J O U R N A L OF B I O L U M I N E S C E N C E AND C H E M I L U M I N E S C E N C E VOL 5 5-10
(1990)
Isoluminol as a Marker in Direct Chemiluminescence Immunoassays for Steroid Hormones J. De Boever", F. Kohen', D. Leyseele and
D.Vandekerckhove
Department of Obstetrics and Gynecology, University Hospital, B-9000 Gent, Belgium 'Department of Hormone Research, The Weizmann institute of Science, IL-76100 Rehovot, Israel
We have used isoluminol steroid hormone conjugates in competitive heterogeneous chemiluminescence immunoassays (CIA) for estradiol, progesterone and estriol in body fluids. The assays did not require prior extraction of the steroid hormone with an organic solvent. Polyclonal or monoclonal antibodies, covalently coupled t o polyacrylamide microspheres or adsorbed onto the plastic surface of microtritation plates via an intermediate second antibody, were used as solid-phase. The latter solid-phase system allows rapid processing of incubation mixtures. Sensitivie and reliable direct ClAs for estradiol in serum, progesterone in serum and in saliva and for estriol in saliva have been developed and their clinical utility has been assessed. Sensitivities ranged from 0.54 t o 0.04 nmol/l, and precision (YOCV) ranged from 10% t o 16%. Keywords: CIA; steroids; isolurninol
INTRODUCTION It is now generally accepted that nonradioisotopic immunoassays (NIIA) with similar sensitivity, reliability and performance as radioimmunoassay (RIA) are possible for a large number of biologically and clinically important molecules. For the elaboration of direct immunoassays for steroid hormones the most successful nonradioisotopic label has been isoluminol. Aminoalkylethyl derivatives of this chemiluminescent molecule were first introduced in a competitive protein-binding, assay for biotin by Schroeder et al. (1976, 1978a), and in immunoassay for steroid hormones by Kohen and colleagues (Kohen et al., 1979; Pazzagli et al., 1981a, b). Oxidation of these *Author for correspondence
0884-39961901010005-06$05 .OO @ 1990 by John Wiley & Sons, Ltd.
isoluminol derivatives under fixed conditions produces light in good quantum yield (1-2%). Measurement of the photons generated during this chemical oxidative reaction can be performed quickly and reliably using a simple photon counter. An advantage of chemiluminescence is that although the quantum yield is low, molecules yielding measurable light are 'seen' in the 10 seconds measuring time. In contrast, in RIA a much smaller fraction of total available counts are measured in the measuring time 1-5 minutes) needed for counting decaying 51- and 3Hradioisotopes. On a theoretical basis this would allow for better sensitivity of CIA compared to RIA. Other important advantages of an isolumino1 derivative is its facile covalent coupling to a variety of molecules (including haptens), the
i
6
J. DE BOEVER, F. KOHEN, D. LEYSEELE AND D. VANDEKERCKHOVE
stability of the label and labelled products, and the lack of health and environmental risks. The first chemiluminescence immunoassay for a steroid sex hormone used aminoalklethyl isoluminol as a label (Kohen et af.,1979). It was a non-separation or ‘homogeneous’ assay with intrinsically less sensitivity (25 pgitube) than the heterogeneous type (below 10 pgitube). Blood serum and plasma components quench the light production in the chemiluminescent reaction (Schroeder et al., 1978a,b), and thus prior extraction of steroid from blood was required. Subsequently other isoluminol-based CIAs for steroid hormones were developed. Most included an extraction step and all were of the more sensitive heterogeneous type. Sensitivities ranged between 2 and 10pgitube (De Boever et af.,1983, 1984; Kim et af., 1982; Klingler et al., 1983; Kohen et af., 1981; Pazzagli et af., 1981c, d , 1982). Except for two assays based on separation of bound and free hormone fractions with dextrancoated charcoal (DCC) (Pazzagli et af., 1981c, d), these assays used solid phase antibodies either coated to tubes or covalently linked to polyacrylamide beads. We have developed several direct CIAs for steroid sex hormones in blood and in saliva, using solid-phase antibodies and a heterogeneous assay type system. Serum and plasma are the most frequently used biological materials in the clinical laboratory for the measurement of steroid hormone concentrations, but in some instances saliva may be a valuable alternative. It reflects the levels of steroids in free condition, i.e. not bound to sex hormone-blinding globulin, SHBG, and corticosteroid-binding globulin, CBG (Smith et af., 1979; Vining et af., 1983), and it can be obtained easily and stress-free even by the patient at home. Saliva is especially attractive if repeated hormone determinations are required for monitoring and follow-up, for example during ovulation induction, to monitor glucocorticoid therapy in congenital adrenal hyperplasia (Vining and McGinley, 1987; Riad-Fahmy et af., 1982) and in pathological pregnancy.
