JOURNAL
OF BIOLUMINESCENCE A N D CHEMILUMINESCENCE
VOL 5 49-52
(1990)
Enhanced Chemiluminescent lmmunoassay for Aldosterone W. Hubl'*, G. H. G. Thorpe', F. Hofmann', D. Meissner' and H. J. Thiele3 'Institute of Clinical Chemistry and Laboratory Diagnostics of Dresden-Friedrichstadt Hospital, Friedrichstrasse 41, DDR-8010 Dresden, GDR 2Department of Clinical Chemistry, Wolfson Research Laboratories, Queen Elizabeth Medical Centre, Birmingham 815 2TH. UK 3Research Institute of Medical Diagnostics, Dresden, GDR
A solid phase immunoassay for aldosterone using enhanced chemiluminescent detection has been developed. Monoclonal antibodies against aldosterone were used for the immune reaction and compared with polyclonal antibodies. Uniform Protein A coated polystyrene tubes were used as solid phase for the monoclonal antibody and second (anti-rabbit) antibody coated tubes for the polyclonal antibody. Horseradish peroxidase was covalently linked t o aldosterone as enzyme label. Optimum conditions were established for the generation and measurement of the luminescent reactions using luminol, p-iodophenol as enhancer and hydrogen peroxide. The advantages of this assay are the high sensitivity with a detection limit of 100fg/tube, the prolonged luminescence signal with a simplification of the measurement (simpler detectors, external start pipetting) and the short measure time with the possibility of repeated measurement. The coefficients of variation were 4.2%-7.3% in the concentration range 140-1 180 pmol/l. The assay showed a significant correlation ( r = 0.91) with the ELISA. The aldosterone concentrations in plasma and saliva of patients with Conn's syndrome were significantly increased, and in patients with Addison's disease were found near the detection limit. Keywords: Aldosterone; enhanced chernilurninescence; irnrnunoassay
INTRODUCTION
The concentration of aldosterone in human plasma in relatively low (picomole-range) and colorimetric enzyme immunoassays for aldosterone using peroxidase labels have been described. Enhanced chemiluminescent detection of peroxidase enables the rapid and more sensitive determination of peroxidase-labelled substances in immunoassays (Kricka et al., 1983; Thorpe et al., 1985a).
In this study we describe the development of a solid phase immunoassay for aldosterone using an optimized enhanced chemiluminescent detection. MATERIALS AND METHODS
Aldosterone and other steroids were purchased from Steraloids (Wilton, USA), p-iodophenol was obtained from Aldrich-Chemie (FRG), horseradish peroxidase was obtained from VEB
*Author for correspondence. 0884-3996/901010049-04$05 .OO @ 1990 by John Wiley & Sons, Ltd.
Received October 1988 Revised February 1989
50 Germed (GDR), luminol and Protein A were purchased from Sigma-Chemie (FRG). Luminol was purified as described by Thorpe et al. (1985b). The Protein A or second antibody coated tubes were prepared as described elsewhere (Hubl et al. 1988). The antibodies against aldosterone were raised in rabbits and mouse, respectively, using aldosterone-18,21-diacetate3(0-carboxymethyl-)oxime bovine serum albumin conjugate, and the aldosterone-horseradish peroxidase conjugate was synthesized using the mixed anhydride reaction (Hubl et al., 1985). Serum or EDTA-plasma were used for specimen analysis.
W. HUBL ETAL.
RESULTS Variations of pH and buffers
Luminescence enhancement by p-iodophenol using the aldosterone-peroxidase conjugate was pH-dependent. In agreement with the results of Kricka and Thorpe (1983 and 1985) we found a maximal intensity with Tris-buffer at pH 8.6 and on the other hand with sodium-borate buffer between 8.0 and 9.5 and with phosphate buffer between 8.5 and 9.2. Luminollpiodophenol ratio
Luminescent enzyme immunoassay procedure
Plasma (0.05 ml) or saliva samples (0.10 ml) were extracted with 1.00 ml dichloromethane. The organic extract was dried under a stream of nitrogen. Extracts were dissolved in O.lOml assay buffer (0.1 mmolll phosphate, p H 7.4, 0.1% gelatine) and 0.05 ml were added to the Protein A (for monoclonal antibody) or second antibody (for the polyclonal antibody) coated tubes. Then 0.05 ml antibody dilution 1:50,000 for the monoclonal and 1:20,000 for the polyclonal antibody) and 0.10 ml aldosterone-peroxidase conjugate (dilution 1:10,000) were added to each of the tubes. The tubes were covered with plastic film and then incubated for 3 h at room temperature. The tubes were then emptied and washed three times with running tap water.
