Clin. Lab.

2004; 50; 11-24

©Copyright

ORIGINAL ARTICLE

Determination of Cortisol in Saliva and Serum by a

Luminescence-Enhanced Enzyme Immunoassay
JÜRGEN WESTERMANN, ANKE DEMIR, VICTOR HERBST
Immuno-Biological Laboratories GmbH, Hamburg,Germany

SUMMARY

A new luminescence-enhanced enzyme immunoassay (LEIA) has been developed and validated for the direct mea-
surement of cortisol in saliva and serum. It has been demonstrated that this LEIA has a very good analytical and
functional sensitivity. There was a good correlation to a commercial RIA. The assessment of diurnal cortisol pro-
files of healthy persons are discussed. Cortisol monitoring is indicated in diseases with abnormal glucocorticoid
production such as Cushing`s syndrome and Addison’s disease. Because of the diurnal fluctuation of cortisol levels
it is necessary to take several samples for an individual cortisol profile or during dynamic tests like the dexa-
methasone-suppression or ACTH stimulation. Salivary sample collection is an alternative method without the
stress of repeated venipuncture. The measurement of cortisol in saliva is advisable in patients with abnormal
cortisol-binding-globulin (CBG) levels such as pregnancy, hypothyroidism, nephrotic syndrome or marked adi-
positas and during the administration of certain drugs, especially oral contraceptives.

KEY WORDS
Cortisol, saliva, luminescence, immunoassay, diurnal
profile, free hormones
INTRODUCTION
Only the non protein bound fraction of the lipophilic
steroid hormones passes quantitatively from the circula-
tion into the saliva (1). This enables scientists to directly
assess the biologically active fraction of these hor-
mones. In addition, using saliva makes it possible to con-
veniently obtain several samples for assessing hormone
profiles. As a result, using saliva as a sample signifi-
cantly extends the application of hormone testing in
such scientific fields as psychology, occupational medi-
cine, sport medicine and pediatrics. (2 - 12).
About 90 % of the cortisol in the circulation is bound to
the corticosteroid-binding globulin (CBG, transcortin),
about 7 % is bound loosely to albumin, and only 1 – 3 %
is unbound. Only cortisol from the two latter fractions is
Manuscript accepted May 8, 2003
released from the salivary glands (1). The normal sali-
vary cortisol concentration in the evening is about 1.5
nmol/l (13). It is therefore necessary to develop an assay
which enables observers to determine such small levels
of cortisol in saliva accurately. In using non-radioiso-
topic immunoasssays it is best to use a chemilumines
cent or a time resolved fluorescence labelled immuno-
assay because of the superior analytical sensitivity (14 -
16).
In saliva there is a diurnal fluctuation of the cortisol
concentration in adult humans, reaching a peak around
30 to 60 minutes after awakening and falling to lower
levels in the afternoon which remains trough the next
morning (13, 17 - 20). The circadian pacemaker is re-
sponsible for this cortisol variation, which also influ-
ences the diurnal melatonin profile, is located in the su-
prachiasmic nuclei of the hypothalamus (13, 21). New-
borns do not exhibit a diurnal cortisol profile. It devel-
ops during the first 20 weeks after birth (22 – 25). Even
in adults large inter- individual differences in the di-
urnal cortisol pattern exists (26). It is advantageous to
take saliva samples in connection with the individual
waking time, rather than blood samples at fixed inter-
vals for the accurate assessment of daily variation.

