows a typical standard curve employing antiserum at an initial titre of 1:600. When normal sheep serum was substituted for the antiserum, it was found that non-specific tracer binding was undetectable at this dilution. (Antisera from the third or fourth bleeds of three of the other immunised sheep had corresponding titres of 1 : 300 and those from the other two animals of 1:150 and 1:90.) Although binding of tracer by antiserum was complete within 5 min, an assay incubation period of 30 min was routinely used for convenience. The dilution of incubation mixtures from 300#1 to 1.5 ml immediately prior to measurement of fluorescence polarisation did not affect the equilibrium position, as judged by stability of the polarisation signal with time. Cross-reactivities, calculated by the method of Abraham[21], in the cortisol PIA system were: l l-deoxycortisol 38%, prednisolone 10%, cortisone 9%, corticosterone 3%, 17ct-hydroxyprogesterone o/ progesterone 1%, and dexamethasone 6%. 3,0, Precision was assessed using three pools of selected patients' sera. Each was measured 10 times in one assay, giving mean results of 47, 225 and 950 nmol/l cortisol, with within-assay CVs of 9.5, 12.6 and 12.0%, respectively. To investigate the contribution of the extraction step to overall imprecision, each
pool was extracted in bulk and the reconstituted extract assayed 10 times, giving CVs of 4.9, 5.0 and o/ 6.4/o, respectively. Assay of each pool on each of 10 different days gave between-assay CVs of 11.0, 13.8 and 14.030, respectively. When the study was repeated using bulk serum extracts as above, between-assay CVs of 6.2, 5.8 and 7.2%, respectively, were found. The sensitivity of the PIA (minimal detectable concentration at the 95% confidence level) was estimated [22] as 18 nmol/l serum cortisol. The minimal detectable dose of cortisol was approx. 2 pmol per tube. Cortisol was added at levels of 30, 60, 150, 300, 600 and 1,200nmol/1 to a pool of patients' sera that contained 60 nmol/l endogenous steroid (as determined by PIA). Analytical recovery of the added cortisol by PIA was 98 + 4~(, ( m e a n _ SD). Assay results for 29 normal or pathological sera determined by PIA (y) and by the Mattingly fluorimetric method (x) were related by the leastsquares regression equation y = 0.88x + 26, with a correlation coefficient (r) of 0.86. PIA results (V) for 73 normal or pathological sera were related to those by RIA (x) by the equation y = 0.92x + 21, with r = 0.97 (Fig. 2).
Polarisation fluoroimmunoassay of salivary cortisol Figure 3 shows a typical standard curve. Two salivary specimens were measured 10 times in one assay, giving mean results of 9.0 and 47 nmol/l cortisol, with within-assay CVs of 3.5 and 4.3~,i, respectively. Assay of the same specimens on each of 10 different days gave between-assay CVs of 5.5 and 6.2%, respectively. The minimal detectable concentration of salivary cortisol was 2.8 nmol/l. The minimal detectable dose was approx. 1 pmol cortisol per tube.
10o0
o v
~, o .E ~ 500 --Y.g_ .~ ~.
0
g-'.° °
| 500
I 1000
Radioimrnunoassay (nmol/I)
Fig. 2. Correlation between cortisol levels in normal or pathological serum specimens determined by polarisation fluoroimmunoassay and by ~251-radioimmunoassay.
Polarisation fluoroimmunoassay of cortisol 0.20
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150
600
Salivary cortisol (nrnol/I)
Fig. 3. Standard curve for polarisation fluoroimmunoassay of salivary cortisol. P1A results (y) for 123 normal morning salivary specimens were related to those obtained by the modified RIA procedure (x) by the equation y = x - 0 . 2 , with r = 0.96 (Fig. 4). Most subjects, including children, had no difficulty in providing adequate volumes of saliva; to collect sufficient (1 ml) for cortisol PIA required typically about 5 min. Circadian variation of salivary cortisol was followed in one adult and three children (Fig. 5). DISCUSSION Gelatin was suitable for use as the detergent component in the PIA diluent buffer, as judged by the low polarisation from free tracer. Other potential detergents tended to bind the tracer themselves as indicated by elevated polarisation signals. The precision of PIA end-point determination was satisfactory when the glass cuvette was used (at least as good as typical counting precision in RIA). Although the use
..-,7
30 oo
o °
of disposable polystyrene cuvettes for both incubation and measurement would have simplified the assays, we found unacceptably high signal variability, presumably a consequence of the polarising properties of moulded and stressed plastic. (Disposable glass test tubes may be used for PIA measurements [9], but our instrument was not suitably adapted.) We found that simple direct PIA of either serum or salivary cortisol could not be achieved because of interferences with the measurement of fluorescence polarisation. Blank correction did not eliminate the interferences, implying that they were largely due to non-specific binding of the tracer by components of the biological fluids. The blocking agents that we tested were ineffective in preventing this binding in our assay system. It is possible, however+ that other blockers or different assay conditions could be found to enable non-extraction assay with blank correction [6], especially for saliva where blank signals are much lower than for serum. We chose to avoid interferences by the extraction of cortisol; it was then possible to complete the assays without the need for blank-correction procedures as required in previous serum cortisol P1A [5, 6]. Direct assay of serum cortisol is possible using solid-phase techniques with fluorescein-labelled antigen[16] or antibodies [20], when interferences are removed at a separation step. However, this requires additional manipulations and these procedures also involve one [16] or three [20] washes of the solid phase. The PIA gave standard curves adequately covering the normal morning ranges for serum and salivary cortisol, the latter having been established as 5 to 28 nmol/1 for our local adult population using the RIA kit method [13]. The serum sample requirement of 500pl is relatively high, however. To attain the necessary extra sensitivity for salivary cortisol assay, the amount of antibody in the assay system was decreased and the volume of specimen doubled. Although 1 ml of saliva is required per assay, most subjects, including children (Fig. 5), have no difficulty in providing adequate volumes.