were purified by thin layer chromatography (Pazzagli et af., 1981a, b). Between 1Opg and lOOpg of the conjugate were added to the tubes or microtitre plate wells. Antibodies (Bauminger et af., 1974; Kohen and Lichter, 1986; Kohen et af., 1975) or their purified gamma globulin fractions (Kim et al., 1982; Weerasekera et al., 1982) were bound in solid phase, either covalently to 5-10,um polyacrylamide beads (De Boever et af., 1983) or to microtitre plates via an intermediary second antibody. For binding monoclonal anti-steroid antibodies to the wells of microtitre plates, purified rabbit anti-mouse immunoglobulins were used as second antibodies (de Boever et af., 1986a; Tallon et af., 1984). After incubation of the reaction mixture and final washing of the antibody-bound fraction, 200 yl of 2 molil NaOH were added to all tubesiwells, followed by incubation at 60°C for 30 minutes. Light measurements were performed in polystyrene tubes, ‘lumacuvettes’, using a simple photon counter (model 2000 or 2010 Biocounter, Lumac, Schaesberg, The Netherlands). After addition of microperoxidase and the oxidant hydrogen peroxide solution, the light emitted during 10s was integrated and recorded as arbitrary light units, ‘counts’. RESULTS
The addition of danazol(2 pmolil) to the antibody, progesterone-isoluminol conjugate and serum (1Oyl) mixture was required for the direct assay of progesterone. Both danazol and serum had an effect on the calibration curve (Fig. l(a) but saliva had no effect (Fig. l(b)). For these assays a polyclonal anti-progesterone antibody, covalently linked to polyacrylamide beads, was used. It allowed separation of bound and free fractions and also additional washings of the bound fraction by simple centrifugation and decantation of the supernatant. However, for estradiol direct CIA, this solid-phase system was inconvenient because the assay required two different sets of washings. Repeated centrifugation and decantation thus became laborious and it MATERIALS AND METHODS increased variability (% CV) considerably. InChemiluminescent marker conjugates of steroid deed, in the presence of serum (501.~1)and a hormones were prepared by covalent linking of sufficient concentration of displacing agents, the steroids with aminoalkylethyl isoluminol deriva- estradiol-isoluminol conjugate bound mainly to tives (Pazzagli et af., 1981a, b). The alkyl chain serum components leaving less than 10% of contained two to six carbon atoms. Conjugates added conjugate bound to the antibody (De
7
ISOLUMINOL-BASED DIRECT CIA'S
loor
a
b
Figure 1. Calibration curves of direct ClAs for progesterone (a) Effect of serum and danazol Solid-phase antibodies bound to polyacrylamide beads, were incubated overnight at 4°C with progesterone and progesterone-isoluminol conjugate in 200 yl of buffer (dotted line) or buffer containing 2ymolil danazol (dashed line) or 2pmolil danazol and lop1 of serum (solid line) (b) Effect of saliva Solid-phase antibodies. bound to polyacrylamide beads together with progesterone and its conjugate were incubated for 90 min at room temperature The incubation mixture, 325 yl. contained 125 111 of saliva An identical curve was obtained when buffer was used instead of saliva
Boever et al., 1986a). Consequently, the competition-type immunoassay was changed into a titration type assay. After a first incubation (30minutes at 37°C) of antibody and serum, a washing step was included to eliminate the serum. Estradiol-isoluminol conjugate was added for a second incubation (30 minutes at 37°C). Finally the unbound fraction was removed by washing. In order to simplify the washing steps we replaced the beads with a microtitre plate as the solidphase. These gave very reproducible results and allowed for easy and fast working. Three consecutive washings could be performed within 3 minutes. As a consequence of the stepwise incubation procedure estradiol, bound to antibody during the first incubation, dissociated from it and was replaced by conjugate during the second incubation. This displacement phenomenon caused higher binding of conjugate than in a normal competitive incubation system (De
Boever et a[., 1986a). As a result relative binding (% BIBo)was high at high estradiol dose (Fig. 2). In the direct CIA for estradiol in saliva no matrix effect was observed and antibody, saliva (1OOp1) and conjugate could be incubated together in the wells of a microtitre plate. The dose-response curve of this non-equilibrium type assay was linear between Opg and lOpg added estradiol (Fig. 2). Sensitivity was below 0.5 pgi well. Microtitre plates as solid-phase are very well suited for the development of direct CIAs for steroid hormones. They were used for the elaboration of two more direct CIAs for salivary steroids: one for estriol, the other for progesterone. The estriol assay was characterized by very fast reaction kinetics (De Boever et al., 1988). Binding of estriol and its conjugate with isolumino1 to the antibody was complete within 2 minutes. For routine assay standardization, in-
8
J. DE BOEVER, F. KOHEN, D. LEYSEELE AND D. VANDEKERCKHOVE cubation time was fixed at 10 minutes. The second assay, for progesterone in saliva, used only 50pl of sample, compared to 1251.11 in the CIA with beads as a solid support for the antibody. Table 1 summarizes and compares assay conditions and characteristics of these assays. Imprecision (% CV) was usually below lo%, and the higher values in Table 1 are for samples with steroid concentrations close to the detection limit of the direct CIAs.