Clin. Lab. 1+2/2004 11

 JÜRGEN WESTERMANN et al.
MATERIALS AND METHODS
Chemicals
All fine chemicals were purchased from Sigma-Aldrich
(Deisenkirchen, Germany) or Merck (Darmstadt, Ger-
many). Cortisol and compounds used for cross-reacti-
vity studies were obtained from Steraloids, Inc. (New-
port, Rhode Island USA).
Reagents
All reagents used in the LEIA kit were provided by the
manufacturer (IBL Hamburg GmbH; Hamburg, Ger-
many).
Microtiter Strips: ready for use; white microtiter plate
wells (Maxisorp, Nunc; Roskilde, Denmark) coated
with goat anti-rabbit IgG and anti-cortisol antibody
(Scantibody Laboratory Inc., Santee, CA, USA and
Prof. Vescei Heidelberg, Germany), and blocked with
BSA (Sigma) and Karion (Merck).
Enzyme Conjugate: ready for use; Cortisol-3-carboxy-
methyloxim was first coupled to horseradish peroxidase
with the mixed anhydride method and then dialyzed
against phosphate-buffered saline. The peroxidase con-
jugate was diluted in Stabilzyme® buffer (Sur Modics
Inc., Eden Prairie, MN, USA).
Saliva Control 1 and 2: ready for use; Saliva was en-
riched with different amounts of cortisol with 0.01 %
thimerosal as a preservative.
Standard A - G: ready for use; contained cortisol in 0.1
mol/l phosphate buffer with 9 g/l NaCl, 0.1 %(w/v)
BSA and 0.01%(w/v) thimerosal. The standard concen-
trations are 0 nmol/l (Standard A, zero standard), 0.83
nmol/l (Standard B), 1.7 nmol/l (Standard C), 5.5 nmol/l
(Standard D), 17 nmol/l (Standard E), 41 nmol/l (Stan-
dard F), 110 nmol/l (Standard G).
Wash Buffer: concentrate; phosphate buffer (0.1 mol/l,
pH 7.3) containing of 9g/l NaCl, 0.1 % (w/v) Tween 80
and 0.01%(w/v) thimerosal. This was diluted 1 to 10
with distilled water prior to use.
Substrate Reagents: Reagent 1 and reagent 2 were ob-
tained from Perbio Science, (Bonn, Germany). Reagent
1 contained luminol and an enhancer. Reagent 2 con-
sisted of a peroxide solution with stabilizers. Reagents 1
and 2 were mixed in equal amounts prior to use.
Adhesive Foil: transparent plastic foils.
All kit reagents were stable for at least 9 months when
stored at 2 - 8 °C. The diluted wash buffer was stable
for up to 4 weeks at 2 - 8 °C and the mixed substrate
reagent is stable for 8 hours at room temperature.
Samples for the diurnal profiles
Saliva was collected with Salivettes® (Sarstedt, Nüm-
brecht, Germany) or with a straw into Polypropylene
tubes (IBL, Hamburg, Germany) every 30 minutes for 4
hours after awakening. Then every hour for 15 hours a
sample was taken. The persons included in this study
were clinically healthy on a normal diet. The samples
12
were collected over a normal working day. Shift wor-
kers were not included in the study. The test persons
were not on drugs which might have affected the corti-
sol level.
Adult Females Adult Males Children
Elderly
subjects
Number 61 39 4 (3f,1m) 10 (4f,6m)
Age
28.8 ± 11.1 32.6 ± 13.0 8.8 ± 5.1 67.5 ± 4.1
(mean ± SD)
Range
17 - 60 17 – 59 3 – 13 62 – 77
(min-max)
Samples for saliva - serum correlation.
Paired saliva and serum samples were obtained from 84
clinically healthy volunteers within 10 minutes of each
other.
Samples for GC/MS correlation of serum samples
As reference method comparison, 20 quality assessment
serum samples from the DGKC (Deutsche Gesellschaft
für klinische Chemie, Bonn, Germany) were used for
hormone measurement were used. These samples were
predetermined by the reference laboratory of the DGKC
(Referenzinstitut für Bioanalytik, Bonn, Germany) us-
ing a GC/MS reference method for determination of
Cortisol.
Quality control samples
Control samples were prepared by adding different
stock solutions with known cortisol concentrations to
individual saliva and serum samples from normal
volunteers. The controls were aliquoted and stored at -
80 °C.
LEIA kit
Handling of samples: Saliva samples were collected by
either using Salivettes® (Sarstedt) without additives or
by transferring saliva with a straw into a glass or poly-
propylene tube (IBL, Hamburg, Germany), after saliva
flow has been stimulated by chewing on a piece of Para-
filmTM (American National Can, Chicago, IL,USA).
Samples with a slightly reddish color, suspected of be-
ing contaminated with blood, were discarded.
It is highly recommended to freeze the samples once at
–20°C prior to the test run in order to precipitate inter-
fering mucoproteins. After thawing and before assay,
the samples were centrifuged at 2000 - 3000 x g to re-
move solids. Repeated freeze-thaw cycles should be
avoided. The saliva samples may be stored for up to 5
days at room temperature or up to 10 days at 2 - 8°C.
For longer periods the saliva should be stored in ali-
quots at –20°C.
Clin. Lab. 1+2/2004
 DETERMINATION OF CORTISOL BY A LUMINESCENCE-ENHANCED ENZYME IMMUNOASSAY

