A Non-Chromatographic Non-Extraction Radioimmunoassay for Serum Aldosterone TOSHIO OGIHARA, KAZUSHIGE IINUMA,* KEIKO NISHI, YUKINOBU ARAKAWA,* ATSUSHI TAKAGI,* KUNIO KURATA,* KIYOSHI MIYAI, AND YUICHI KUMAHARA Department of Medicine and Geriatrics, Central Laboratory for Clinical Investigation, Osaka University Medical School, Osaka, Japan, and Dainabot Radioisotope Laboratories,* Tokyo, Japan method using tritiated aldosterone, 2) a method using dichloromethane extraction before assay, and 3) a commercial kit method. The intra-assay coefficient of variation was 6.9%, and the inter-assay coefficient of variation was 10.7%. The values found in normal human serum were comparable with those reported using other methods. The present radioimmunoassay eliminates both extraction of aldosterone from serum and chromatographic separation and requires only 0.1 ml of serum for assay. (J Clin Endocrinol Metab 45: 726, 1977)
ABSTRACT. A direct radioimmunoassay for serum aldosterone was developed using a highly specific antibody and 8-anilino-l-naphthalene sulfonic acid as a blocking agent to inhibit the binding of aldosterone to serum proteins. 125I-labeled aldosterone was used as the labeled antigen and polyethylene glycol was used to separate antibody-bound and free aldosterone. The minimum detectable concentration was 1.5 pg/tube. There were excellent correlations between the present method and other methods, i.e., 1) a
D
ETERMINATION of aldosterone in plasma has greatly advanced since the development of radioimmunoassays by Mayes et al. (1) and by Bayard et al. (2) in 1970. Many simplified methods for aldosterone radioimmunoassay have been reported during the last few years (3-8). The assays reported by McKenzie et al. (7) and by Pham-Huu-Trung et al. (8) are the most simplified methods at present; however, they still require extraction of the sample by organic solvents before assay. In the present study we report a further simplification of radioimmunoassay of serum aldosterone in which both extraction and chromatography can be eliminated by using a highly specific antiserum, an agent to inhibit binding of aldosterone to serum proteins, 125I-aldosterone, and polyethylene glycol. Received March 1, 1977. Supported in part by grants from the Ministry of Education, Japan. Correspondence should be addressed to: Toshio Ogihara, M.D., Department of Medicine and Geriatrics, Osaka University Hospital, Fukushima-ku, Osaka, 553, Japan.
Materials and Methods Reagents All steroids were purchased from Sigma Chemical Co. except spironolactone and canrenone (Searle Laboratory) and used without further purification. Bovine gamma globulin was obtained from Miles Co., calf serum from Chiba Kessei Laboratory, 8-anilino-l-naphthalene-sulfonic acid (ANS) from Eastman Kodak Co., complete Freund's adjuvant from Difco, and polyethylene glycol 6000 (PEG) from Wako Pure Chemical. Ethanol and dichloromethane (Wako Pure Chemical) were spectrograde and were used without purification. l,2-3H-aldosterone was purchased from New England Nuclear Co., Boston, Mass., and was used after checking purity by paper chromatography (1). Na125I solution (1117 mCi/jag I) was obtained from Radiochemical Centre, London. A radioimmunoassay kit for aldosterone using l,2-3H-aldosterone was obtained from Cea Ire Sorin (C.I.S.), France. Antigen and immunization Aldosterone-3-oxime was prepared and conjugated with bovine gamma globulin by the method described by McKenzie and Clements (7). Five New Zealand female rabbits were immunized
726
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 02 June 2015. at 15:05 For personal use only. No other uses without permission. . All rights reserved.