•
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j
.+
el
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!
i
!
10
20
30
Radioimmu noassay (nmol/I)
Fig. 4. Correlation between cortisol levels in normal morning salivary specimens determined by polarisation fluoroimmunoassay and by 1251-radioimmunoassay.
0
i
i
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05.00 09.00
I
13.00 17.00
I
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Time (hoursl
Fig. 5. Circadian variation of salivary cortisol levels in one adult (broken line) and three children (solid lines).
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A . A . K . AL-ANSARI et al.
W i t h specimens from n o r m a l volunteers and subjects u n d e r g o i n g adrenal stimulation tests, results by serum cortisol PIA correlated acceptably with those by established fluorimetric a n d R1A methods, and the salivary PIA agreed well with the modified RIA. The a n t i s e r u m used, even t h o u g h it was raised against a 3-(O-carboxymethyl)oxime i m m u n o g e n [14, 15], showed high cross-reactivity with l l-deoxycortisol and with d e x a m e t h a s o n e . F o r assay of m e t y r a p o n e test or d e x a m e t h a s o n e - s u p p r e s s i o n test specimens, substitution of an a n t i s e r u m with more a p p r o p r i a t e specificity would be advisable to ensure optimal accuracy. The precision of the serum cortisol PIA was comparable with t h a t of the existing PIA m e t h o d s [5, 6] a n d of the solid-phase assay employing labelled antibodies[20]. However, the solid-phase fluoroi m m u n o a s s a y [16] has better precision t h a n any of these. W e showed that the extraction step m a d e a m a j o r c o n t r i b u t i o n to the overall imprecision of the serum cortisol PIA. The salivary PIA, in c o n t r a s t to the serum assay, h a d good precision indicating that cortisol may be extracted with greater ease and reproducibility from saliva as c o m p a r e d with serum. This finding agrees with the recovery results using tritiated cortisol, and p r e s u m a b l y reflects the lower level o f binding proteins in saliva. The availability of salivary cortisol assays opens up m a n y new possibilities [11, 13], notably in studies of children where serum sampling is i n a p p r o p r i a t e for physiological investigations a n d unsatisfactory (because of the risk of stress stimulation of cortisol secretion) for pathophysiological investigations. A preliminary study (Fig. 5) suggested that the circ a d i a n variation o f salivary cortisol in children is similar to that in adults; c o m p a r a b l e findings have been reported independently [23]. A. A. K. A1-Ansari gratefully acknowledges support by the Ministry of Health, Kuwait. We thank Corning Medical for the provision of RIA kits. Acknowledgements
REFERENCES
1. Dandliker W. B., Kelly R. J., Dandliker J., Farquhar J. and Levin J.: Fluorescence polarization immunoassay. Theory and experimental method, lmmunochemistry 10 (1973) 219 227. 2. Spencer R. D., Toledo F. B., Williams B. T. and Yoss N. L.: Design, construction, and two applications for an automated flow-cell polarization fluorometer with digital readout: enzyme-inhibitor (antitrypsin) assay and antigen-antibody (insulin-insulin antiserum) assay. Clin. Chem. 19 (1973) 838 844. 3. Dandliker W. B.: Investigation of immunochemical reactions by fluorescence polarization. In lmmunochemistry o f Proteins (Edited by M. Z. Atassi). Plenum Press, New York, Vol. 1 (1977) pp. 231-261. 4. Jolley M. E.: Fluorescence polarization immunoassay for the determination of therapeutic drug levels in human plasma. J. analyt. Toxicol. 5 (1981) 23(~240.