Table 1: Specificity of the anti-cortisol antibody used in the described LEIA

Compound Cross-reactivity [%]

Cortisol, hydrocortisone (4-Pregnen-11β,17α,21-triol-3,20-dion) 100
Prednisolone (1,4-Pregnadien-11β,17α,21-triol-3,20-dion) 57
11-Deoxycortisol (4-Pregnen-17α,21-diol-3,20-dion 12
Corticosterone (4-Pregnen-11β,21-diol-3,20-dion) 2.5
Cortisone (4-Pregnen-17α,21-diol-3,11,20-trion) 2
Prednisone (1,4-Pregnadien-17α,21-diol-3,11,20-trion) 1
17α –Hydroxyprogesterone (4-Pregnen-17αol-3,20-dion) 0.5
Deoxycorticosterone (4-Pregnen-21-ol-3,20-dion) 0.3
6α-Methyl-17α-Hydroxyprogesterone (6α-Methyl-4-pregnen-17α-ol-3,20-dion) 0.1
Progesterone (4-Pregnen-3,20-dion) <0.05
Dexamethasone (9α-Fluor-16α-methyl-prednisolone) <0.05
17α–Hydroxypregnolene (5-Pregnen-3β,17α-diol-20-on) <0.01
Dehydroisoandrosterone (5-Androsten-3β-ol-17-on) <0.01
Androstenedione (4-Androstene-3,17-dion) <0.01
Oestriol (1,3,5(10)-Estratrien-3,16α,17β-triol) <0.01
6α-Methyl-17α-Hydroxyprogesteroneacetate (6α-Methyl-4-pregnen-3,17-acetate) <0.01
Pregnenolone (5-Pregnen-3β-ol-20-on) <0.01
Oestrone (1,3,5(10)-Estratrien-3-ol-17-on) <0.01
Testosterone (4-Androstene-17β-ol-3-on) <0.01
17α-Hydroxyprogesterone-17 sulphate (4-Pregnen-17α-ol-3,20-dion sulphate) <0.01
Androsterone sulphate (5α-Androstene-3α-ol-17-on sulphate) <0.01
Testosterone sulphate (4-Androstene-17β-ol-3-on sulphate) <0.01
Cholesteryl sulphate (5-Cholesten-3β-ol sulphate) <0.01
17β-Oestradiol-17 sulphate (1,3,5(10)-Estratrien-3,17β-diol 17-sulphate) <0.01
DHEA-S (5-Androstene-3β-ol-17-on sulphate) <0.01