100-
\ 90
80
70
60
-
\
0
c
g
.
50-
\
40-
\
20 -
\
30
10
DISCUSSION
\ \
\
\ \
-
\ \ ,
.2
.5
I
1
1
2
1
1
,
5
l0
20
50
\ 1 100
ESTRADIOL (pg/well)
Figure 2. Calibration curves of direct CIAs for estradiol curve of the direct CIA for estradiol in serum After a first ) wells of the incubation of antibody and serum ( 5 0 ~ 1 the microtitre plate are emptied and washed Then conjugate is added During the second incubation antibody-bound estradiol is displaced by estradiol-isoluminol conjugate This causes relatively high binding at elevated estradiol dose levels - - - curve of the direct CIA for estradiol in saliva Antibody, saliva (100bI) and the conjugate were incubated together ~
Direct CIAs for steroid hormones in saliva can be developed without pronounced difficulties because there is very little if any matrix effect. CIAs for steroids in plasma or serum, however, are problematical because of matrix effects, lack of suitable reagents for displacement of steroid hormones from steroid-binding proteins in serum/ plasma, and the unexplained preferential binding of the steroid-isoluminol conjugate to serum components. We have shown that isoluminol can advantageously be used as a label for the development of simple and sensitive direct CIAs for steroid sex hormones (see Table 1). Care must be taken to eliminate serum or plasma components before the chemiluminescent reaction is started, because these components interfere with the production of light (Schroeder et al., 1978a, b).
Table 1. Direct ClAs for sexual steroid hormones Precision ( % CV)
Steroid
In
Sample Solid size (PI) phase
Progesterone
serum
10
Progesterone Estradiol Estradiol Estriol Progesterone
saliva serum saliva saliva saliva
125 50 100 50 50
Incubation
Beads overnight 4°C 90 min R T ~ Beads 90 min RT MTP" 60 rnin RT MPT 60 min RT MTP 10 rnin RT 30 min RT MPT
Sensitivity (nmolll)
within assay
between assays
References De Boever e t a / . ,(1984)
0.54
6.5-12.0'
8.3-14.7'
0.04 0.18 0.01 0.31 0.32
6.8- 8.8 5.0-12.4
6.9- 9.3 De Boever et a/., (1986b) 6.5-12.7 De Boever e t a / . ,(1986a) De Boever era/.,(1988) 6.9-16.0
5.0-11.8
MTP, microtitre plate. RT, room temperature. 'The higher CVs are for samples with steroid hormone concentrations around the level of sensitivity of the assay. a
ISOLUMINOL-BASED DIRECT CIA'S
Another disadvantage, related to the use of isoluminol as a label, is the obligatory treatment with sodium hydroxide during 30 minutes at 60 "C prior to the chemical reaction. Currently alternative methods for direct and efficient light production are under study. With a plate reader and an efficient system for direct light production up to 40 unknown saliva or serum samples could be routinely analysed within 2 to 2.5 hours with direct isoluminol-based CIAs as described here. Acknowledgements We are grateful to Dr Mackintosh and Mr Vossen of Lumac BV, Landgraaf, The Netherlands, for the loan of a Biocounter M2000 luminometer. We thank Mrs S. Lichter, B. Gayer, and J. Ausher, Mr X. Geenens and G . Van Maele for technical assistance, Mr H . Chretien for artworks and Mrs S. Hoste for typing the manusctript. Part of this work was supported by a grant from the Belgian National Fund for Scientific Research (NFWO grant No 3.0030.86 to JDB) and by a grant from the Binational Science Foundation, Jerusalem (to FK).