RADIOIMMUNOASSAY FOR SERUM ALDOSTERONE according to the method described by Vaitukaitis et al. (9). Approximately 100 /xg of aldosterone-bovine gamma globulin conjugate emulsified with Freund's complete adjuvant was used for the initial injection in each rabbit. Four boosters of 200 /Ag were injected at monthly intervals for 4 months. Blood was drawn one week after the final injection. 125
I-aldosterone solution
A phenol ring was coupled to aldosterone as a 3-oxime as reported by Nars and radioiodinated by the method of Hunter and Greenwood (10, 11). The reaction mixture was purified by thin layer chromatography (benzene:methanol, 7:3). After 2 h development the 125I-aldosterone fraction was eluted with ethanol. The specific activity of the purified 125I-aldosterone was 10002000 mCi/mg. One-tenth ml of 125I-aldosterone in ethanol was diluted with 500 ml of a 358: 642 mixture of 0.1M citrate and 0.2M disodium phosphate buffers, pH 6.0, containing 0.2% bovine gamma globulin. This solution was kept frozen at —20 C until immediately before use. 125 I-aldosterone in this condition was stable and useful for radioimmunoassay for at least 4 weeks. Aldosterone-free calf serum A charcoal column (1 x 40 cm) was prepared by filling a glass column with charcoal suspension which was washed with distilled water. Two hundred ml of calf serum was passed through the column unrestrictedly. The initial 40 ml was discarded and the following eluate was collected and used as aldosterone-free calf serum.
727
The methods with extraction and with 3Haldosterone were similar to the direct method. However, in the extraction method, 0.2 ml volumes of serum samples and standard solutions were extracted with 2 ml of dichloromethane and 1 ml of the organic aliquot was used for assay after evaporation. In the method using 3Haldosterone, after separation of bound and free aldosterone 0.5 ml volumes of supernatant were transferred to counting vials containing 10 ml of Bray's solution and counted with a liquid scintillation counter. The C.I.S. kit was used as indicated in the brochure. The method of this kit was essentially based on the method of Mckenzie et al. (7).
Results Standard curve The standard curve of the assay is shown in Fig. 1. The minimal amount of aldosterone which could be measured with 95% confidence {i.e., 2 x SD at zero) was 1.5 pg and the measurable range was 1.5 to 160 pg. Aldosterone levels of samples from two adrenalectomized patients and one patient with Addison's disease were assayed to examine the blank. All of these samples had aldosterone values less than the sensitivity 60 r
Measurement of serum aldosterone One-tenth ml serum samples or standard aldosterone solutions (0 pg/ml to 1600 pg/ml) in aldosterone-free calf serum, 0.1 ml of the 125I-aldosterone solution (about 10,000 cpm), and 0.5 ml of a 1:5,000 dilution of the antiserum in 0 . 1 M borate buffer, pH 8.6, containing ANS (960 /ug/ml) were mixed with a Vortex in a disposable glass tube. After 20 h incubation at 4 C, 1 ml of 25% polyethylene glycol in 0.1M borate buffer, pH 8.6, was added. All tubes were mixed with a Vortex for 5 to 10 sec, and centrifuged at 2264 g for 15 min. The supernatant was aspirated and the 125I in the precipitate was counted using a scintillation counter.
i 20 "
20
40
80
160
aldosterone (pg)
FIG. 1. Standard curve of aldosterone radioimmunoassay. Points are mean values and brackets indicate one standard deviation (n = 10).
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 02 June 2015. at 15:05 For personal use only. No other uses without permission. . All rights reserved.
728
OGIHARA ET AL.
level. Standard curves obtained with aldosterone added to charcoal treated calf serum and with aldosterone added to serum from adrenalectomized patient were superimposable. Non-specific binding was less than 5% in all 10 samples from different subjects.
JCE & M • 1977 Vol 45 • No 4
150
Specificity The specificity of the antibody was tested and expressed according to the method described by Abraham (12). The results are summarized in Table 1. Dilution study Four serum samples containing different concentrations of aldosterone were diluted serially with charcoal treated calf serum and their aldosterone levels assayed. A linear relation was obtained between the sample dilution and the aldosterone concentrations for each sample (Fig. 2).
50
FIG. 2. Dilution study. Four sera were diluted with charcoal treated calf serum.