All blood samples were obtained without an anticoagu-
lant, stored at room temperature for 30 minutes until
coagulation was complete, and then centrifuged for
10 min at 2000 x g. Serum was separated and stored at
-20 °C.
LEIA test principle: The assay procedure of this corti-
sol LEIA kit follows the basic principles of a competi-
tive immunoassay. The wells of a microtiter plate were
first coated with goat anti-rabbit antibodies followed by
rabbit anti-cortisol antibodies. An unknown amount of
cortisol present in the sample and a fixed amount of per-
oxidase-labelled cortisol compete for the binding sites
of the antigen-specific coated antibodies. After an incu-
bation time the wells were washed to stop the competi-
tion reaction. The added luminescence substrate solu-
tion was converted to a light emitting component by the
bound peroxidase conjugate. The measured relative lu-
minescence units (RLU) are inversely proportional to
the cortisol concentration.
LEIA test procedure: 20 µl of each standard, control,
and sample (saliva undiluted, serum diluted 1:50 in
Zero Standard) were pipetted in duplicate into the wells
of the white microtiter strips and 100 µl of cortisol-
horseradish peroxidase conjugate added. After covering
with adhesive foil the plate was incubated for
3 hours at room temperature (18 - 24°C). All wells
were then washed four times with 250 µl wash buffer.
Luminescence was read 10 minutes after addition of
50 µl substrate solution. An MPL2 luminometer from
Berthold Detection Systems (Pforzheim,Germany) was
used for measurement. For calculation of the standard
curves with four-parameter logistics, the Microwin 2000
software was used (Mikrotek Laborsysteme GmbH,
Overath, Germany).

Clin. Lab. 1+2/2004 13

 JÜRGEN WESTERMANN et al.

100
90
80
70
RLU/RLUmax [%]
60
50
40
30
20
10
0
0 1 10 100 1000
Cortisol [nmol/l]

Figure 1: Typical standard curve of the cortisol LEIA

Comparison methods Specificity of the antibodies

The radioimmunoassay from Diagnostic Systems Labo-
ratories (DSL, Webster, Texas, USA) was used for
comparison. The RIA was modified according to the
method described by M. Gröschl et al (27). Regression
analysis was performed according to Bablock et al. (28).
Shelf-life evaluation
Both accelerated and real-time stability testing of the
reagents, standards and controls was performed to deter-
mine the expiration date (29).
Statistical methods
Statistical calculations were performed according to the
recommendations of Krouwer and Rabinowitz (30). The
software used for calculation of statistical data was
Winstat (Kalmia Co. Inc.) and SigmaPlot 2001 (SPSS
Inc.). The slope of the calibration curves was calculated
according to Rodbard (31).
RESULTS
The analytical performance of the newly developed
LEIA kit was determined by evaluating analytical speci-
ficity and sensitivity, precision, accuracy (recovery,
linearity) and comparison with other established im-
munoassay kits.
The specificity of the anti-cortisol antibody used in our
LEIA was evaluated for cross-reactivity to various com-
pounds (Table 1) that may potentially interfere with the
assay.
A 57% cross-reactivity for prednisolone (difference
from cortisol is one double bond in position 1) was
seen. 11-deoxycortisol showed cross reactivity of 12%,
while all other compounds showed cross-reactivity less
than 2.5%.
LEIA standard curve and sensitivity
A typical standard curve is shown in Figure 1. A cali-
bration curve was established by using cortisol stan-
dards from 0.83 to 110 nmol/l. The measuring range of
the LEIA covers the range of normal and elevated cor-
tisol concentrations in human saliva and in 1:50 diluted
serum.
The analytical sensitivity, calculated from the mean mi-
nus 3 standard deviations (SD) of 15 replicates of the
zero standard is 0.38 nmol/l. Functional sensitivity was
determined from the between-lot (n=6) assay variation
coefficient of very low saliva samples. The lowest corti-
sol concentration which could be measured with a co-
efficient of variation below 20% is 0.52 nmol/l.
Precision
The intra-assay variation, the inter-assay variation and
the between-lot variation for saliva samples were deter-

14 Clin. Lab. 1+2/2004

 DETERMINATION OF CORTISOL BY A LUMINESCENCE-ENHANCED ENZYME IMMUNOASSAY

Table 2: Precision of the cortisol LEIA for saliva samples

A. Determination of intra-assay variation of 6 different saliva samples

Saliva 1 Saliva 2 Saliva 3 Saliva 4 Saliva 5 Saliva 6

20 each in one run
[nmol/l] [nmol/l] [nmol/l] [nmol/l] [nmol/l] [nmol/l]