5. Kobayashi Y., Amitani K., Watanabe F. and Miyai K.: Fluorescence polarization immunoassay for cortisol. Clin. chim. Acta 92 (1979) 241-247. 6. Kobayashi Y., Miyai K., Tsubota N. and Watanabe F.: Direct fluorescence polarization immunoassay of serum cortisol. Steroids 34 (1979) 829-834. 7. Smith D. S., Hassan M. and Nargessi R. D.: Principles and practice of fluoroimmunoassay procedures. In Modern Fluorescence Spectroscopy (Edited by E. L. Wehry). Plenum Press, New York, Vol. 3 (1981) pp. 143-191. 8. Maeda H., Nakayama M., lwaoka D. and Sato T.: Assay of angiotensin I by fluorescence polarization method. In Kinins-ll: Biochemistry, Pathophysiology, and Clinical Aspects (Edited by S. Fujii, H. Moriya and T. Suzuki). Plenum Press, New York (1979) pp. 203 211. 9. Popelka S. R.. Miller D. M., Holen J. T. and Kelso D. M.: Fluorescence polarization immunoassay II. Analyzer for rapid, precise measurement of fluorescence polarization with use of disposable cuvettes. Clin. Chem. 27 (1981) 1198 1201. 10. Jolley M. E., Stroupc S. D., Schwenzer K. S., Wang C. J.. Lu-Steffes M., Hill H. D., Popelka S. R., Holen J. T. and Kelso D. M.: Fluorescence polarization immunoassay 1II. An automated system for therapeutic drug determination. Clin. Chem. 27 (1981 ) 1575-1579. 11. Riad-Fahmy D., Read G. F. and Walker R. F.: Salivary steroid assays for screening endocrine function. Postgrad. reed. J. 56, Suppl. I (1980) 75 78. 12. Jenkins G. N.: The Physiology and Biochemistry q / t h e Mouth. Blackwell, Oxford (1978) pp. 284-359. 13. Al-Ansari A. A. K., Perry L. A., Smith D. S. and Landon J.: Salivary cortisol determination: adaptation of a commercial serum cortisol kit. Ann. clin. Biochem. 19 (1982) 163 166. 14. Fahmy D., Read G. F. and Hillier S. G.: Some observations on the determination of cortisol in human plasma by radioimmunoassay using antisera against cortisol-3-BSA. Steroids 26 (1975) 267-280. 15. Hasler M. J., Painter K. and Niswender G. D.: An ~:SI-labeled cortisol radioimmunoassay in which serum binding proteins are enzymatically denatured. Clin. Chem. 22 (1976) 185~1854. 16. Pourfarzaneh M., White G. W., Landon J. and Smith D. S.: Cortisol directly determined in serum by fluoroimmunoassay with magnetizable solid phase. Clin. Chem. 26 (1980) 730-733. 17. Hamman B. 1. and Martin M. M.: Direct spectrophotometric quantitation of steroid chromatograms I. The measurement of corticosteroids. Analvt. Biochem. 20 (1967) 423 43 I. 18. Sidki A, M., Pourfarzaneh M., Rowell F. J. and Smith D. S.: Direct determination of phenobarbital in serum or plasma by polarization fluoroimmunoassay. Ther, Drug. Monit. 4 (1982) 397-403. 19. Mattingly D.: A simple fluorimetric method lbr the estimation of tYee I I-hydroxycorticoids in human plasma. J. olin. Path. 15 (1962) 374~379. 20. Kobayashi Y., Yahata M., Watanabe F. and Miyai K.: A solid phase fluoroimmunoassay of serum cortisol. J. steroid Biochem. 16 (1982) 521-524. 21. Abraham G. E.: Solid-phase radioimmunoassay of estradiol-17fl. J. olin. Endocr. 29 (1969) 866-870. 22. Rodbard D.: Statistical estimation of minimal detectable concentration ("sensitivity") for radioligand assays. AnaO, t. Biochem. 90 (1978) l q 2 . 23. Hiramatsu R.: Direct assay of cortisol in human saliva by solid phase radioimmunoassay and its clinical applications. Clin. chim. Acta 117 (t981) 239 249.
0022-4731/83 $3.00+0.00 Copyright .~'~ 1983 Pergamon Press Ltd
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POLARISATION F L U O R O I M M U N O A S S A Y OF SERUM A N D SALIVARY CORTISOL A. A. K. AL-ANSARI*, D. S. SMITH and J. LANDON Department of Chemical Pathology, St. Bartholomew's Hospital, London ECI, U.K. (Received 8 December 1982)
Summary Polarisation fluoroimmunoassays for serum and salivary cortisol were developed using the steroid labelled at the 3-position with fluorescein and antiserum raised in sheep. To avoid interfering factors present in the biological fluids, cortisol was extracted with dichloromethane prior to assay, which was then accomplished without the need for any further separation or blank-correction procedures. The methods were sufficiently sensitive to cover the nomml morning ranges for serum and salivary cortisoI and correlated acceptably with established fluorimetric and radioimmunoassay techniques. The salivary cortisol assay was more precise than the serum assay, reflecting the greater ease with which cortisol may be extracted from saliva. The potential usefulness of salivary cortisol assays was illustrated by a preliminary study showing that the circadian variation in three children was similar to that in an adult.