Figure 1 demonstrates the dependence of enhancement on different luminollp-iodophenol ratios with different buffer systems. The maximal luminescence was found at the ratio between 0.38 and 0.75 with the highest intensity using sodium borate buffer. Decreasing the Na-luminollp-iodophenol ratio to 0.25 produced lower photon counts.
60
lo
45
f2 X
v)
I-
z 3
Chemiluminescent measurement. Bound aldosterone-peroxidase conjugate was determined by adding 0.40 ml substrate solution which contained luminol (0.50 mmol/l), p-iodophenol (0.91 mmol/l) and hydrogen peroxide (1.8 mmol/l) in 0.13mol/l borate buffer (pH 8.6). The chemiluminescent reaction was measured between 10 and 25 min after initiation. Standard curve. Aldosterone standards (2770; 1390; 690; 345; 172; 86; 43; Opmolll) were added (0.050 ml) to the polystyrene tubes as the dissolved extracts and assayed as described above.
0
0
30
2
E
Pn 15
0
0:5 1.'o 1.I5 210 Na-luminol (mol/l) / p-iodophenol (rnol/l)
Figure 1. The influence of IuminoVpiodophenol ratio on light emission using different buffer systems
ALDOSTERONE IMMUNOASSAY
51
Table 1. Cross-reactivities of aldosterone-antisera Polyclonal Monoclonal Aldosterone Cortisol 17-OH-Progesterone Corticosterone Progesterone 1I-Deoxycortisol 11-Deoxycorticosterone Testosterone 18-OH-Deoxycorticosterone Tetra hydroaldosterone Spironolactone
100 0.0002 0.04 0.004 0.0001 0.04 0.0005 0.0001 0.005 1.o 0.0005
100 0.0004 0.0008 0.005 0.002 0.0009 0.01 0.0001 0.05 0.5 0.0001
Analytical recovery of added aldosterone
Addition of aldosterone 150 pmol/l produced a recovery of 92.5% (SD 1.8%, n = 20), at 320pmoVl the recovery was 90.6% (SD = 2.3%, n = 20).
Specificity of aldosterone-antisera
The cross-reactivities of different steroid hormones with the polyclonal and monoclonal antibodies are shown in Table 1. Both antisera are highly specific and enable the aldosterone determination in biological fluids without chromatography.
Kinetics of light emission
Different dilutions of aldosterone-peroxidase show a relatively constant light emission between 10 and 25min. The light emission of the conjugates with the lower concentrations used under immunoassay conditions (1:200,000) was constant for a longer time, between 10 and 40 min .
Detection limit
The detection limit was calculated by means of the 3 SD of the Bo and was found to be 100 fg/tube.
Precision studies
Comparison of aldosterone-LEIA with aldosterone-ELISA
Intra-assay variations ( n = 30) between 4.2% and 6.8% were found with low, medium and high concentrations of aldosterone (140-1 180 pmol/l). The inter-assay variations ( n = 20) were 4.5% to 7.3%.
Results obtained on 30 serum specimens using the aldosterone-ELISA (Hub1 et al., 1985) were compared with these from the aldosterone-LEIA. A correlation coefficient of 0.91 was found (y = 0.85 x 42).
+
Table 2. Reference intervals and clinical results of aldosteroneLElA Aldosterone (pmolll) mean (2 SD) Plasma Saliva Healthy adults ( n = 120) supine posture upright posture Conn's syndrome ( n = 4) Addison's Disease ( n = 4)
240 485 3460 62
(80-400) (180-790) (247C-4450) (21-103)
85 195 2080 24
(10-160) (70-320) (1350-2810)
(8-40)
Blood was taken from the Conn's and Addison's patients in an upright position.