Precision Accuracy The recovery of various amounts of aldosterone added to two different pooled samples of serum was 101.2 ± 8.3% (mean ± SD). The correlations of aldosterone recovered and aldosterone added were linear and the y axis intercept of the each regression line was close to the value obtained on assay of each original pooled serum (Fig. 3). TABLE 1. Cross-reactivity of the antiserum with various steroids Steroids
Cross-reaction
Aldosterone Corticosterone Deoxycorticosterone Testosterone Progesterone Canrenone Cortisol Cortisone DHEA DHEA-sulfate Spironolactone Tetrahydrocortisol Tetrahydrocortisone
100 0.0019 0.0009 0.0003 0.0002
ABSTRACT. A direct radioimmunoassay for serum aldosterone was developed using a highly specific antibody and 8-anilino-l-naphthalene sulfonic acid as a blocking agent to inhibit the binding of aldosterone to serum proteins. 125I-labeled aldosterone was used as the labeled antigen and polyethylene glycol was used to separate antibody-bound and free aldosterone. The minimum detectable concentration was 1.5 pg/tube. There were excellent correlations between the present method and other methods, i.e., 1) a
D
ETERMINATION of aldosterone in plasma has greatly advanced since the development of radioimmunoassays by Mayes et al. (1) and by Bayard et al. (2) in 1970. Many simplified methods for aldosterone radioimmunoassay have been reported during the last few years (3-8). The assays reported by McKenzie et al. (7) and by Pham-Huu-Trung et al. (8) are the most simplified methods at present; however, they still require extraction of the sample by organic solvents before assay. In the present study we report a further simplification of radioimmunoassay of serum aldosterone in which both extraction and chromatography can be eliminated by using a highly specific antiserum, an agent to inhibit binding of aldosterone to serum proteins, 125I-aldosterone, and polyethylene glycol. Received March 1, 1977. Supported in part by grants from the Ministry of Education, Japan. Correspondence should be addressed to: Toshio Ogihara, M.D., Department of Medicine and Geriatrics, Osaka University Hospital, Fukushima-ku, Osaka, 553, Japan.
Materials and Methods Reagents All steroids were purchased from Sigma Chemical Co. except spironolactone and canrenone (Searle Laboratory) and used without further purification. Bovine gamma globulin was obtained from Miles Co., calf serum from Chiba Kessei Laboratory, 8-anilino-l-naphthalene-sulfonic acid (ANS) from Eastman Kodak Co., complete Freund's adjuvant from Difco, and polyethylene glycol 6000 (PEG) from Wako Pure Chemical. Ethanol and dichloromethane (Wako Pure Chemical) were spectrograde and were used without purification. l,2-3H-aldosterone was purchased from New England Nuclear Co., Boston, Mass., and was used after checking purity by paper chromatography (1). Na125I solution (1117 mCi/jag I) was obtained from Radiochemical Centre, London. A radioimmunoassay kit for aldosterone using l,2-3H-aldosterone was obtained from Cea Ire Sorin (C.I.S.), France. Antigen and immunization Aldosterone-3-oxime was prepared and conjugated with bovine gamma globulin by the method described by McKenzie and Clements (7). Five New Zealand female rabbits were immunized
726
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 02 June 2015. at 15:05 For personal use only. No other uses without permission. . All rights reserved.
RADIOIMMUNOASSAY FOR SERUM ALDOSTERONE according to the method described by Vaitukaitis et al. (9). Approximately 100 /xg of aldosterone-bovine gamma globulin conjugate emulsified with Freund's complete adjuvant was used for the initial injection in each rabbit. Four boosters of 200 /Ag were injected at monthly intervals for 4 months. Blood was drawn one week after the final injection. 125
I-aldosterone solution
A phenol ring was coupled to aldosterone as a 3-oxime as reported by Nars and radioiodinated by the method of Hunter and Greenwood (10, 11). The reaction mixture was purified by thin layer chromatography (benzene:methanol, 7:3). After 2 h development the 125I-aldosterone fraction was eluted with ethanol. The specific activity of the purified 125I-aldosterone was 10002000 mCi/mg. One-tenth ml of 125I-aldosterone in ethanol was diluted with 500 ml of a 358: 642 mixture of 0.1M citrate and 0.2M disodium phosphate buffers, pH 6.0, containing 0.2% bovine gamma globulin. This solution was kept frozen at —20 C until immediately before use. 125 I-aldosterone in this condition was stable and useful for radioimmunoassay for at least 4 weeks. Aldosterone-free calf serum A charcoal column (1 x 40 cm) was prepared by filling a glass column with charcoal suspension which was washed with distilled water. Two hundred ml of calf serum was passed through the column unrestrictedly. The initial 40 ml was discarded and the following eluate was collected and used as aldosterone-free calf serum.