INTRODUCTION
Polarisation fluoroimmunoassay (PIA), introduced in 1973 by the groups of Dandliker[l] and Spencer[2], is one of the simplest immunoassay techniques. It is based on the increase in polarisation of fluorescence that occurs when a relatively small fluorescentlabelled antigen is b o u n d by a large antibody molecule [3] and hence is best suited to the assay of haptens such as drugs [4] and steroids[5, 6]. PIA requires no separation of free and bound fractions of the labelled antigen and employs stable and hazardfree reagents. PIA has not been widely applied in routine clinical practice for two main reasons. First, its sensitivity is limited by interfering factors that may be present intrinsically or abnormally in serum or plasma; these include fluorophores, haemoglobin and lightscattering materials that may interfere spectroscopically, and proteins (notably albumin) that may bind tracers non-specifically [7]. Secondly, suitable polarisation fluorimeters for precise and convenient end-point measurement have not been readily available. However, both manual[8,9] and fully automated [10] instrument systems have recently been described. Miyai, Watanabe and their colleagues have reported PIA methods for serum cortisol that employ steroid labelled at the 21-position with fluorescein and rabbit antibodies raised against a cortisol-21hemi-succinate-protein conjugate. In one system [5] interfering serum proteins are precipitated with methanol in a pre-treatment step, while in another [6] sodium dodecyl sulphate (SDS) is used as a blocking agent to prevent non-specific serum-protein binding *Present address: Endocrinology Unit, Sabah Hospital, P.O. Box 4078~ Safat, Kuwait. sl~ 19/4
(~
of the tracer and enable direct (non-extraction) assay. In both cases, correction for residual serum interferences (blank signals) is necessary and is achieved by making polarisation measurements both before and after the addition of antibody to each assay mixture. There are potential advantages in the measurement of steroids in saliva as opposed to serum or plasma[l 1]. First, salivary levels generally reflect those of the free (non-protein-bound) steroid fraction in the circulation, which is believed to be directly related to physiological effects. Secondly, saliva may be easily collected non-invasively and without stress and hence is suitable for studies on children and in diagnostic investigations requiring serial sampling. Thirdly, saliva contains relatively little protein, of which only traces are of plasma origin[12], thus protein matrix effects can be expected to be less of a problem in salivary immunoassays. Salivary cortisol levels average only about 50,~, of total serum levels and therefore relatively more sensitive assays are required. We have described an adaptation of a commercially-available radioimmunoassay (RIA) kit method for serum or plasma cortisol that permits direct assay of salivary specimens[13]. To study the PIA of serum and salivary cortisol, we raised antiserum against cortisol conjugated to carrier protein at the 3-position because this has generally been found to result in better specificity than when 21-1inked immunogens are used [14, 15]. Since antiserum consumption in PIA is high (relative to that in RIA) we used sheep as experimental animals rather than rabbits. MATERIALS
Cortisol and other steroids, N-hydroxysuccinimide, l-ethyl-3(3-dimethylaminopropyl) carbo-
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diimide hydrochloride (EDC), tetrazolium blue and bovine albumin (type A4503) were obtained from Sigma, Poole, Dorset, U.K.: organic solvents (all Analar grade and used without further purification), gelatin, sodium salicylate, SDS and Triton X-100 from BDH, Poole, Dorset, U.K.: silica-gel thin-layer chromatography (TLC) sheets (DC-Alufotien Kieselgel 60 F254, Art. 5554) from Merck, Darmstadt, F.R.G.: keyhole limpet hemocyanin (KLH), 133 g/l in 50!}i;glycerol, from Calbiochem, San Diego, California, U.S.A.: complete and incomplete Freund's adjuvant from Difco, West Molesey, Surrey, U.K.; activated charcoal from Hopkin and Williams, Chadwell Heath, Essex, U.K.; pooled normal human serum from ILS, Newbury Street, London ECI, U.K.; and [1,2,6,7-3H] cortisol from Amersham International, Amersham, Buckinghamshire, U.K. Glass centrifuge tubes ( l l 0 x 17ram) were obtained from Coming, Stone, Staffordshire, U.K.; glass test tubes (76 × 16mm) from Hoslab, llford, Essex, U.K.: polystyrene test tubes type LP3 (63.5 x 9.5 mm) from Luckham, Burgess Hill, Sussex, U.K.; polystyrene Universal containers (90 x 24 mm) from Sterilin, Richmond, Surrey, U.K.; and polystyrene fluorimeter cuvettes No. 67.754 (10 x 10 ram) from Sarstedt, Leicester, Leicestershire, U.K. Cortisol solid-phase ~2SI-RIA (Immophase ~M) kits were provided by Coming Medical Halstead, Essex, U.K, METHODS
Cortisol immunogen
Anti-eortiso[ serum
Six mature female Border-Leicester cross sheep were each immunised with 2.5 mg of the cortisolKLH conjugate in 8 ml of an emulsion of water and complete Freund's adjuvant (1:3 parts by volume). The immunogen was divided between several intramuscular and subcutaneous sites. The animals were given booster injections 20 days later in the same way, except that half the amount of immunogen was used and the adjuvant was incomplete Freund's. Subsequently the sheep were boosted every 20 days and bleeds taken 10 days after alternate boosts. Antiserum obtained from the fourth bleed of one sheep was used in the studies reported in detail below. Fluoreseein-labelled eortisol
Fluoresceinthiocarbamyl ethylenediamine, prepared by reaction of fluorescein isothiocyanate with ethytenediamine, was conjugated with cortisol-3-(Ocarboxymethyl) oxime by an active-ester procedure and the product purified by TLC on silica gel as described previously [16]. Labelled cortisol, eluted from silica gel with methanol, was stored at - 2 0 C . Its concentration was estimated spectrophotometrically [16]. Cortisol-l?ee human serum
Pooled normal human serum was mixed with activated charcoal (100 g/l) by gentle rotation for 18 h at 37 C, followed by centrifugation for 45 min at 4 C (10,000 rpm). Charcoal fines were removed from the supernatant by pressure filtration through membranes with a series of pore sizes (2, 1, 0.8, 0.45 and 0.22/~m) using equipment from Sartorius, Belmont, Surrey, U.K. When a trace amount of tritiated cortisol was equilibrated with pooled normal human serum for 4 h at room temperature prior to treatment, it was found that 99.6'},] of the cortisol was removed by the charcoal. When tested by the RIA kit method, no cortisol was detected in the charcoal-treated serum.
