Instructions for use

Cortisol Saliva ELISASA E-6000R

FreeCortisol Saliva ELISA Free
1

INTRODUCTION

1.1

Intended Use

Enzyme immunoassay for the quantitative measurement of active free cortisol in saliva.
It is intended only for research use.
1.2

Summary and explanation

Cortisol (hydrocortisone) is the major glucocorticoid produced in the adrenal cortex. Cortisol is a potent stresshormone and the secretion is regulated by the Hypothalamic-Pituitary-Adrenal-axis (HPA-axis).
The secretion of cortisol has a specific circadian rhythm with a curve presenting a sharp peak in the earlymorning and a gradually decrease over the day with a nadir in the evening (7). The position of this peak-value
is strongly influenced by the average wake-up time during the past weeks. It is not dependent on the actual
wake-up time of the specific day of sample collection (if different from the average wake-up time of the pastweek).
The loss of circadian rhythm with absence of a late-night cortisol nadir is a consistent abnormality in donors
with Cushing´s syndrome. This difference forms the basis for measurement of late-night salivary cortisol (4).
Studies show that salivary cortisol concentration reflects the serum unbound cortisol concentration throughout
the physiological concentration range (7, 8, 12). In serum, 90-95% of cortisol is bound to protein while insaliva cortisol appears mainly in its free, metabolic active form. The salivary cortisol concentration is
independent of saliva flow rate as well as of the serous and mucous content (12). Spontaneous increases incortisol concentration during the day may occur commonly due to stress or food intake. Changed patterns ofCortisol levels have been observed in connection with abnormal ACTH levels, clinical depression, psychologicalstress, and various physiological stressors as hypoglycemia, illness, fever, trauma, surgery, or pain.
2

PRINCIPLE

The Cortisol free in Saliva ELISA Kit is a solid phase enzyme-linked immunosorbent assay (ELISA), based on
the principle of competitive binding. The microtiter wells are coated with a polyclonal rabbit antibody directedagainst the cortisol molecule. The samples are dispensed in the coated wells and incubated with the enzyme
conjugate (cortisol conjugated to horseradish peroxidase). During incubation endogenous cortisol of a sample
competes with the enzyme conjugate for binding to the coated antibody. The unbound conjugate is removed bywashing the wells.
Subsequently, the substrate solution is added and the color development is stopped after a defined time. The
intensity of the color formed is inversely proportional to the concentration of cortisol in the sample. The
absorbance is measured at 450 nm with a microtiter plate reader.
3

WARNINGS AND PRECAUTIONS

1.

This kit is for research use only.

2.

Before starting the assay, read the instructions completely and carefully. Use the valid version of the
package insert provided with the kit. Be sure that everything is understood.

3.

The microplate contains snap-off strips. Unused wells must be stored at 2-8 °C in the sealed foil pouch andused in the frame provided.

4.

Pipetting of samples and reagents must be done as quickly as possible and in the same sequence for each
step.

5.

Use reservoirs only for single reagents. This especially applies to the substrate reservoirs. Using a reservoir
for dispensing a substrate solution that had previously been used for the conjugate solution turn solution
colored. Do not pour reagents back into vials as reagent contamination may occur.

6.

Mix the contents of the microplate wells thoroughly to ensure good test results. Do not reuse microwells.

7.

Do not let wells dry during assay; add reagents immediately after completing the rinsing steps.

8.

Allow the reagents to reach room temperature (18-25°C) before starting the test. Temperature will affectthe absorbance readings of the assay.

9.

Never pipet by mouth and avoid contact of reagents and specimens with skin and mucous membranes.

10. Do not smoke, eat, drink or apply cosmetics in areas where specimens or kit reagents are handled.
11. Wear disposable latex gloves when handling specimens and reagents. Microbial contamination of reagentsor specimens may give false results.
12. Handling should be done in accordance with the procedures defined by an appropriate national biohazard
safety guideline or regulation.
13. Do not use reagents beyond expiry date as shown on the kit labels.
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14. All indicated volumes have to be performed according to the protocol. Optimal test results are only
obtained when using calibrated pipettes and microtiter plate readers.
15. Do not mix or exchange components from kits with different lot numbers. It is advised not to exchange
wells of different plates even of the same lot. The kits may have been shipped or stored under different
conditions and the binding characteristics of the plates may differ slightly.
16. Avoid contact with Stop Solution. It may cause skin irritation and burns.
17. Chemicals and prepared or used reagents have to be treated as hazardous waste according to the national
biohazard safety guideline or regulation.
18. For information please refer to Material Safety Data Sheets. Safety Data Sheets for this product areavailable upon request directly from the manufacturer.
4
4.1

REAGENTS
Reagents provided
SA E-6031 Microtiterwells

12x8 (break apart) strips, 96 wells; wells coated with an anti-cortisol antibody (polyclonal).
Standards
ready to use
Cat. no.

