Enzyme-Labelled Immunoassay for Plasma Cortisol TOSHIO OGIHARA, KIYOSHI MIYAI,* KEIKO NISHI,* KAICHIRO ISHIBASHI,** AND YUICHI KUMAHARA Department of Medicine and Geriatrics, the Central Laboratory for Clinical Investigation,' Osaka University Medical School, Osaka and Eiken Immunochemical Institute,** Tokyo, Japan ABSTRACT. An enzyme-labelled immunoassay for plasma cortisol was developed. For this alkalinephosphatase was conjugated through 21-hemisuccinate of cortisol using water-soluble carbodiimide. An antibody was raised in rabbits against cortdsol21-hemisuccinate bovine serum albumin. Before assay 10 fi\ samples of plasma were mixed with glutamate buffer, pH 3.3, and boiled to denature endogenous cortisol-binding globulin and alkalinephosphatase. Separation of "bound and free" cortisol was done by the double antibody method. A linear

relationship was obtained between the volume of plasma and the amount of cortisol. The minimal detectable level of plasma cortisol was 1 /xg/dl and the coefficients of variation were 2.7%-4.4% (within assays) and 4.7%-6.0% (between assays). Cortisol values determined by the present method correlated well with those determined by radioimmunoassay. The present method of enzyme-labelled immunoassay is suitable for routine clinical analysis of plasma cortisol. (J Clin Endocrinol Metab 44: 91,



metho-p-toluenesulphonate from Aldrich Chemicals, Inc. Alkaline phosphatase from Escherichia coli (Grade I) was obtained as a suspension of 5 mg of protein/ml in 3.2M ammonium sulphate from Boehringer, Mannheim. "K-phos-Eiken®," a kit for the Kind-King method of measuring alkaline phosphatase and "Cortisol kit Eiken®," a kit for cortisol RIA were supplied from Eiken Chemical Co. and Eiken Immunochemical Co., respectively, Tokyo, Japan. Heparinized blood was obtained from patients with various diseases for determination of plasma cortisol. "Cortisol poor plasma" was obtained from normal subjects who had received 2 mg of dexamethasone the previous night. "Cortisol poor plasma" was used to dilute standard cortisol after confirming that its cortisol level were less than that detectable by RIA.

METHOD for radioimmunoassay (RIA) of cortisol was developed by Ruder and co-workers (1), and subsequently modified by various groups (2-5). The number of compounds measured by RIA is increasing with a concomitant increase in radiation hazard and so non-radioisotopic immunoassays, such as viroimmunoassay (6) or enzyme-labelled immunoassay (7) have been developing in many hormone assay. Enzyme as a tracer of immunoassay also has the advantages of low cost and maintenance. The present paper reports a practical enzyme-labelled immunoassay for plasma cortisol. Materials and Methods

Preparation conjugate

Reagents and samples Cortisol and cortisol-21-hemisuccinate were purchased from Sigma, bovine serum albumin (BSA) Cohn fraction V from Armour, and 1-cyclohexyl - 3 - (2 - morpholinoethyl) - carbodiimide

of cortisol-alkaline


Cortisol-21-hemisuccinate-alkaline phosphatase conjugate was prepared using watersoluble carbodiimide as follows. A suspension of alkaline phosphatase (1 mg/200 (A) was centrifuged and the precipitate was dissolved in 0.7 ml of water and mixed with 10 mg of 1-cyclohexyl - 3 - (2 - morpholinoethyl) - carbodiimide metho-p-toluenesulphonate and 20 fjd of cortisol21-hemisuccinate dimethylformamide solution (60 mg/100 fjd). The mixture was gently stirred overnight at room temperature and the resultant emulsion was applied to a Sephadex G-200

Received April 26, 1976. Supported by grants from the Ministry of Education and the Ministry of Health and Welfare, Japan. Correspondence should be addressed to: Kiyoshi Miyai, M.D., The Central Laboratory for Clinical Investigation, Osaka University Hospital, Fukushima-ku, Osaka 553, Japan.