Cortisol-3-(O-carboxymethyl)oxime, prepared by reaction of cortisol with carboxymethoxylamine hemihydrochloride as described previously [16], was added (35 mg) to a solution of N-hydroxysuccinimide ( l l . 5 m g ) and EDC (15.3rag) in 3.5ml of dimethylformamide. After 45 min at room temperature, TLC of a small aliquot of the reaction mixture on silica gel developed with ethyl acetate methanol-glacial acetic acid (90:8:2 by vol) indicated the disappearance of the oxime starting material (Rt 0.35 ) and its conversion to the active ester (R~- Cortisol serum standards 0.75). Steroid-containing spots were visualized on the Cortisol dissolved in methanol (1 mmol/l) was adTLC plate by spraying with tetrazolium blue reagent [17] to give the blue colour characteristic of ded to cortisol-free human serum. The standards the dihydroxyacetone group. The reaction mixture were stored in aliquots at - 2 0 C. was diluted with dimethylformamide (I.5 ml) and Specimen collection distilled water (5 ml), then immediately added to a Normal sera were obtained from volunteers in the well-stirred solution of KLH (133 mg diluted to 6 ml with distilled water). The mixture was stirred for 24 h Departments of Chemical Pathology and Reat room temperature, dialysed against four changes productive Physiology, St. Bartholomew's Hospital, of 21 of distilled water over 48 h at 4 C , and the and sera from subjects undergoing adrenal stimulation tests from the Department of Endocrinology, product freeze-dried A degree of incorporation of 300 steroid residues St. Bartholomew's Hospital and the Endocrinology per protein molecule (assuming a mol. wt of Unit, Sabah Hospital, Kuwait. Whole, mixed saliva was collected. Subjects were 3,000,000 for KLH) was estimated for the cortisol immunogen by absorption measurements made in instructed to rinse their mouths with tap water and, 15 min later, to salivate into a polystyrene Universal 2 mol/l aqueous potassium hydroxide.
Polarisation fluoroimmunoassay of cortisol container. Salivary specimens were stored frozen (-20c~C) until required, when they were thawed, centrifuged for 10 rain (2,000 rpm) to sediment any debris or aggregated material, and the supernatants taken for assay. Specimens were obtained between 09.00 and 10.30 from normal volunteers in the Departments of Chemical Pathology and Reproductive Physiology, St. Bartholomew's Hospital. Saliva was also collected at various times during the course of one day from one normal adult and three children (ages 3, 4 and 6 years).
Polarisation fluorimeter A Model 4000 polarisation fluorimeter (SLM Instruments, Urbana, Illinois, U.S.A.), as described in detail elsewhere [18], was used. All measurements of fluorescence polarisation were made at ambient room temperature after transfer of sample solutions (l.5ml) from test tubes into a glass fluorimeter cuvette (10 × 10 ram) from Hellma, Westcliff-on-Sea, Essex, U.K. A single measurement based on total fluorescence emission was made in each case, and no corrections for background or blank signals were applied.
Assay diluent buffer Sodium phosphate buffer (100retool/I, pH 8.0) containing gelatin (1 g/l) and sodium azide (1 g/l) was used to prepare all working assay reagents.
Polarisation fluoroimmunoassay of serum cortisol Serum specimens (or standards) were pipetted (500pl) into glass centrifuge tubes and thoroughly vortexed with 4.5 ml of dichloromethane for 2 rain. The tubes were stood for l h then centrifuged for 10rain (2,000rpm). Aliquots (3 ml) of the organic (lower) layer were transferred to glass test tubes, the solvent evaporated under vacuum (Searle Vortex Evaporator, Buchler Instruments, Fort Lee, New Jersey, U.S.A.), and the residues taken up in 300/~1 of diluent buffer. Aliquots (100#1) of the reconstituted serum extracts were pipetted in duplicate into polystyrene test tubes, followed by 100 pl of fluorescein-labelled cortisol (40nmol/l), then 100/~1 of anti-cortisol serum (diluted 1: 600). After incubation for 30 rain at room temperature, 1.2 ml of diluent buffer was added and fluorescence polarisation measured.
Polarisation .fluoroimmunoassay of salivary cortisol Salivary specimens (or standards prepared in diluent buffer) were pipetted (1 ml) into glass centrifuge tubes and extracted with 5 ml of dichloromethane as described above. Aliquots (4.2 ml) of the organic layer after centrifugation were evaporated as above and taken up in 700/~1 of diluent buffer. Aliquots (300 kel) of the reconstituted salivary or buffer extracts were pipetted in duplicate into polystyrene test tubes, followed by 100~1 of fluorescein-labelled cortisol (40 nmol/l), then 100 ~tl
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of anti-cortisol serum (diluted 1 : 1,000). After incubation for 30min at room temperature, l ml of diluent buffer was added and fluorescence polarisation measured.
Established assay methods Serum cortisol assay by the Mattingly fluorimetric method[19] was performed in the Department of Chemical Pathology, St. Bartholomew's Hospital. Using the ~:SI-RIA kit, direct assay of serum cortisol was performed according to the manufacturer's instructions and direct assay of salivary cortisol by a modified procedure [13].
RESULTS
Choice of diluent b~4ffer Fluorescein-labelled cortisol at 2.67nmol/l (the final assay concentration) in sodium phosphate buffer containing only sodium azide gave a fluorescence polarisation signal of 0.060. In the presence of gelatin (1 g/l), bovine albumin (1 g/l), Triton X-100 detergent (1 ml/l), SDS (1 g/l) or a mixture of Triton X-100 and SDS (1 ml/I and 1 g/l) signals of 0.069, 0.105, 0.133, 0.199 and 0.103, respectively, were recorded. Diluent buffer containing gelatin was chosen for assay use.