W. HUBL ETAL.
Clinical applications
Acknowledgement
Table 2 presents the results of aldosterone determinations in plasma as well as in saliva samples of healthy adults and of patients with Conn’s syndrome and Addison’s disease.
The investigations are supported by the research project of Medizinische Laboratoriuccdiagnostik of the Ministry Of GDR.
DISCUSSION
The optimized enhanced chemiluminescent system allows the sensitive determination of aldosterone in biological fluids. We found a very low detection limit of 100fg/tube in comparison with the ELISA of 1pg/tube. The marked increase in luminescent signal leads to a further simplification of the equipment for the measurement without internal start pipetting and the application of a microtitreplate reader. We observed a significant linear correlation of the LEIA and the aldosterone-ELISA. The clinical application of the aldosteroneLEIA shows significantly increased concentrations in patients with Conn’s syndrome and very low aldosterone levels in patients with Addison’s disease. Furthermore, the sensitivity of the enhanced luminescent immunoassay allowed the aldosterone determination in saliva, too.
REFERENCES Hubl, W. (1985). Aldosterone. In Methods of Enzymatic Analysis, Bermeyer, H. U . (Ed.), VCH Verlagsgesellschaft, Weinheim, FRG. pp. 256266. Hubl, W., Daxenbichler, G., Meissner, D. and Thiele, H. J . (1988). An improved solid phase enzyme and luminescent immunoassay system for steroidhormones and digoxin. Clin. Chem., 34, 2521-2523. Kricka, L. J., Thorpe, G. H. G . and Whitehead, T. P. (1983). Enhanced luminescent or lurninometric assay. European Patent Publication, 116-454. Thorpe, G. H. G., Kricka, L. J., Moseley, S . B. and Whitehead, T. P. (1985a). Phenols as enhancers of the chemiluminescent horseradish peroxidase - luminol hydrogen peroxide reaction: application in luminescence monitored enzyme immunoassays. C h . Chem., 31, 1335-1341. Thorpe, G. H. G . , Williams, L. A., Kricka, L. J., Whitehead, T. P., Evans, H. and Stanworth, D. R. (1985b). A rapid luminescently monitored enzyme immunoassay for IgE. J . Immunol. Methods, 79, 51-63.
OF BIOLUMINESCENCE A N D CHEMILUMINESCENCE
VOL 5 49-52
(1990)
Enhanced Chemiluminescent lmmunoassay for Aldosterone W. Hubl'*, G. H. G. Thorpe', F. Hofmann', D. Meissner' and H. J. Thiele3 'Institute of Clinical Chemistry and Laboratory Diagnostics of Dresden-Friedrichstadt Hospital, Friedrichstrasse 41, DDR-8010 Dresden, GDR 2Department of Clinical Chemistry, Wolfson Research Laboratories, Queen Elizabeth Medical Centre, Birmingham 815 2TH. UK 3Research Institute of Medical Diagnostics, Dresden, GDR
A solid phase immunoassay for aldosterone using enhanced chemiluminescent detection has been developed. Monoclonal antibodies against aldosterone were used for the immune reaction and compared with polyclonal antibodies. Uniform Protein A coated polystyrene tubes were used as solid phase for the monoclonal antibody and second (anti-rabbit) antibody coated tubes for the polyclonal antibody. Horseradish peroxidase was covalently linked t o aldosterone as enzyme label. Optimum conditions were established for the generation and measurement of the luminescent reactions using luminol, p-iodophenol as enhancer and hydrogen peroxide. The advantages of this assay are the high sensitivity with a detection limit of 100fg/tube, the prolonged luminescence signal with a simplification of the measurement (simpler detectors, external start pipetting) and the short measure time with the possibility of repeated measurement. The coefficients of variation were 4.2%-7.3% in the concentration range 140-1 180 pmol/l. The assay showed a significant correlation ( r = 0.91) with the ELISA. The aldosterone concentrations in plasma and saliva of patients with Conn's syndrome were significantly increased, and in patients with Addison's disease were found near the detection limit. Keywords: Aldosterone; enhanced chernilurninescence; irnrnunoassay
INTRODUCTION
The concentration of aldosterone in human plasma in relatively low (picomole-range) and colorimetric enzyme immunoassays for aldosterone using peroxidase labels have been described. Enhanced chemiluminescent detection of peroxidase enables the rapid and more sensitive determination of peroxidase-labelled substances in immunoassays (Kricka et al., 1983; Thorpe et al., 1985a).