REFERENCES Bauminger, S., Kohen, F. and Lindner, H . R. (1974). Steroids as haptens: optimal design of antigens for the formation of antibodies to steroid hormones. J. Steroid Bochem., 5, 739-747. D e Boever, J . , Kohen, F. and Vandekerckhove, D. (1983). Solid-phase chemiluminescence immunoassay for plasma estradiol-17P during gonadotropin therapy compared with two radioimmunoassays. Clin. Chem., 29, 2068-2072. D e Boever, J., Kohen, F . , Vandekerckhove, D. and Van Maele, G . (1984). Solid-phase chemiluminescence immunoassay for progesterone in unextracted serum. Clin. Chem., 30, 1637-1641. D e Boever, J . , Kohen, F . , Usanachitt, C . , Vandekerckhove, D . , Leyseele, D . and Vandewalle, L. (1986a). Direct chemiluminescence immunoassay for estradiol in serum. Clin. Chem., 32, 1895-1900. D e Boever, J . , Kohen, F. and Vandekerckhove, D. (1986b). Direct solid-phase chemiluminescence immunoassay for salivary progesterone. Clin. Chem., 32, 763-767. D e Boever, J . , Kohen, F., Ulrix, W. and Vandekerckhove, D. (1988). Anal. Chim. Acta, 205, 215-222. Kim, J . B., Barnard, G . J . , Collins, W . P., Kohen, F. Lindner, H. R. and Eshhar, Z. (1982). Measurement of plasma estradiol-17P by solid-phase chemiluminescence immunoassay. Clin. Chem., 28, 1120-1124. Klingler, W., Haupt, O., Von Postel, G . and Knuppen, R. (1983). Immunoassay of unconjugated estriol in serum of pregnant women monitored by chemiluminescence. Steroids, 42, 123-136. Kohen, F. and Lichter, S. (1986). Monoclonal antibodies to steroid hormones. In Proc. Int. Symp. Monoclonal
9 Antibodies: Basic Principles, Experimental and Clinical Applications in Endocrinology, Forti, G . , Lipsett, M.B. and Serio, M. (Eds), Raven Press, New York, pp. 87-95. Kohen, F., Bauminger, S. and Lindner, H . R . (1975). Preparation of antigenic steroid-protein conjugates. In Steroid Immunoassay, Proceedings Fifth Tenovus Workshop, Cameron, E.H.D., Hillier, S.G. and Griffiths, K. (Eds), Alpha Omega Publishing, Cardiff, pp. 11-32. Kohen, F., Kim, J . B., Lindner, H. R. and Collins, W. P. (1981). Development of a solid-phase chemiluminescence immunoassay for plasma progesterone. Steroids, 38, 73-1 09. Kohen, F., Pazzagli, M . , Kim, J . B., Lindner, H . R. and Boguslaski, R . C . (1979). An assay procedure for plasma progesterone based on antibody-enhanced luminescence. FEBS Letters, 104, 201-205. Pazzagli, M., Kim, J . B . , Messeri, G., Kohen F. Bolelli. G . F., Tommasi, A . , Salerno, R., Moneti, G . and Serio, M. (1981a). Luminescent immunoassay (LIA) of cortisol. 1. Synthesis and evaluation of two chemiluminescent labels of cortisol. J . Steroid Biochem., 14, 1005-1012. Pazzagli, M., Kim, J . B., Messeri, G . , Martinazzo, G . , Kohen, F., Franceschetti, F., Moneti, G . , Salerno, R.. Tommasi, A . and Serio, M. (1981b). Evaluation of different progesterone-isoluminol conjugates for chemiluminescence immunoassay. Clin.Chim. Acta, 115,277-286. Pazzagli, M., Kim, J . B . , Messeri, G . , Kohen, F., Bolelli, G . F., Tommasi, A , , Salerno, R. and Serio, M. ( 1 9 8 1 ~ ) . Luminescent immunoassay (LIA) of cortisol. 2. Development and validation of the immunoassay monitored by chemiluminescence. J . Steroid Biochem., 14, 1181-1 187. Pazzagli, M., Kim, J. B . , Messeri, G . , Martinazzo, G . . Kohen, F . , Franceschetti, F., Tommasi, A., Salerno, R. and Serio, M. (1981d). Luminescent immunoassay (LIA) for progesterone in a heterogeneous system. Clin. Chim. Acta, 115, 287-296. Pazzagli, M., Serio, M., Munson, P. and Rodbard, D. (1982). A chemiluminescent immunoassay (LIA) for testosterone. In Radioimmunoassay and Related Procedures in Medicine, International Atomic Energy Agency (Ed.), I A E A , Vienna, pp. 747-755. Riad-Fahmy, D., Read, G . F., Walker, R. F. and Griffiths, K . (1982). Steroids in saliva for assessing endocrine function. Endocr. Rev., 3, 367-395. Schroeder, H . R., Vogelhut P. O., Carrico, R . J . , Boguslaski, R . C . and Buckler, R. T. (1976). Competitive protein binding assay for biotin monitored by chemiluminescence. Anal. Chem., 48, 1933-1937. Schroeder, H. R. and Yeager, F. M . (1978a). Chemiluminescence yields and detection limits for some isoluminol derivatives in various oxidation systems. Anal. Chem., 50, 111 4 1120. Schroeder, H. R . , Boguslaski, R . C., Carrico, R. J . and Buckler, R. T. (1978b). Monitoring specific proteinbinding reactions with chemiluminescence. In Methods in Enzymology, Vol. 57, Bioluminescence and Chemilurninescence, De Luca, M. A. (Ed.), pp. 424445. Smith, R. G., Besh, P. K., Dill, B. and Bottram, V. C. (1979). Saliva as a matrix for measuring free androgens: comparison with serum androgens in polycystic ovarian disease. Fertil. Steril., 31, 513-517. Tallon, D. F., Gosling, J . P . , Buckley, P. M., Dooley, M. M . , Cleere, W. F., O'Dwyer, E . M. and Fottrell, P. F. (1984). Direct solid-phase enzyme immunoassay of progesterone in saliva. Clin. Chem., 30, 1507-1511.