Polarisation fluorimetrv The intensity of the fluorescence emission from 2.67 nmol/l labelled cortisol in diluent buffer, measured in the Model 4000 instrument[18], was 14.2 times that of the background from diluent buffer alone. Replicate immunoassay mixtures that gave an average polarisation signal of 0.138 were used in assessments of the precision of end-point determination in PIA. Twenty measurements made on one mixture in the glass cuvette gave a within-sample coefficient of variation (CV) of 0 8°4, while measurements made on 10 different mixtures gave a between-sample CV of 1.2'~. When similar assay mixtures were measured in 10 different polystyrene fluorimeter cuvettes, a between-sample CV of 16.5% was found.
Effects o[ serum and saliva The presence of 100#1 of cortisol-free human serum in a mixture (1.5 ml total volume) of labelled cortisol (final concentration 2.67 nmol/1) and anticortisol serum (final dilution 1:9,000) was sufficient to raise the measured polarisation from 0.143 to 0.270. Similarly, the polarisation of labelled cortisol in diluent buffer (1.5 ml total volume) increased from 0.069 to 0.090 when 300/~1 of normal saliva was present. Neither SDS[6] nor sodium salicylate [16, 20] at concentrations up to 40g/1 in diluent buffer significantly blocked the effects of serum or saliva in the PIA system. Correction for blank signals was also ineffective in eliminating the interference.
A . A . K . AL-ANSAR]et al.
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I 150
I 600
I 3000
Serum cortisol (nmol/I)
Fig. 1. Standard curve for polarisation fluoroimmunoassay of serum cortisol.
Extraction of cort&ol fi'om serum and saliva Trace amounts of tritiated cortisol were added to normal human sera or salivary specimens, or to diluent buffer, and allowed to equilibrate for at least 4 h at room temperature. Recovery after extraction with dichloromethane ranged from 86 to 99% (mean 92%) for serum and from 92 to 105% (mean 99%) for saliva or buffer. The serum cortisol PIA was accordingly calibrated with standards prepared in cortisolfree human serum and the salivary assay with standards prepared in diluent buffer.
Polarisation fluoroimmunoassay o f serum cortisol Figure 1 shows a typical standard curve employing antiserum at an initial titre of 1:600. When normal sheep serum was substituted for the antiserum, it was found that non-specific tracer binding was undetectable at this dilution. (Antisera from the third or fourth bleeds of three of the other immunised sheep had corresponding titres of 1 : 300 and those from the other two animals of 1:150 and 1:90.) Although binding of tracer by antiserum was complete within 5 min, an assay incubation period of 30 min was routinely used for convenience. The dilution of incubation mixtures from 300#1 to 1.5 ml immediately prior to measurement of fluorescence polarisation did not affect the equilibrium position, as judged by stability of the polarisation signal with time. Cross-reactivities, calculated by the method of Abraham[21], in the cortisol PIA system were: l l-deoxycortisol 38%, prednisolone 10%, cortisone 9%, corticosterone 3%, 17ct-hydroxyprogesterone o/ progesterone 1%, and dexamethasone 6%. 3,0, Precision was assessed using three pools of selected patients' sera. Each was measured 10 times in one assay, giving mean results of 47, 225 and 950 nmol/l cortisol, with within-assay CVs of 9.5, 12.6 and 12.0%, respectively. To investigate the contribution of the extraction step to overall imprecision, each
pool was extracted in bulk and the reconstituted extract assayed 10 times, giving CVs of 4.9, 5.0 and o/ 6.4/o, respectively. Assay of each pool on each of 10 different days gave between-assay CVs of 11.0, 13.8 and 14.030, respectively. When the study was repeated using bulk serum extracts as above, between-assay CVs of 6.2, 5.8 and 7.2%, respectively, were found. The sensitivity of the PIA (minimal detectable concentration at the 95% confidence level) was estimated [22] as 18 nmol/l serum cortisol. The minimal detectable dose of cortisol was approx. 2 pmol per tube. Cortisol was added at levels of 30, 60, 150, 300, 600 and 1,200nmol/1 to a pool of patients' sera that contained 60 nmol/l endogenous steroid (as determined by PIA). Analytical recovery of the added cortisol by PIA was 98 + 4~(, ( m e a n _ SD). Assay results for 29 normal or pathological sera determined by PIA (y) and by the Mattingly fluorimetric method (x) were related by the leastsquares regression equation y = 0.88x + 26, with a correlation coefficient (r) of 0.86. PIA results (V) for 73 normal or pathological sera were related to those by RIA (x) by the equation y = 0.92x + 21, with r = 0.97 (Fig. 2).
Polarisation fluoroimmunoassay of salivary cortisol Figure 3 shows a typical standard curve. Two salivary specimens were measured 10 times in one assay, giving mean results of 9.0 and 47 nmol/l cortisol, with within-assay CVs of 3.5 and 4.3~,i, respectively. Assay of the same specimens on each of 10 different days gave between-assay CVs of 5.5 and 6.2%, respectively. The minimal detectable concentration of salivary cortisol was 2.8 nmol/l. The minimal detectable dose was approx. 1 pmol cortisol per tube.