Mean value 2.65 4.33 5.96 10.82 24.01 71.46

SD 0.19 0.33 0.36 0.30 1.08 5.88
CV [%] 7.3 7.6 6.0 2.8 4.5 8.2

B. Determination of interassay variation of 6 different saliva samples

Duplicates in 20 runs Saliva 1 Saliva 2 Saliva 3 Saliva 4 Saliva 5 Saliva 6

[nmol/l] [nmol/l] [nmol/l] [nmol/l] [nmol/l] [nmol/l]

Mean value 2.07 3.67 5.71 9.52 18.82 66.54

SD 0.25 0.28 0.44 0.61 1.16 6.51
CV [%] 12.0 7.5 7.7 6.4 6.2 9.8

C. Determination of interlot variation of 6 different saliva samples

Saliva 1 Saliva 2 Saliva 3 Saliva 4 Saliva 5 Saliva 6

Duplicates in 7 different Kit Lots
[nmol/l] [nmol/l] [nmol/l] [nmol/l] [nmol/l] [nmol/l]

Mean value 1.96 3.48 5.93 9.69 19.13 64.34

SD 0.17 0.30 0.44 0.94 1.74 6.79
CV [%] 8.5 8.7 7.4 9.7 9.1 10.6

120
Cortisol Saliva IBL LEIA [nmol/l]

100

80

60

40

20
y = 1,38x - 0,88
2
R = 0,96

0
0 10 20 30 40 50 60 70 80

RIA [nmol/l]

Figure 2: Comparison of cortisol measured by immunoassays RIA (x) and IBL LEIA (y)

Clin. Lab. 1+2/2004 15

 JÜRGEN WESTERMANN et al.

Table 3: Analytical recovery for saliva samples

Added Expected Found Recovery

Sample
[nmol/l] [nmol/l] [nmol/l] [%]

Saliva without added cortisol: 6.7

1.3 8.0 7.8 98
2.6 9.2 8.5 92
Saliva 1 5.2 11.9 11.0 92
10.4 17.0 16.9 99
20.7 27.4 26.6 97
41.4 48.1 50.8 106
Saliva without added cortisol: 10.7
1.3 12.0 11.2 93
2.6 13.3 11.8 89
Saliva 2 5.2 15.9 14.1 89
10.4 21.1 20.3 96
20.7 31.4 32.9 105
41.4 52.1 55.2 106
Saliva without added cortisol: 11.9
1.3 13.2 13.2 100
2.6 14.5 13.1 90
Saliva 3 5.2 17.1 15.7 92
10.4 22.3 21.8 98
20.7 32.7 35.3 108
41.4 53.4 60.8 114

25

20
Saliva [nmol/l]

15

10

0
0 200 400 600 800
Serum [nmol/l]

Figure 3: Cortisol values of simultaneously taken saliva and serum samples, measured with the cortisol LEIA.

16 Clin. Lab. 1+2/2004

 DETERMINATION OF CORTISOL BY A LUMINESCENCE-ENHANCED ENZYME IMMUNOASSAY

Table 4: Serial dilution of saliva samples in the cortisol LEIA

Measured Expected Recovery

Saliva Dilution
conc.[nmol/L] conc.[nmol/L] [%]

neat 86.08 - 100

1:2 41.05 43.04 95
1:4 19.64 21.52 91
1
1:8 9.57 10.76 89
1:16 4.52 5.38 84
1:32 2.51 2.69 93

neat 63.18 - 100

1:2 36.23 31.59 115

1:4 16.75 15.80 106
2
1:8 7.50 7.90 95
1:16 3.75 3.95 95

1:32 1.99 1.97 101

neat 60.45 - 100

1:2 30.49 30.22 101
1:4 15.15 15.11 100
3
1:8 7.09 7.56 94
1:16 3.39 3.78 90
1:32 1.93 1.89 102