10
J. DE BOEVER, F. KOHEN, D. LEYSEELE AND D. VANDERKERCKHOVE
Vining, R. F. and McGinley, R. A . (1987). The measurement of hormones in saliva: possibilities and pitfalls. J . Steroid Biochem., 27, 81-94. Vining, R. F., McGinley, R. A . and Symons, R. G . (1983). Hormones in saliva: mode of entry and consequent implications for clinical interpretation. Clin. Chem.. 29.
1752-1756. Weerasekera, D. A , , Kim, J . B . , Barnard, G. J . , Collins, W . P., Kohen, F. and Lindner, H. R. (1982). Monitoring ovarian function by a solid-phase chemiluminescence immunoassay. Acra Endocrinol., 101, 254263.
(1990)
Isoluminol as a Marker in Direct Chemiluminescence Immunoassays for Steroid Hormones J. De Boever", F. Kohen', D. Leyseele and
D.Vandekerckhove
Department of Obstetrics and Gynecology, University Hospital, B-9000 Gent, Belgium 'Department of Hormone Research, The Weizmann institute of Science, IL-76100 Rehovot, Israel
We have used isoluminol steroid hormone conjugates in competitive heterogeneous chemiluminescence immunoassays (CIA) for estradiol, progesterone and estriol in body fluids. The assays did not require prior extraction of the steroid hormone with an organic solvent. Polyclonal or monoclonal antibodies, covalently coupled t o polyacrylamide microspheres or adsorbed onto the plastic surface of microtritation plates via an intermediate second antibody, were used as solid-phase. The latter solid-phase system allows rapid processing of incubation mixtures. Sensitivie and reliable direct ClAs for estradiol in serum, progesterone in serum and in saliva and for estriol in saliva have been developed and their clinical utility has been assessed. Sensitivities ranged from 0.54 t o 0.04 nmol/l, and precision (YOCV) ranged from 10% t o 16%. Keywords: CIA; steroids; isolurninol
INTRODUCTION It is now generally accepted that nonradioisotopic immunoassays (NIIA) with similar sensitivity, reliability and performance as radioimmunoassay (RIA) are possible for a large number of biologically and clinically important molecules. For the elaboration of direct immunoassays for steroid hormones the most successful nonradioisotopic label has been isoluminol. Aminoalkylethyl derivatives of this chemiluminescent molecule were first introduced in a competitive protein-binding, assay for biotin by Schroeder et al. (1976, 1978a), and in immunoassay for steroid hormones by Kohen and colleagues (Kohen et al., 1979; Pazzagli et al., 1981a, b). Oxidation of these *Author for correspondence
0884-39961901010005-06$05 .OO @ 1990 by John Wiley & Sons, Ltd.
isoluminol derivatives under fixed conditions produces light in good quantum yield (1-2%). Measurement of the photons generated during this chemical oxidative reaction can be performed quickly and reliably using a simple photon counter. An advantage of chemiluminescence is that although the quantum yield is low, molecules yielding measurable light are 'seen' in the 10 seconds measuring time. In contrast, in RIA a much smaller fraction of total available counts are measured in the measuring time 1-5 minutes) needed for counting decaying 51- and 3Hradioisotopes. On a theoretical basis this would allow for better sensitivity of CIA compared to RIA. Other important advantages of an isolumino1 derivative is its facile covalent coupling to a variety of molecules (including haptens), the
i
6
J. DE BOEVER, F. KOHEN, D. LEYSEELE AND D. VANDEKERCKHOVE
stability of the label and labelled products, and the lack of health and environmental risks. The first chemiluminescence immunoassay for a steroid sex hormone used aminoalklethyl isoluminol as a label (Kohen et af.,1979). It was a non-separation or ‘homogeneous’ assay with intrinsically less sensitivity (25 pgitube) than the heterogeneous type (below 10 pgitube). Blood serum and plasma components quench the light production in the chemiluminescent reaction (Schroeder et al., 1978a,b), and thus prior extraction of steroid from blood was required. Subsequently other isoluminol-based CIAs for steroid hormones were developed. Most included an extraction step and all were of the more sensitive heterogeneous type. Sensitivities ranged between 2 and 10pgitube (De Boever et af.,1983, 1984; Kim et af., 1982; Klingler et al., 1983; Kohen et af., 1981; Pazzagli et af., 1981c, d , 1982). Except for two assays based on separation of bound and free hormone fractions with dextrancoated charcoal (DCC) (Pazzagli et af., 1981c, d), these assays used solid phase antibodies either coated to tubes or covalently linked to polyacrylamide beads. We have developed several direct CIAs for steroid sex hormones in blood and in saliva, using solid-phase antibodies and a heterogeneous assay type system. Serum and plasma are the most frequently used biological materials in the clinical laboratory for the measurement of steroid hormone concentrations, but in some instances saliva may be a valuable alternative. It reflects the levels of steroids in free condition, i.e. not bound to sex hormone-blinding globulin, SHBG, and corticosteroid-binding globulin, CBG (Smith et af., 1979; Vining et af., 1983), and it can be obtained easily and stress-free even by the patient at home. Saliva is especially attractive if repeated hormone determinations are required for monitoring and follow-up, for example during ovulation induction, to monitor glucocorticoid therapy in congenital adrenal hyperplasia (Vining and McGinley, 1987; Riad-Fahmy et af., 1982) and in pathological pregnancy.