10o0
o v
~, o .E ~ 500 --Y.g_ .~ ~.
0
g-'.° °
| 500
I 1000
Radioimrnunoassay (nmol/I)
Fig. 2. Correlation between cortisol levels in normal or pathological serum specimens determined by polarisation fluoroimmunoassay and by ~251-radioimmunoassay.
Polarisation fluoroimmunoassay of cortisol 0.20
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0.10 ' ..~i" I
0
3.2
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i
i
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150
600
Salivary cortisol (nrnol/I)
Fig. 3. Standard curve for polarisation fluoroimmunoassay of salivary cortisol. P1A results (y) for 123 normal morning salivary specimens were related to those obtained by the modified RIA procedure (x) by the equation y = x - 0 . 2 , with r = 0.96 (Fig. 4). Most subjects, including children, had no difficulty in providing adequate volumes of saliva; to collect sufficient (1 ml) for cortisol PIA required typically about 5 min. Circadian variation of salivary cortisol was followed in one adult and three children (Fig. 5). DISCUSSION Gelatin was suitable for use as the detergent component in the PIA diluent buffer, as judged by the low polarisation from free tracer. Other potential detergents tended to bind the tracer themselves as indicated by elevated polarisation signals. The precision of PIA end-point determination was satisfactory when the glass cuvette was used (at least as good as typical counting precision in RIA). Although the use
..-,7
30 oo
o °
of disposable polystyrene cuvettes for both incubation and measurement would have simplified the assays, we found unacceptably high signal variability, presumably a consequence of the polarising properties of moulded and stressed plastic. (Disposable glass test tubes may be used for PIA measurements [9], but our instrument was not suitably adapted.) We found that simple direct PIA of either serum or salivary cortisol could not be achieved because of interferences with the measurement of fluorescence polarisation. Blank correction did not eliminate the interferences, implying that they were largely due to non-specific binding of the tracer by components of the biological fluids. The blocking agents that we tested were ineffective in preventing this binding in our assay system. It is possible, however+ that other blockers or different assay conditions could be found to enable non-extraction assay with blank correction [6], especially for saliva where blank signals are much lower than for serum. We chose to avoid interferences by the extraction of cortisol; it was then possible to complete the assays without the need for blank-correction procedures as required in previous serum cortisol P1A [5, 6]. Direct assay of serum cortisol is possible using solid-phase techniques with fluorescein-labelled antigen[16] or antibodies [20], when interferences are removed at a separation step. However, this requires additional manipulations and these procedures also involve one [16] or three [20] washes of the solid phase. The PIA gave standard curves adequately covering the normal morning ranges for serum and salivary cortisol, the latter having been established as 5 to 28 nmol/1 for our local adult population using the RIA kit method [13]. The serum sample requirement of 500pl is relatively high, however. To attain the necessary extra sensitivity for salivary cortisol assay, the amount of antibody in the assay system was decreased and the volume of specimen doubled. Although 1 ml of saliva is required per assay, most subjects, including children (Fig. 5), have no difficulty in providing adequate volumes.
•
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j
.+
el
•
1479
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•
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!
i
!
10
20
30
Radioimmu noassay (nmol/I)
Fig. 4. Correlation between cortisol levels in normal morning salivary specimens determined by polarisation fluoroimmunoassay and by 1251-radioimmunoassay.
0
i
i
I
05.00 09.00
I
13.00 17.00
I
21.00
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Time (hoursl
Fig. 5. Circadian variation of salivary cortisol levels in one adult (broken line) and three children (solid lines).
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A . A . K . AL-ANSARI et al.
W i t h specimens from n o r m a l volunteers and subjects u n d e r g o i n g adrenal stimulation tests, results by serum cortisol PIA correlated acceptably with those by established fluorimetric a n d R1A methods, and the salivary PIA agreed well with the modified RIA. The a n t i s e r u m used, even t h o u g h it was raised against a 3-(O-carboxymethyl)oxime i m m u n o g e n [14, 15], showed high cross-reactivity with l l-deoxycortisol and with d e x a m e t h a s o n e . F o r assay of m e t y r a p o n e test or d e x a m e t h a s o n e - s u p p r e s s i o n test specimens, substitution of an a n t i s e r u m with more a p p r o p r i a t e specificity would be advisable to ensure optimal accuracy. The precision of the serum cortisol PIA was comparable with t h a t of the existing PIA m e t h o d s [5, 6] a n d of the solid-phase assay employing labelled antibodies[20]. However, the solid-phase fluoroi m m u n o a s s a y [16] has better precision t h a n any of these. W e showed that the extraction step m a d e a m a j o r c o n t r i b u t i o n to the overall imprecision of the serum cortisol PIA. The salivary PIA, in c o n t r a s t to the serum assay, h a d good precision indicating that cortisol may be extracted with greater ease and reproducibility from saliva as c o m p a r e d with serum. This finding agrees with the recovery results using tritiated cortisol, and p r e s u m a b l y reflects the lower level o f binding proteins in saliva. The availability of salivary cortisol assays opens up m a n y new possibilities [11, 13], notably in studies of children where serum sampling is i n a p p r o p r i a t e for physiological investigations a n d unsatisfactory (because of the risk of stress stimulation of cortisol secretion) for pathophysiological investigations. A preliminary study (Fig. 5) suggested that the circ a d i a n variation o f salivary cortisol in children is similar to that in adults; c o m p a r a b l e findings have been reported independently [23]. A. A. K. A1-Ansari gratefully acknowledges support by the Ministry of Health, Kuwait. We thank Corning Medical for the provision of RIA kits. Acknowledgements
REFERENCES
1. Dandliker W. B., Kelly R. J., Dandliker J., Farquhar J. and Levin J.: Fluorescence polarization immunoassay. Theory and experimental method, lmmunochemistry 10 (1973) 219 227. 2. Spencer R. D., Toledo F. B., Williams B. T. and Yoss N. L.: Design, construction, and two applications for an automated flow-cell polarization fluorometer with digital readout: enzyme-inhibitor (antitrypsin) assay and antigen-antibody (insulin-insulin antiserum) assay. Clin. Chem. 19 (1973) 838 844. 3. Dandliker W. B.: Investigation of immunochemical reactions by fluorescence polarization. In lmmunochemistry o f Proteins (Edited by M. Z. Atassi). Plenum Press, New York, Vol. 1 (1977) pp. 231-261. 4. Jolley M. E.: Fluorescence polarization immunoassay for the determination of therapeutic drug levels in human plasma. J. analyt. Toxicol. 5 (1981) 23(~240.