mined by repeated measurements of control samples.
The results for the saliva samples are shown in Tables
2. A, B and C. For saliva (range 2 - 70 nmol/l) the mean
intra-assay variation CV% was 6.13 %, the mean inter-
assay variation CV% was 8.13 % and the mean CV%
for the between-lot variation of seven different lots was
8.93 %.
For serum (range 50-2000 nmol/l), the mean intra-assay
variation CV% was 7.70 %, the mean inter-assay varia-
tion CV% was 8.83 % and the mean CV% for the be-
tween-lot variation of four different lots was 8.14 %.
To demonstrate the assay accuracy by another method,
linearity studies were performed. Saliva samples with
high basal cortisol concentrations were serially diluted
with cortisol-free medium (standard A), and the cortisol
concentration was assayed by the LEIA. The results are
presented in Table 4. To cover the whole standard range
5 dilutions were made of each sample. The ratio be-
tween concentration and dilution of sera did not signifi-
cantly deviate from linearity across the concentration
range studied (mean recovery 97%, range 84-115%).
In a dilution study of two serum samples we found a
mean recovery of 94% (range 83- 99%).

Accuracy
We estimated the analytical recovery of cortisol in the
LEIA at six different concentrations added to three Sali-
va samples (Table 3). Increasing amounts of cortisol
were added to saliva samples with various initial corti-
sol concentrations. Cortisol concentrations were mea-
sured, and the percentage of recovery was calculated.
The mean recovery of cortisol from all three saliva sam-
ples was 98% (range 89 - 114%).
In a spike experiment with serum we found a mean re-
covery of 97% (range 90 -107%).
Method comparison
The LEIA was compared with a radioimmunoassay. 384
saliva and 106 serum samples were assayed for cortisol
concentrations, both by the comparison methods (x-
axis) and by the LEIA (y-axis). The results of linear re-
gression analysis of saliva samples showed excellent
correlation, with a correlation coefficient r = 0.98. The
slope of the regression lines was 1.38 and the intercept
was 0.88 nmol/l. Figure 2 demonstrates the results of
the method comparison for saliva samples.
The method comparison data for serum samples are:
IBL LEIA = 0.94*RIA + 0.171 ; r = 0.95; n = 106

Clin. Lab. 1+2/2004 17

 JÜRGEN WESTERMANN et al.

Table 5: Interferences of blood in saliva (bold samples show increased cortisol levels)

Final blood Measured conc. Recovery

Sample
concentration [%] v/v [nmol/l] [%]
0.000 23.73 100
0.060 24.56 103
0.125 23.45 99
0.250 22.62 95
1
0.500 26.49 112
0.625 45.52 192
1.250 68.98 291
2.500 114.50 483
0.000 61.25 100
0.060 69.53 114
0.125 77.53 127
0.250 62.63 102
2
0.500 61.80 101
0.625 87.46 143
1.250 113.39 185
2.500 126.09 206

40

35

30
Salivary Cortisol [nmol/l]

25

20

15

10

0
4 6 8 10 12 14 16 18
Time of day [h]