were purified by thin layer chromatography (Pazzagli et af., 1981a, b). Between 1Opg and lOOpg of the conjugate were added to the tubes or microtitre plate wells. Antibodies (Bauminger et af., 1974; Kohen and Lichter, 1986; Kohen et af., 1975) or their purified gamma globulin fractions (Kim et al., 1982; Weerasekera et al., 1982) were bound in solid phase, either covalently to 5-10,um polyacrylamide beads (De Boever et af., 1983) or to microtitre plates via an intermediary second antibody. For binding monoclonal anti-steroid antibodies to the wells of microtitre plates, purified rabbit anti-mouse immunoglobulins were used as second antibodies (de Boever et af., 1986a; Tallon et af., 1984). After incubation of the reaction mixture and final washing of the antibody-bound fraction, 200 yl of 2 molil NaOH were added to all tubesiwells, followed by incubation at 60°C for 30 minutes. Light measurements were performed in polystyrene tubes, ‘lumacuvettes’, using a simple photon counter (model 2000 or 2010 Biocounter, Lumac, Schaesberg, The Netherlands). After addition of microperoxidase and the oxidant hydrogen peroxide solution, the light emitted during 10s was integrated and recorded as arbitrary light units, ‘counts’. RESULTS
The addition of danazol(2 pmolil) to the antibody, progesterone-isoluminol conjugate and serum (1Oyl) mixture was required for the direct assay of progesterone. Both danazol and serum had an effect on the calibration curve (Fig. l(a) but saliva had no effect (Fig. l(b)). For these assays a polyclonal anti-progesterone antibody, covalently linked to polyacrylamide beads, was used. It allowed separation of bound and free fractions and also additional washings of the bound fraction by simple centrifugation and decantation of the supernatant. However, for estradiol direct CIA, this solid-phase system was inconvenient because the assay required two different sets of washings. Repeated centrifugation and decantation thus became laborious and it MATERIALS AND METHODS increased variability (% CV) considerably. InChemiluminescent marker conjugates of steroid deed, in the presence of serum (501.~1)and a hormones were prepared by covalent linking of sufficient concentration of displacing agents, the steroids with aminoalkylethyl isoluminol deriva- estradiol-isoluminol conjugate bound mainly to tives (Pazzagli et af., 1981a, b). The alkyl chain serum components leaving less than 10% of contained two to six carbon atoms. Conjugates added conjugate bound to the antibody (De
7
ISOLUMINOL-BASED DIRECT CIA'S
loor
a
b
Figure 1. Calibration curves of direct ClAs for progesterone (a) Effect of serum and danazol Solid-phase antibodies bound to polyacrylamide beads, were incubated overnight at 4°C with progesterone and progesterone-isoluminol conjugate in 200 yl of buffer (dotted line) or buffer containing 2ymolil danazol (dashed line) or 2pmolil danazol and lop1 of serum (solid line) (b) Effect of saliva Solid-phase antibodies. bound to polyacrylamide beads together with progesterone and its conjugate were incubated for 90 min at room temperature The incubation mixture, 325 yl. contained 125 111 of saliva An identical curve was obtained when buffer was used instead of saliva
Boever et al., 1986a). Consequently, the competition-type immunoassay was changed into a titration type assay. After a first incubation (30minutes at 37°C) of antibody and serum, a washing step was included to eliminate the serum. Estradiol-isoluminol conjugate was added for a second incubation (30 minutes at 37°C). Finally the unbound fraction was removed by washing. In order to simplify the washing steps we replaced the beads with a microtitre plate as the solidphase. These gave very reproducible results and allowed for easy and fast working. Three consecutive washings could be performed within 3 minutes. As a consequence of the stepwise incubation procedure estradiol, bound to antibody during the first incubation, dissociated from it and was replaced by conjugate during the second incubation. This displacement phenomenon caused higher binding of conjugate than in a normal competitive incubation system (De
Boever et a[., 1986a). As a result relative binding (% BIBo)was high at high estradiol dose (Fig. 2). In the direct CIA for estradiol in saliva no matrix effect was observed and antibody, saliva (1OOp1) and conjugate could be incubated together in the wells of a microtitre plate. The dose-response curve of this non-equilibrium type assay was linear between Opg and lOpg added estradiol (Fig. 2). Sensitivity was below 0.5 pgi well. Microtitre plates as solid-phase are very well suited for the development of direct CIAs for steroid hormones. They were used for the elaboration of two more direct CIAs for salivary steroids: one for estriol, the other for progesterone. The estriol assay was characterized by very fast reaction kinetics (De Boever et al., 1988). Binding of estriol and its conjugate with isolumino1 to the antibody was complete within 2 minutes. For routine assay standardization, in-
8
J. DE BOEVER, F. KOHEN, D. LEYSEELE AND D. VANDEKERCKHOVE cubation time was fixed at 10 minutes. The second assay, for progesterone in saliva, used only 50pl of sample, compared to 1251.11 in the CIA with beads as a solid support for the antibody. Table 1 summarizes and compares assay conditions and characteristics of these assays. Imprecision (% CV) was usually below lo%, and the higher values in Table 1 are for samples with steroid concentrations close to the detection limit of the direct CIAs.