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JCE & M • 1977 Vol 44 • No 1

obtained from Endocrine Sciences, Tarzana, California. Second antibody (purified anti-rabbit •y-globulin goat serum) was obtained from Eiken Immunochemical Co.



Procedure of enzyme-labelled c 0.6

0.1 0.25 as i 2 4 Cortisol concentration (ng)

FIG. 1. The standard curve for cortisol enzyme-labelled immunoassay. Each point shows mean ± SD from 10 replicate determinations.

column (1 x 47 cm). The column was eluted with 10 ml of 0.05M phosphate buffer, pH 8.0, and fractions of 0.5 ml of eluate were collected. Determination of the alkaline-phosphatase activity in 0.01 ml of each fraction showed that the enzyme was eluted as a single peak. The 3 fractions with highest activity were combined, adjusted to 10 ml with the same buffer, and stored at 4 C. The enzyme activity and immunoreactivity of this conjugate were stable for at least 3 month at 4 C. This stock solution was diluted 800-fold with 0.05M phosphate buffer, pH 8.0, containing 0.5% normal rabbit serum (NRS) and 0.25% BSA immediately before use. Cortisol standard A stock solution of 100 /xg/ml of cortisol in ethanol was serially diluted with cortisol poor human plasma and 0.1 ml volumes of the standard solutions obtained (0 to 80 /xg/dl) were stored in a deep freezer. These standard solutions were stable for at least 6 months. Antiserum for cortisol immunoassay Antiserum to cortisol-21-hemisuccinate BSA raised in rabbits was used for both enzymelabelled assay and radioimmunoassay. The specificity of this antiserum was described elsewhere (8). Antiserum against cortisol 3-oxime was


Volumes of 10 /xl of standards or plasma were mixed with 200 fi\ of 0.6% glutamate buffer with 0.002% KCN, pH 3.3, in glass assay tubes. After thorough mixing, the solutions were heated in a boiling water bath for 15 min. The mixtures were then cooled and 500 fA of 1:4000 dilution of cortisol antiserum solution (final dilution, 1:6400) in 0.05M phosphate buffer, pH 8.0 and 100 ix\ of diluted enzyme-labelled cortisol solution were added with thorough mixing. The solution was then incubated for 60 min at room temperature. Then, 100 fi\ of second antisera (1:40) were added and incubation was continued for 16 h at 4 C. Then, 2 ml of water were added and the mixtures were centrifuged at 2000 x g The resulting precipitates were washed with 2 ml of water by centrifugation and aspiration, and dissolved in 1 ml of "substrate buffered reagent" containing phenylphosphate disodium salt (0.215 g/200 ml of 0.05M bicarbonate buffer, pH 10.15) as substrate and 4-aminoantipyrine (0.09 g/200 ml) as a reagent for quinone formation. The mixtures were incubated at 37 C for 30 min, and 1.0 ml of "color reagent" of the kit, containing potassium ferricyanide (0.38 g/200 ml of 0.2M borate) was added. This stopped the reaction and resulted in formation of a red quinone by coupling of 4-aminoantipyrine with phenol produced from the substrate. The absorbance was measured at 500 nm after 10 to 60 min using a Beckman SPECTA 30 spectrophotometer. A standard curve was obtained by plotting the O.D. values of standards directly on semi-log paper. All assays were done in duplicate. Radioimmunoassay for cortisol Cortisol RIA was done using a commercial kit from Eiken Immunochemical Co. (8). The method used was a modification of that of Foster and Dunn (2). 125I-cortisol (21-hemisuccinate tyrosine methyl ester) was used as a labelled antigen. Antiserum for cortisol 21-hemisuccinate BSA was used at 1:6400 dilution in this assay. "Free and bound" cortisol were separated with polyethylene glycol.