Figure 4: Typical diurnal profiles of persons waking up at different times

18 Clin. Lab. 1+2/2004

 DETERMINATION OF CORTISOL BY A LUMINESCENCE-ENHANCED ENZYME IMMUNOASSAY

Table 6: Data of the diurnal profile study

Cortisol peak maximum Delay (decimal) Peak time (decimal) Wake up time (decimal) Age
[nmol/l] [h] [h] [h] [years]
Senior Citizens n=10
MEAN 11.56 1.13 7.98 6.72 67.5
MEDIAN 11.32 1.00 8.25 6.67 67.0
SD 4.08 0.62 0.87 0.67 4.14
MIN 5.08 0.00 6.00 5.50 62
MAX 19.46 2.00 9.00 7.83 77
Children n=4
MEAN 7.17 1.21 8.83 7.63 8.75
MEDIAN 7.38 1.13 8.88 7.50 9.50
SD 4.14 1.10 1.06 0.25 5.06
MIN 2.61 0.00 7.50 7.50 3
MAX 11.29 2.58 10.08 8.00 13
Females n=61
MEAN 28.1 0.83 7.81 6.98 28.8
MEDIAN 28.1 0.75 7.50 6.75 24.0
SD 13.3 0.55 1.60 1.60 11.1
MIN 6.8 0.00 4.50 4.00 17
MAX 78.4 2.50 12.75 12.25 61
Males n=39
MEAN 26.2 0.82 8.39 7.57 32.6
MEDIAN 24.3 0.50 8.00 7.00 27.5
SD 10.2 0.64 1.90 2.06 13.0
MIN 10.3 0.00 4.75 3.75 17
MAX 62.0 3.00 12.33 12.30 59
All n=110 (without children)
MEAN 27.6 0.85 8.06 7.17 33.8
MEDIAN 26.6 0.75 7.83 6.92 26.0
SD 12.1 0.59 1.71 1.74 15.8
MIN 6.8 0.00 4.50 3.75 17
MAX 78.5 3.00 12.75 12.30 77

Comparison of saliva and serum samples
Figure 3 shows the correlation in simultaneously taken
saliva and serum samples from 84 healthy volunteers.
Both samples were measured with the LEIA. A non-
linear correlation between saliva and serum cortisol was
observed, with correlation coefficient r=0.951, because
of the saturation of the binding proteins with high cor-
tisol levels in serum. The linear correlation coefficient
was r = 0.823.
Comparison to serum samples from the DGKC
For comparison the 20 serum samples from the DGKC
were diluted 1:100 in zero standard and then measured
in the LEIA. Figure 7 shows excellent correlation to the
GC/MS reference method values and underlining the
sensitivity of the assay.
The method comparison data for GC/MS predetermined
serum samples are:
IBL LEIA=0.958*GC/MS+57.8; r = 0.987; n = 20
Stability of the kit
The stability of the developed cortisol LEIA kit was in-
vestigated by real-time stability studies. The kit was
found to be stable for at least 9 months when stored at 2
to 8 °C (data not shown).

Clin. Lab. 1+2/2004 19

 JÜRGEN WESTERMANN et al.

100

80
Peak maximum [%]
60

40

20

0 2 4 6 8 10 12 14
Time after awakening [h]

Figure 5: Mean diurnal cortisol in saliva profile of 110 persons.

The black squares indicate the mean and the bars the ± 2SD range.

40

30
number

20

10

0
0,0 0,5 1,0 1,5 2,0 2,5 3,0

peak delay [hours]

Figure 6: Time intervals of maximum cortisol peak of 110 persons after waking up.

20 Clin. Lab. 1+2/2004

 DETERMINATION OF CORTISOL BY A LUMINESCENCE-ENHANCED ENZYME IMMUNOASSAY

Table 7: Normal Ranges

Hours after awakening [hours] Salivary Cortisol Ranges [nmol/l]

decimal 5% Percentile Median 95% Percentile
0 - 1.5 5.1 18.9 40.2
1.5 - 3.0 3.6 11.8 28.4
3.0 - 6.0 2.1 6.7 15.7
6.0 - 9.0 1.8 5.5 12.1
9.0 - 15.0 0.9 3.3 9.2

1400

1200
IBL Cortisol LEIA [nmol/l]

1000

800

600

400
y = 0,958x + 57,798
200 R = 0,987; n=20

0
0 200 400 600 800 1000 1200 1400
GC/MS reference value [nmol/l]

Figure 7: LEIA comparison to GC/MS reference values of 20 serum samples

Interferences of blood in saliva Salivary cortisol determinations in healthy persons

Quality control samples with known concentrations of
cortisol were enriched with amounts of blood (all results
given in nmol/l). Saliva samples with blood contamina-
tion greater than 0.2% (V/V) are visually reddish in co-
lor and therefore must be excluded. An increased level
of more than 10% was observed due to higher cortisol
concentrations in blood. The results are shown in
Table 5.
For estimating reference ranges, basal cortisol concen-
trations were determined in saliva samples from a total
of 110 normal volunteers (see Material and Methods).
Figure 4 shows typical diurnal profiles of the cortisol
concentration in saliva. The wake-up time is indicated
with an open circle. The overall results and those of the
different subgroups are shown in Table 6. We found no
correlation between age and peak concentration (r=
0.234) or between peak time and peak concentration