In this study we describe the development of a solid phase immunoassay for aldosterone using an optimized enhanced chemiluminescent detection. MATERIALS AND METHODS
Aldosterone and other steroids were purchased from Steraloids (Wilton, USA), p-iodophenol was obtained from Aldrich-Chemie (FRG), horseradish peroxidase was obtained from VEB
*Author for correspondence. 0884-3996/901010049-04$05 .OO @ 1990 by John Wiley & Sons, Ltd.
Received October 1988 Revised February 1989
50 Germed (GDR), luminol and Protein A were purchased from Sigma-Chemie (FRG). Luminol was purified as described by Thorpe et al. (1985b). The Protein A or second antibody coated tubes were prepared as described elsewhere (Hubl et al. 1988). The antibodies against aldosterone were raised in rabbits and mouse, respectively, using aldosterone-18,21-diacetate3(0-carboxymethyl-)oxime bovine serum albumin conjugate, and the aldosterone-horseradish peroxidase conjugate was synthesized using the mixed anhydride reaction (Hubl et al., 1985). Serum or EDTA-plasma were used for specimen analysis.
W. HUBL ETAL.
RESULTS Variations of pH and buffers
Luminescence enhancement by p-iodophenol using the aldosterone-peroxidase conjugate was pH-dependent. In agreement with the results of Kricka and Thorpe (1983 and 1985) we found a maximal intensity with Tris-buffer at pH 8.6 and on the other hand with sodium-borate buffer between 8.0 and 9.5 and with phosphate buffer between 8.5 and 9.2. Luminollpiodophenol ratio
Luminescent enzyme immunoassay procedure
Plasma (0.05 ml) or saliva samples (0.10 ml) were extracted with 1.00 ml dichloromethane. The organic extract was dried under a stream of nitrogen. Extracts were dissolved in O.lOml assay buffer (0.1 mmolll phosphate, p H 7.4, 0.1% gelatine) and 0.05 ml were added to the Protein A (for monoclonal antibody) or second antibody (for the polyclonal antibody) coated tubes. Then 0.05 ml antibody dilution 1:50,000 for the monoclonal and 1:20,000 for the polyclonal antibody) and 0.10 ml aldosterone-peroxidase conjugate (dilution 1:10,000) were added to each of the tubes. The tubes were covered with plastic film and then incubated for 3 h at room temperature. The tubes were then emptied and washed three times with running tap water.
Figure 1 demonstrates the dependence of enhancement on different luminollp-iodophenol ratios with different buffer systems. The maximal luminescence was found at the ratio between 0.38 and 0.75 with the highest intensity using sodium borate buffer. Decreasing the Na-luminollp-iodophenol ratio to 0.25 produced lower photon counts.
60
lo
45
f2 X
v)
I-
z 3
Chemiluminescent measurement. Bound aldosterone-peroxidase conjugate was determined by adding 0.40 ml substrate solution which contained luminol (0.50 mmol/l), p-iodophenol (0.91 mmol/l) and hydrogen peroxide (1.8 mmol/l) in 0.13mol/l borate buffer (pH 8.6). The chemiluminescent reaction was measured between 10 and 25 min after initiation. Standard curve. Aldosterone standards (2770; 1390; 690; 345; 172; 86; 43; Opmolll) were added (0.050 ml) to the polystyrene tubes as the dissolved extracts and assayed as described above.
0
0
30
2
E
Pn 15
0
0:5 1.'o 1.I5 210 Na-luminol (mol/l) / p-iodophenol (rnol/l)
Figure 1. The influence of IuminoVpiodophenol ratio on light emission using different buffer systems
ALDOSTERONE IMMUNOASSAY
51
Table 1. Cross-reactivities of aldosterone-antisera Polyclonal Monoclonal Aldosterone Cortisol 17-OH-Progesterone Corticosterone Progesterone 1I-Deoxycortisol 11-Deoxycorticosterone Testosterone 18-OH-Deoxycorticosterone Tetra hydroaldosterone Spironolactone
100 0.0002 0.04 0.004 0.0001 0.04 0.0005 0.0001 0.005 1.o 0.0005
100 0.0004 0.0008 0.005 0.002 0.0009 0.01 0.0001 0.05 0.5 0.0001
Analytical recovery of added aldosterone
Addition of aldosterone 150 pmol/l produced a recovery of 92.5% (SD 1.8%, n = 20), at 320pmoVl the recovery was 90.6% (SD = 2.3%, n = 20).