727
The methods with extraction and with 3Haldosterone were similar to the direct method. However, in the extraction method, 0.2 ml volumes of serum samples and standard solutions were extracted with 2 ml of dichloromethane and 1 ml of the organic aliquot was used for assay after evaporation. In the method using 3Haldosterone, after separation of bound and free aldosterone 0.5 ml volumes of supernatant were transferred to counting vials containing 10 ml of Bray's solution and counted with a liquid scintillation counter. The C.I.S. kit was used as indicated in the brochure. The method of this kit was essentially based on the method of Mckenzie et al. (7).
Results Standard curve The standard curve of the assay is shown in Fig. 1. The minimal amount of aldosterone which could be measured with 95% confidence {i.e., 2 x SD at zero) was 1.5 pg and the measurable range was 1.5 to 160 pg. Aldosterone levels of samples from two adrenalectomized patients and one patient with Addison's disease were assayed to examine the blank. All of these samples had aldosterone values less than the sensitivity 60 r
Measurement of serum aldosterone One-tenth ml serum samples or standard aldosterone solutions (0 pg/ml to 1600 pg/ml) in aldosterone-free calf serum, 0.1 ml of the 125I-aldosterone solution (about 10,000 cpm), and 0.5 ml of a 1:5,000 dilution of the antiserum in 0 . 1 M borate buffer, pH 8.6, containing ANS (960 /ug/ml) were mixed with a Vortex in a disposable glass tube. After 20 h incubation at 4 C, 1 ml of 25% polyethylene glycol in 0.1M borate buffer, pH 8.6, was added. All tubes were mixed with a Vortex for 5 to 10 sec, and centrifuged at 2264 g for 15 min. The supernatant was aspirated and the 125I in the precipitate was counted using a scintillation counter.
i 20 "
20
40
80
160
aldosterone (pg)
FIG. 1. Standard curve of aldosterone radioimmunoassay. Points are mean values and brackets indicate one standard deviation (n = 10).
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 02 June 2015. at 15:05 For personal use only. No other uses without permission. . All rights reserved.
728
OGIHARA ET AL.
level. Standard curves obtained with aldosterone added to charcoal treated calf serum and with aldosterone added to serum from adrenalectomized patient were superimposable. Non-specific binding was less than 5% in all 10 samples from different subjects.
JCE & M • 1977 Vol 45 • No 4
150
Specificity The specificity of the antibody was tested and expressed according to the method described by Abraham (12). The results are summarized in Table 1. Dilution study Four serum samples containing different concentrations of aldosterone were diluted serially with charcoal treated calf serum and their aldosterone levels assayed. A linear relation was obtained between the sample dilution and the aldosterone concentrations for each sample (Fig. 2).
50
FIG. 2. Dilution study. Four sera were diluted with charcoal treated calf serum.
Precision Accuracy The recovery of various amounts of aldosterone added to two different pooled samples of serum was 101.2 ± 8.3% (mean ± SD). The correlations of aldosterone recovered and aldosterone added were linear and the y axis intercept of the each regression line was close to the value obtained on assay of each original pooled serum (Fig. 3). TABLE 1. Cross-reactivity of the antiserum with various steroids Steroids
Cross-reaction
Aldosterone Corticosterone Deoxycorticosterone Testosterone Progesterone Canrenone Cortisol Cortisone DHEA DHEA-sulfate Spironolactone Tetrahydrocortisol Tetrahydrocortisone
100 0.0019 0.0009 0.0003 0.0002