Clin. Lab. 1+2/2004 21

 JÜRGEN WESTERMANN et al.
(r=0.229). The maximum cortisol concentration in sali-
va was reached with a peak mean of 0.85h = 51 minutes
after wake up (see Figure 6). The cortisol concentration
maximum in saliva from normal volunteers covered a
broad range of 10.5 – 52.7nmol/l (5-95% percentiles).
The mean value for the whole group was 27.6nmol/l
(SD = 12.1 nmol/l). Figure 5 shows the diurnal profile
of all volunteers. To normalize the overall ranges, the
percentage of the peak maximum for all profiles is
plotted against time after awakening. The bars indicate
two standard deviations of the mean (black squares).
Table 7 shows normal values depending on awakening
time.
DISCUSSION
The aim of this study was the development of a non-
radioactive, sensitive and reproducible immunoassay
which accurately measures cortisol in saliva. The devel-
oped LEIA has an analytical sensitivity of 0.38 nmol/l
cortisol, which is sufficient to detect even decreased
salivary cortisol levels. It is important to mention the
functional sensitivity in order to estimate the reproduci-
bility of cortisol results in saliva samples. The func-
tional sensitivity of the described LEIA is 0.52 nmol/l.
The assessment of salivary cortisol is a common method
in various scientific fields. It is well known that the sali-
vary cortisol level is not dependent on the saliva flow
rate (1, 12). Moreover, it has been proven that there is a
good correlation between the salivary and the non-
protein bound plasma cortisol concentration (1). When
the protein bound plasma cortisol is compared to the
salivary cortisol level, it must be taken into account that
there is a nonlinear correlation because of the saturation
of the cortisol binding globulin at higher cortisol con-
centrations. Therefore, we obtained a good correlation
(r = 0.95; n = 84) with a hyperbolic regression function.
Even in recent literature a poor correlation has been
described due to the use of linear regression analysis
(32, 33).
As requirement for further studies we established nor-
mal ranges for cortisol with the cortisol LEIA (Table 7).
It is necessary to establish normal cortisol profiles be-
cause of the diurnal fluctuation of this hormone. As de-
scribed it is important to refer the cortisol values to the
wake-up time of the individuals and not to the time of
day. Even in newer studies this fact has not been taken
into account (22, 34 – 37).
In accordance with the literature, the established day
profiles in this study show a peak within 60 minutes
after awakening (13, 17 – 20). Compared to the plasma
cortisol morning peak, the peak concentration in saliva
is more significantly elevated and decreases more rapid-
ly (39).
The developed assay is specific and does not cross-react
with analogues of cortisol except the drug prednisolone
and 11-desoxycortisol.
22
The precision of the LEIA has demonstrated by
the intra- (2.9-7.7%) and inter- (6.2-11.5%) assay coef-
ficients of variation. The assay can measure cortisol
concentrations accurately in both serum and saliva
samples. Our LEIA shows a good agreement between
the expected and measured concentrations in recovery
tests and good linearity in dilution studies. The results
are consistent with those obtained with other immuno-
assay kits (RIA). High aberration in regression slope to
the RIA (Figure 2, slope=1.38) is due to used diluted
serum standards, while cited literature (27) used self
made saliva standards. Using the LEIA standards for the
RIA leads to a regression equation of:
LEIA=1.14*RIA-2.85, r = 0.974
The excellent correlation using GC/MS reference serum
samples, measured in a 1:100 dilution demonstrates the
good calibration of the standards.
In summary, the LEIA kit described here is useful for
the routine determination of cortisol as well as for basic
research, where a highly sensitive and reproducible as-
say is required.
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Correspondence: Jürgen Westermann
Immuno-Biological Laboratories GmbH
Flughafenstraße 52a
D-22335 Hamburg
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