5. Kobayashi Y., Amitani K., Watanabe F. and Miyai K.: Fluorescence polarization immunoassay for cortisol. Clin. chim. Acta 92 (1979) 241-247. 6. Kobayashi Y., Miyai K., Tsubota N. and Watanabe F.: Direct fluorescence polarization immunoassay of serum cortisol. Steroids 34 (1979) 829-834. 7. Smith D. S., Hassan M. and Nargessi R. D.: Principles and practice of fluoroimmunoassay procedures. In Modern Fluorescence Spectroscopy (Edited by E. L. Wehry). Plenum Press, New York, Vol. 3 (1981) pp. 143-191. 8. Maeda H., Nakayama M., lwaoka D. and Sato T.: Assay of angiotensin I by fluorescence polarization method. In Kinins-ll: Biochemistry, Pathophysiology, and Clinical Aspects (Edited by S. Fujii, H. Moriya and T. Suzuki). Plenum Press, New York (1979) pp. 203 211. 9. Popelka S. R.. Miller D. M., Holen J. T. and Kelso D. M.: Fluorescence polarization immunoassay II. Analyzer for rapid, precise measurement of fluorescence polarization with use of disposable cuvettes. Clin. Chem. 27 (1981) 1198 1201. 10. Jolley M. E., Stroupc S. D., Schwenzer K. S., Wang C. J.. Lu-Steffes M., Hill H. D., Popelka S. R., Holen J. T. and Kelso D. M.: Fluorescence polarization immunoassay 1II. An automated system for therapeutic drug determination. Clin. Chem. 27 (1981 ) 1575-1579. 11. Riad-Fahmy D., Read G. F. and Walker R. F.: Salivary steroid assays for screening endocrine function. Postgrad. reed. J. 56, Suppl. I (1980) 75 78. 12. Jenkins G. N.: The Physiology and Biochemistry q / t h e Mouth. Blackwell, Oxford (1978) pp. 284-359. 13. Al-Ansari A. A. K., Perry L. A., Smith D. S. and Landon J.: Salivary cortisol determination: adaptation of a commercial serum cortisol kit. Ann. clin. Biochem. 19 (1982) 163 166. 14. Fahmy D., Read G. F. and Hillier S. G.: Some observations on the determination of cortisol in human plasma by radioimmunoassay using antisera against cortisol-3-BSA. Steroids 26 (1975) 267-280. 15. Hasler M. J., Painter K. and Niswender G. D.: An ~:SI-labeled cortisol radioimmunoassay in which serum binding proteins are enzymatically denatured. Clin. Chem. 22 (1976) 185~1854. 16. Pourfarzaneh M., White G. W., Landon J. and Smith D. S.: Cortisol directly determined in serum by fluoroimmunoassay with magnetizable solid phase. Clin. Chem. 26 (1980) 730-733. 17. Hamman B. 1. and Martin M. M.: Direct spectrophotometric quantitation of steroid chromatograms I. The measurement of corticosteroids. Analvt. Biochem. 20 (1967) 423 43 I. 18. Sidki A, M., Pourfarzaneh M., Rowell F. J. and Smith D. S.: Direct determination of phenobarbital in serum or plasma by polarization fluoroimmunoassay. Ther, Drug. Monit. 4 (1982) 397-403. 19. Mattingly D.: A simple fluorimetric method lbr the estimation of tYee I I-hydroxycorticoids in human plasma. J. olin. Path. 15 (1962) 374~379. 20. Kobayashi Y., Yahata M., Watanabe F. and Miyai K.: A solid phase fluoroimmunoassay of serum cortisol. J. steroid Biochem. 16 (1982) 521-524. 21. Abraham G. E.: Solid-phase radioimmunoassay of estradiol-17fl. J. olin. Endocr. 29 (1969) 866-870. 22. Rodbard D.: Statistical estimation of minimal detectable concentration ("sensitivity") for radioligand assays. AnaO, t. Biochem. 90 (1978) l q 2 . 23. Hiramatsu R.: Direct assay of cortisol in human saliva by solid phase radioimmunoassay and its clinical applications. Clin. chim. Acta 117 (t981) 239 249.