100-
\ 90
80
70
60
-
\
0
c
g
.
50-
\
40-
\
20 -
\
30
10
DISCUSSION
\ \
\
\ \
-
\ \ ,
.2
.5
I
1
1
2
1
1
,
5
l0
20
50
\ 1 100
ESTRADIOL (pg/well)
Figure 2. Calibration curves of direct CIAs for estradiol curve of the direct CIA for estradiol in serum After a first ) wells of the incubation of antibody and serum ( 5 0 ~ 1 the microtitre plate are emptied and washed Then conjugate is added During the second incubation antibody-bound estradiol is displaced by estradiol-isoluminol conjugate This causes relatively high binding at elevated estradiol dose levels - - - curve of the direct CIA for estradiol in saliva Antibody, saliva (100bI) and the conjugate were incubated together ~
Direct CIAs for steroid hormones in saliva can be developed without pronounced difficulties because there is very little if any matrix effect. CIAs for steroids in plasma or serum, however, are problematical because of matrix effects, lack of suitable reagents for displacement of steroid hormones from steroid-binding proteins in serum/ plasma, and the unexplained preferential binding of the steroid-isoluminol conjugate to serum components. We have shown that isoluminol can advantageously be used as a label for the development of simple and sensitive direct CIAs for steroid sex hormones (see Table 1). Care must be taken to eliminate serum or plasma components before the chemiluminescent reaction is started, because these components interfere with the production of light (Schroeder et al., 1978a, b).
Table 1. Direct ClAs for sexual steroid hormones Precision ( % CV)
Steroid
In
Sample Solid size (PI) phase
Progesterone
serum
10
Progesterone Estradiol Estradiol Estriol Progesterone
saliva serum saliva saliva saliva
125 50 100 50 50
Incubation
Beads overnight 4°C 90 min R T ~ Beads 90 min RT MTP" 60 rnin RT MPT 60 min RT MTP 10 rnin RT 30 min RT MPT
Sensitivity (nmolll)
within assay
between assays
References De Boever e t a / . ,(1984)
0.54
6.5-12.0'
8.3-14.7'
0.04 0.18 0.01 0.31 0.32
6.8- 8.8 5.0-12.4
6.9- 9.3 De Boever et a/., (1986b) 6.5-12.7 De Boever e t a / . ,(1986a) De Boever era/.,(1988) 6.9-16.0
5.0-11.8
MTP, microtitre plate. RT, room temperature. 'The higher CVs are for samples with steroid hormone concentrations around the level of sensitivity of the assay. a
ISOLUMINOL-BASED DIRECT CIA'S
Another disadvantage, related to the use of isoluminol as a label, is the obligatory treatment with sodium hydroxide during 30 minutes at 60 "C prior to the chemical reaction. Currently alternative methods for direct and efficient light production are under study. With a plate reader and an efficient system for direct light production up to 40 unknown saliva or serum samples could be routinely analysed within 2 to 2.5 hours with direct isoluminol-based CIAs as described here. Acknowledgements We are grateful to Dr Mackintosh and Mr Vossen of Lumac BV, Landgraaf, The Netherlands, for the loan of a Biocounter M2000 luminometer. We thank Mrs S. Lichter, B. Gayer, and J. Ausher, Mr X. Geenens and G . Van Maele for technical assistance, Mr H . Chretien for artworks and Mrs S. Hoste for typing the manusctript. Part of this work was supported by a grant from the Belgian National Fund for Scientific Research (NFWO grant No 3.0030.86 to JDB) and by a grant from the Binational Science Foundation, Jerusalem (to FK).