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Results Standard curve The standard curve for enzyme-labelled immunoassay of cortisol is shown in Fig. 1. The minimal amount of cortisol which could be measured with 95% confidence (i.e., 2 x SD at zero concentration) in the assay was 0.1 /Ag and the measurable range was 1 to 80 fxg/d\, using 10 /xl plasma samples. In Fig. 2 the standard curve for enzymelabelled immunoassay is replotted taking Bo as 100% for comparison with the standard curve for RIA. The standard curve for enzyme-labelled immunoassay is slightly steeper than that for RIA when compared at the 50% intercept. Little binding was observed using antiserum for cortisol-3-oxime BSA, and no displacement of the curve was observed even with 10 times more concentrated antiserum than that used for RIA. Dilution of samples Three plasma samples containing various concentrations of cortisol were diluted serially with cortisol poor plasma and their cortisol levels were determined by enzyme-

~ 40

g 30 g o _


o O xVs


x'/2 Dilution

FIG. 3. Effect of dilution of 3 plasma samples.

labelled immunoassay. As shown in Fig. 3, a linear relation was obtained between the dilution and the cortisol concentration for each sample. Specificity Cross-reactivity of other steroids in this enzyme-labelled immunoassay system were: corticosterone, 16.0%; desoxycortisol, 2.5%: progesterone, 4.3%; deoxycorticosterone, 0.003% at a level of 50% bound. And those in RIA system with the same antisera were: 16.8%, 3.4%, 4.0%, and 0.003%, respectively. Accuracy


100 RIA EnIA


The recovery of various amounts (2.5 to 50 /xg/dl) of cortisol added to three different pooled samples of plasma was 97.5 ± 2.0% (mean ± SEM) (Fig. 4). Comparison of enzyme-labelled assay and RIA



The cortisol levels in 28 plasma samples were determined by RIA and enzymelabelled immunoassay. Figure 5 shows that there was a good correlation between the values determined by the two methods.




V A-

1 Cortisol









FIG. 2. Comparison of the standard curves for enzymelabelled immunoassay (EnIA) and RIA.

Intra- and inter-assay assessed by assays on samples with low and high The intra-assay coefficient

variations were pooled plasma cortisol contents. of variation (CV)

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JCE & M • 1977 Vo! 44 • No 1



method for hapten immunoassay of morphine using lysozyme as label and VanWeemen and Schuurs reported an enzymelabelled immunoassay for estradiol using peroxidase (7). Subsequently, methods of enzyme-labelled immunoassay for other steroids have been reported (16,17). In general, non-isotopic immunoassays have low sensitivity, but a clinical method for assay of cortisol need not be very sensitive, because there is a relatively high concentration of cortisol in human blood. The method described in this paper has the same sensitivity and specificity as that in RIA, and the amount of antiserum required is the same as that for RIA. The accuracy and precision of this method are also quite sufficient for clinical purposes. Moreover, this method requires only a normal spectrophotometer, not special equipment, such as a liquidscintillation counter, a gamma-counter in RIA. Thus this method should be useful for routine analysis for human plasma corti0 10 20 30 40 sol. Cortisol added jjg/dl An antiserum against cortisol-3-oxime did FIG. 4. Study of accuracy of the method. not react in this system, possibly due to a difference in the relation of the site of conjufor analyses of 5 samples from pools of gation and the antigenicity of hapten, as high (mean 16.6 /u,g/dl) and low (mean 5.6 described by Midgley et al. (18). It is im/Ltg/dl) cortisol concentrations were 2.7% and 50 4.4%, respectively. The values for interY=1.0X-0.12 n0.99 n:28 P 2 0









FIG. 5. Comparison of plasma cortisol levels determined by enzyme-labelled immunoassay and RIA.

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CORTISOL ENZYME IMMUNOASSAY portant to ensure that the antigenic groups which are exposed upon the steroid-protein conjugate employed for immunization are also available in the enzyme-coupled steroid complex. To use the same site of the steroid molecule for combination with both carrier protein and enzyme is probably the most certain way to achieve this; however, it is not the only possibility. Acknowledgments We are grateful to Dr. and Mrs. A. Ichihara for their help in preparing the manuscript in English. We are also grateful to Dr. R. Osawa for his active interest in this work and to Ms. E. Kuroyama and T. Kawano for secretarial assistances.

