Clinica Chimica Acta, 208 (1992) 233-235 0 1992 Elsevier Science Publishers B.V. All rights reserved.

233 0009~8981/92/$05.00

CCA 05313

Letter to the Editor

Enzyme immunoassay for serum cortisol (Received

25 September

1991; revision

Key words; Plasma



5 May 1992; accepted


12 May 1992)


Dear Editor, Various investigators have demonstrated a good correlation between radioimmunoassays (RIA) and enzyme immunoassays (EIA) for blood cortisol [l-3]. Because chronobiologic studies require repeat assays, we felt it interesting to use EIA to investigate the circadian rhythm of cortisol. Two female patients on antidepressant medication (a 43-year-old patient with secondary depression and a 74-year-old patient with unipolar endogenous depression as defined by DSM III criteria) and one control (a 47-year-old man) were investigated. EIA data were compared to results obtained with RIA. Blood sampling was begun at 09:OOand was continued every hour until 08:OOthe following morning. After centrifugation at 4°C the samples were frozen at -20°C until assayed. Serum cortisol was measured by RIA and EIA, in duplicate, for all three subjects. All samples from an individual subject were measured in the same run. Both methods were direct, without deproteinization. RIA consisted in competition between the labelled cortisol and cortisol in the sample for the cortisol antibody binding sites on coated tubes (Cat0 method) (CIA assay; Commissariat a 1’Energie Atomique, Gif-sur-Yvette, France). EIA involved competition between &galactosidase-labelled cortisol coupled by succinidization and cortisol in samples for the cortisol antibody. The immune complexes were then immobilized on magnetic particle goat anti-rabbit antibody and P-galactosidase activity was measured in the presence of the specific enzyme substrate ONPG (ortho-nitrophenyl-&galactopyranoside) (Magnelia Cortisol, Institut Pasteur, Marnes la Coquette, France). The coefficients of variation of the two control sera (215 and 860 nM) assayed in duplicate in each serum sample were, respectively, 5 f 2 and 4 f 1.5% for RIA and Correspondence to: Dr. Mirande Candito, Laboratoire voie Romaine, 06031 Nice Cedex, France.

de Biochimie,



30, avenue de la

234 TABLE I 24-h mean, standard deviation, minimum and maximum RIA and EIA values (nmol/l) for the three study subjects Subject



41 43




Psychiatric state

Antidepressant medication

Control No Secondary Yes depression Unipolar Yes depression



24 h SD. mean



24 h SD. mean



212 218

155 85

30 81

552 373

342 265

137 85

152 124

607 442









8.5 f 3.5 and 7.2 f 2.1% for EIA. The thresholds of detection were 15 nmol/l for RIA and 20 nmol/l for EIA. Analysis of variance by the Fisher test did not reveal any significant difference in the mean values of the depressed patients under antidepressant treatment and the control (Table I). The wide standard deviations and differences between the maximum and minimum levels can be explained by the existence of a circadian rhythm. EIA and RIA values reflected the clear circadian rhythm described in the literature: morning acrophase, progressive decline throughout the afternoon and nadir at night. However, while the serum cortisol values given by the two methods were similar for the two depressed patients, the two techniques were not as well correlated for the control (general equation of the line of correlation = y = 94 + 0.90 x; R = 0.80, where y = RIA value and x = EIA value). In this study, the control’s RIA and EIA values gave the same acrophase and the same amplitude, but differed for the bathyphase: this subject’s RIA levels remained elevated whereas his EIA levels dropped. Both EIA and RIA were performed with rabbit polyclonal anticortisol prepared against cortisol-21-hemisuccinate (CHS) coupled with bovine serum albumin (BSA). The two immune sera presented different cross-reactions: 37% with 1 1-deoxycortisol in EIA (1.8% for RIA); 51% with prednisolone in RIA (12% for EIA). However, none of our subjects was receiving metyrapone or prednisolone. In RIA, [‘25-I]cortisol labelling modifies the kinetics between the labeled cortisol and the cortisol in the test sample. Similarly, the mode of fl-galactosidase conjugation in position 21 can alter the sensitivity of EIA. Although the conjugates showed elevated immunoreactivity with the antibody, they are readily displaced by the cortisol to be assayed; this might explain the lower cortisol levels dosed by EIA in the control [4]. The kinetics of EIA immune complex formation may be disturbed by the high steroid concentration of the enzymaticallylabelled cortisol. By contrast, in RIA, a state of equilibrium is rapidly reached between the antibody and a small hapten [5,6]. This situation, together with the fact that shortening of the incubation time in immunologic reactions plays a role in both


association and dissociation of immune complexes [7] and what Pratt termed the ‘first come, first served’ effect, could result in a loss of specificity [8]. This loss would be greater in EIA, where more time is required for conjugate displacement. The circadian rhythm of cortisol demonstrated by RIA and EIA for the control subject and the two depressed patients is the same as that described in the literature for this time of the year (month of May). The unipolar depressed patient presented a non-significant tendency for advance of maximum secretion (advance of the minimum level described in the literature) [9]. While cortisol levels in this study varied considerably owing to variations in secretion related to the time of day or pathology, the good correlation noted between the two methods allowed detection of hypo- or hypersecretion. However, the punctual differences observed in cortisol levels require comparison with control levels regardless of the method utilized. Mirande Canditoa, Bernard Krebsb and Pierre Chambona uLaboratoire de Biochimie. H6pital Pasteur, 30, avenue de la voie Romaine, 06031 Nice Cedex and bCentre Antoine-Lacassagne, 36, voie Romaine, 06054 Nice Cedex (France)

References Izquierdo JM, Quiros A, Alvarez-Uria J, Sotorrio P. Homogeneous tisol with a centrifugal analyser. Clin Chem 1984;30:1824.

enzyme immunoassay

for cor-

Dhar TK, Voss E, Schoneshijfer M. Enzyme immunoassay of serum cortisol using a new transferrable needle lid technique. Clin Chim Acta 1986;157:231. Dona V. Simple, direct coated-tube enzyme immunoassay of cortisol in serum. Clin Chem 1987;33:422. Monji N, Gomez NO, Kawashima H, Ali H, Castro A. Practical enzyme immunoassay for plasma cortisol using /3-galactosidase as enzyme label. J Clin Endocrinol Metab 1980;50:355. Kominami G, Fujisaka I, Yamauchi A, Kono M. A sensitive enzyme immunoassay for plasma cortisol. Clin Chim Acta 1980;103:381. Bressot N, Zanca M, Bordebat C, Silhol MJ. Etude comparative du cortisol plasmatique. Trait d’Union 1987;7:27. Vining RF, Compton P, McGinley R. Steroid radioimmunoassay time on specificity. Clin Chem 1981;27:910.

de differentes -


Effect of shortened

de dosage incubation

Pratt JJ, Woldring MG. Radioimmunoassay specificity and the first-come, first-served effect. Clin Chim Acta 1976;698:87. Linkowski P, Medlewicz J, Leclercq R, Brisseur M, Hubain P, Golstein J, Copinschi G, Van Cauter E. The 24-hour profile of adrenocorticotropin and cortisol in major depressive illness, J Clin Endocrinol



Enzyme immunoassay for serum cortisol.

Clinica Chimica Acta, 208 (1992) 233-235 0 1992 Elsevier Science Publishers B.V. All rights reserved. 233 0009~8981/92/$05.00 CCA 05313 Letter to t...
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