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9 Antibodies: Basic Principles, Experimental and Clinical Applications in Endocrinology, Forti, G . , Lipsett, M.B. and Serio, M. (Eds), Raven Press, New York, pp. 87-95. Kohen, F., Bauminger, S. and Lindner, H . R . (1975). Preparation of antigenic steroid-protein conjugates. In Steroid Immunoassay, Proceedings Fifth Tenovus Workshop, Cameron, E.H.D., Hillier, S.G. and Griffiths, K. (Eds), Alpha Omega Publishing, Cardiff, pp. 11-32. Kohen, F., Kim, J . B., Lindner, H. R. and Collins, W. P. (1981). Development of a solid-phase chemiluminescence immunoassay for plasma progesterone. Steroids, 38, 73-1 09. Kohen, F., Pazzagli, M . , Kim, J . B., Lindner, H . R. and Boguslaski, R . C . (1979). An assay procedure for plasma progesterone based on antibody-enhanced luminescence. FEBS Letters, 104, 201-205. Pazzagli, M., Kim, J . B . , Messeri, G., Kohen F. Bolelli. G . F., Tommasi, A . , Salerno, R., Moneti, G . and Serio, M. (1981a). Luminescent immunoassay (LIA) of cortisol. 1. Synthesis and evaluation of two chemiluminescent labels of cortisol. J . Steroid Biochem., 14, 1005-1012. Pazzagli, M., Kim, J . B., Messeri, G . , Martinazzo, G . , Kohen, F., Franceschetti, F., Moneti, G . , Salerno, R.. Tommasi, A . and Serio, M. (1981b). Evaluation of different progesterone-isoluminol conjugates for chemiluminescence immunoassay. Clin.Chim. Acta, 115,277-286. Pazzagli, M., Kim, J . B . , Messeri, G . , Kohen, F., Bolelli, G . F., Tommasi, A , , Salerno, R. and Serio, M. ( 1 9 8 1 ~ ) . Luminescent immunoassay (LIA) of cortisol. 2. Development and validation of the immunoassay monitored by chemiluminescence. J . Steroid Biochem., 14, 1181-1 187. Pazzagli, M., Kim, J. B . , Messeri, G . , Martinazzo, G . . Kohen, F . , Franceschetti, F., Tommasi, A., Salerno, R. and Serio, M. (1981d). Luminescent immunoassay (LIA) for progesterone in a heterogeneous system. Clin. Chim. Acta, 115, 287-296. Pazzagli, M., Serio, M., Munson, P. and Rodbard, D. (1982). A chemiluminescent immunoassay (LIA) for testosterone. In Radioimmunoassay and Related Procedures in Medicine, International Atomic Energy Agency (Ed.), I A E A , Vienna, pp. 747-755. Riad-Fahmy, D., Read, G . F., Walker, R. F. and Griffiths, K . (1982). Steroids in saliva for assessing endocrine function. Endocr. Rev., 3, 367-395. Schroeder, H . R., Vogelhut P. O., Carrico, R . J . , Boguslaski, R . C . and Buckler, R. T. (1976). Competitive protein binding assay for biotin monitored by chemiluminescence. Anal. Chem., 48, 1933-1937. Schroeder, H. R. and Yeager, F. M . (1978a). Chemiluminescence yields and detection limits for some isoluminol derivatives in various oxidation systems. Anal. Chem., 50, 111 4 1120. Schroeder, H. R . , Boguslaski, R . C., Carrico, R. J . and Buckler, R. T. (1978b). Monitoring specific proteinbinding reactions with chemiluminescence. In Methods in Enzymology, Vol. 57, Bioluminescence and Chemilurninescence, De Luca, M. A. (Ed.), pp. 424445. Smith, R. G., Besh, P. K., Dill, B. and Bottram, V. C. (1979). Saliva as a matrix for measuring free androgens: comparison with serum androgens in polycystic ovarian disease. Fertil. Steril., 31, 513-517. Tallon, D. F., Gosling, J . P . , Buckley, P. M., Dooley, M. M . , Cleere, W. F., O'Dwyer, E . M. and Fottrell, P. F. (1984). Direct solid-phase enzyme immunoassay of progesterone in saliva. Clin. Chem., 30, 1507-1511.
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J. DE BOEVER, F. KOHEN, D. LEYSEELE AND D. VANDERKERCKHOVE
Vining, R. F. and McGinley, R. A . (1987). The measurement of hormones in saliva: possibilities and pitfalls. J . Steroid Biochem., 27, 81-94. Vining, R. F., McGinley, R. A . and Symons, R. G . (1983). Hormones in saliva: mode of entry and consequent implications for clinical interpretation. Clin. Chem.. 29.
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