References 1. Ruder, H. J., L. G. Robert, and M. B. Lipsett, A radioimmunoassay for cortisol in plasma and urine, J Clin Endocrinol Metab 35: 219, 1972. 2. Foster, L. B., and R. T. Dunn, Single-antibody technique for radioimmunoassay of cortisol in unextracted serum or plasma, Clin Chem 20: 365, 1974. 3. Tiang, N. S., D. Machacek, and O. P. Wadel, Comparison of clinical assays for serum corticosteroids, Clin Chem 21: 387, 1975. 4. Kao, M., S. Voina, A. Nichols, and R. Horton, Parallel radioimmunoassay for plasma cortisol and 11-deoxycortisol, Clin Chem 21: 1644, 1975. 5. Dash, R. J., B. G. England, A. R. Midgley, Jr., and G. D. Niswender, A specific, non-chromatographic radioimmunoassay for human plasma cortisol, Steroids 26: 647, 1975. 6. Andrieu, J. M., S. Mamas, and F. Dray, Viroimmunoassay of steroids, In Cameron, E. H. D., S. G. Hillier, and K. Griffiths (eds.), Method and Principles in Steroid Immunoassay, Alpha Omega Publishing Co., Ltd., Wales) 1975 p. 189.


7. Van Weemen, B. K., and A. H. W. M. Shuurs, Immunoassay using hapten enzyme conjugate, FEBS Letters 24: 77, 1972. 8. Nishi, K., T. Ogihara, K. Miyai, T. Kumahara, and K. Ishibashi, Plasma cortisol radioimmunoassay with 125I-cortisol and polyethylene glycol, Jap J Nucl Med 13: 469, 1976. 9. Landon, J., J. Grookall, and A. McGregor, Enzyme-labelled immunoassays for steroids in steroid immunoassay, In Cameron, E. H. D., S. G. Hillier, and K. Giffith (eds.), Methods and Principles in Steroid Immunoassay, Alpha Omega Publishing Co., Ltd., (Wales), 1975 p. 183. 10. Engvall, E., and P. Perlmann, Enzyme-linked immunosorbent assay (ELISA), Quantitative assay of immunoglobulin G., Immunochemistry 8: 871, 1971. 11. Van Weemen, B. D., and A. H. W. M. Schuurs, Immunoassay using antigen-enzyme conjugate, FEBS Letters 15: 232, 1971. 12. Ishikawa, E., Enzyme immunoassay of insulin by fluorimetry of the insulin-glucoamylase complex, J Biochem (Tokyo) 73: 1319, 1973. 13. Miyai, K., K. Ishibashi, and Y. Kumahara, Enzymelinked immunoassay of thyrotropin, Clin Chim Ada 67: 263, 1976. 14. Kitagawa, T., and T. Aikawa, Enzyme coupled immunoassay of insulin using a novel coupling reagent, 7 Biochem (Tokyo) 79: 233, 1976. 15. Rubenstein, K. E., R. S. Schneider, and E. F. Ullman, Homogeneous enzyme immunoassay. A new immunochemical technique, Biochem Biophys Res Commun 47: 846, 1972. 16. Dray, F., J. A. Andrieu, and F. Renaud, Enzyme immunoassay of progesterone at the picogram level using /3-galactosidase as label, Biochim Biophys Ada 403: 131, 1975. 17. Comoglio, S., and F. Celada, An immuno-enzymatic assay of cortisol using E. coli /3-galactosidase as label,/ Immunol Methods 10: 161, 1976. 18. Midgley, A. R., Jr., and G. D. Niswender, Radioimmunoassay of steroids, Ada Endocrinol [Suppl] (Kbh) 147: 320, 1971.

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Enzyme-labelled immunoassay for plasma cortisol.

Enzyme-Labelled Immunoassay for Plasma Cortisol TOSHIO OGIHARA, KIYOSHI MIYAI,* KEIKO NISHI,* KAICHIRO ISHIBASHI,** AND YUICHI KUMAHARA Department of...
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