Specificity of aldosterone-antisera
The cross-reactivities of different steroid hormones with the polyclonal and monoclonal antibodies are shown in Table 1. Both antisera are highly specific and enable the aldosterone determination in biological fluids without chromatography.
Kinetics of light emission
Different dilutions of aldosterone-peroxidase show a relatively constant light emission between 10 and 25min. The light emission of the conjugates with the lower concentrations used under immunoassay conditions (1:200,000) was constant for a longer time, between 10 and 40 min .
Detection limit
The detection limit was calculated by means of the 3 SD of the Bo and was found to be 100 fg/tube.
Precision studies
Comparison of aldosterone-LEIA with aldosterone-ELISA
Intra-assay variations ( n = 30) between 4.2% and 6.8% were found with low, medium and high concentrations of aldosterone (140-1 180 pmol/l). The inter-assay variations ( n = 20) were 4.5% to 7.3%.
Results obtained on 30 serum specimens using the aldosterone-ELISA (Hub1 et al., 1985) were compared with these from the aldosterone-LEIA. A correlation coefficient of 0.91 was found (y = 0.85 x 42).
+
Table 2. Reference intervals and clinical results of aldosteroneLElA Aldosterone (pmolll) mean (2 SD) Plasma Saliva Healthy adults ( n = 120) supine posture upright posture Conn's syndrome ( n = 4) Addison's Disease ( n = 4)
240 485 3460 62
(80-400) (180-790) (247C-4450) (21-103)
85 195 2080 24
(10-160) (70-320) (1350-2810)
(8-40)
Blood was taken from the Conn's and Addison's patients in an upright position.
W. HUBL ETAL.
Clinical applications
Acknowledgement
Table 2 presents the results of aldosterone determinations in plasma as well as in saliva samples of healthy adults and of patients with Conn’s syndrome and Addison’s disease.
The investigations are supported by the research project of Medizinische Laboratoriuccdiagnostik of the Ministry Of GDR.
DISCUSSION
The optimized enhanced chemiluminescent system allows the sensitive determination of aldosterone in biological fluids. We found a very low detection limit of 100fg/tube in comparison with the ELISA of 1pg/tube. The marked increase in luminescent signal leads to a further simplification of the equipment for the measurement without internal start pipetting and the application of a microtitreplate reader. We observed a significant linear correlation of the LEIA and the aldosterone-ELISA. The clinical application of the aldosteroneLEIA shows significantly increased concentrations in patients with Conn’s syndrome and very low aldosterone levels in patients with Addison’s disease. Furthermore, the sensitivity of the enhanced luminescent immunoassay allowed the aldosterone determination in saliva, too.
REFERENCES Hubl, W. (1985). Aldosterone. In Methods of Enzymatic Analysis, Bermeyer, H. U . (Ed.), VCH Verlagsgesellschaft, Weinheim, FRG. pp. 256266. Hubl, W., Daxenbichler, G., Meissner, D. and Thiele, H. J . (1988). An improved solid phase enzyme and luminescent immunoassay system for steroidhormones and digoxin. Clin. Chem., 34, 2521-2523. Kricka, L. J., Thorpe, G. H. G . and Whitehead, T. P. (1983). Enhanced luminescent or lurninometric assay. European Patent Publication, 116-454. Thorpe, G. H. G., Kricka, L. J., Moseley, S . B. and Whitehead, T. P. (1985a). Phenols as enhancers of the chemiluminescent horseradish peroxidase - luminol hydrogen peroxide reaction: application in luminescence monitored enzyme immunoassays. C h . Chem., 31, 1335-1341. Thorpe, G. H. G . , Williams, L. A., Kricka, L. J., Whitehead, T. P., Evans, H. and Stanworth, D. R. (1985b). A rapid luminescently monitored enzyme immunoassay for IgE. J . Immunol. Methods, 79, 51-63.