Standard

Concentration

Volume/Vial

SA E-6001

Standard A (0)0 ng/ml

2 ml

SA E-6002

Standard B (1)0.1 ng/ml

0.5 mlSA E-6003

Standard C (2)0.4 ng/ml

0.5 mlSA E-6004

Standard D (3)1.7 ng/ml

0.5 mlSA E-6005

Standard E (4)7.0 ng/ml

0.5 mlSA E-6006

Standard F (5)30 ng/ml

0.5 mlConversion factor: 1 ng/ml = 2.76 nmol/l+

SA E-6051 + SA E-6052 Control 1 (low) / Control 2 (high)2 vials, 0.5 ml each, ready to use; for control values and ranges please refer QC-Datasheet
SA E-6040 Enzyme Conjugate
1 vial, 7 ml, ready to use; Cortisol conjugated to horseradish peroxidase
AR E-0055 Substrate Solution
1 vial, 22 ml each, ready to use; Tetramethylbenzidine (TMB)
Hazards
identification:
H360D May damage the unborn child.
AR E-0080 Stop Solution
1 vial, 7.0 ml, ready to use; contains 2 N acidic solution
Avoid contact with the stop solution. It may cause skin irritations and burns.
Hazards
identification:
H290 May be corrosive to metals.
H314 Causes severe skin burns and eye damage.
H335 May cause respiratory irritation.
AR E-0030 Wash Solution
1 vial, 50 ml (10X concentrated);see „Preparation of Reagents“.
All reagents contain azide-free and mercury-free preservatives.

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4.2
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4.3

Material required but not provided
Microcentrifuge
A calibrated microtiter plate calibrated reader (450±10 nm)
Microplate mixer operating at about 600 - 900 rpm, optionallyVortex mixer
Calibrated variable precision micropipettes (50 µl, 100 µl, 200 µl)
Absorbent paper
Distilled or deionized water
Timer
Semi logarithmic graph paper or software for data reduction
Storage conditions

When stored at 2-8 °C unopened reagents will be stable until expiration date. Do not use reagents beyond thisdate. Opened reagents must be stored at 2-8 °C. After first opening the reagents are stable for 30 days if usedand stored properly.
Microtiter wells must be stored at 2-8°C. Take care that the foil bag is sealed tightly.
4.4

Reagents preparations

Allow the reagents and the required number of wells to reach room temperature (18–25 °C) before starting thetest.
Wash Solution
Add deionized water to the 10X concentrated Wash Solution.
Dilute 50 ml of concentrated Wash Solution with 450 ml deionized water to a final volume of 500 ml.
The diluted Wash Solution is stable for 12 weeks at room temperature (18-25 °C).
4.5

Disposal of the kits

The disposal of the kit must be made according to the national regulations. Special information for this productis given in the Material Safety Data Sheet.
4.6

Damaged test kits

In case of any severe damage of the test kit or components, the manufacturer has to be informed written,
latest one week after receiving the kit. Severely damaged single components should not be used for a test run.
They have to be stored until a final solution has been found. After this, they should be disposed according tothe official regulations.
5

SPECIMEN COLLECTION AND PREPARATION

Samples containing sodium azide must not be used in the assay. The saliva samples should be completely
colorless. Even the slightest red color shows blood contamination. Such blood contamination will result in falsely
elevated concentration values. In case of visible blood contamination the donor should discard the sample, rinse
the collection device with water, also rinse the mouth with (preferably) cold water, wait for 10 minutes and takea new sample. Chewing anything during the sampling period must be avoided. Any pressure on the teeth may
result in falsely elevated measurements due to an elevated content of gingival liquid in the saliva sample.
5.1

Specimen Collection

For the correct collection of saliva we are recommending to only use appropriate devices made from ultra-purepolypropylene (PP). Do not use any PE devices or Salivettes for sampling; in most cases this will result insignificant interferences. Glass tubes can be used as well, but in this case special attention is necessary for
excluding any interference caused by the stopper.
As the Cortisol secretion in saliva as well in serum shows an obvious secretion pattern throughout the day, it isimportant to care for a proper sample timing of the sampling. The morning peak normally appears during thefirst two hours after the average wake-up time. Therefore we recommend taking 5 separate samples within aperiod of two hours (multiple sampling) directly after the usual wake-up time (e.g. 1 min, 30 min, 60 min,90 min and 120 min). It is important to know that the timing of the morning peak is not related to the absolute
time or day light. It is just related to the wake-up habits of the donor. If possible the volume of each single
sample should be a minimum of 0.5 ml (better 1 ml).
As food might contain significant amounts of steroid hormones samples preferably should be taken while
fasting. Do not collect samples within 60 minutes after eating a major meal, 12 hours after consuming alcoholor 60 minutes after brushing teeth. Rinse mouth with water 10 minutes prior to specimen collection.
Furthermore please avoid any strenuous physical exercises and intense stress situations.
The collection for the evening sample has to be done during the late evening (at best between 10 and 12 PM).
Also in this case we recommend collecting 5 samples in intervals of at least 30 minutes. If only 5 sampling
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devices are available for the collection of a day profile, sampling also can be done as follows. 30 min, 60 min,and 90 minutes after the usual wake-up time for the morning value, followed by 2 samples in the late evening
collected during the last hour prior to regular bed time.
5.2

Specimen Storage and Preparation

In general saliva samples are stable at ambient temperature for several days. Therefore mailing of such
samples by ordinary mail without cooling will not create a problem. Whenever possible, samples preferable
should be kept at a temperature of -20°C. We recommend avoiding multiple freeze-thaw cycles.
Each sample has to be frozen, thawed, and centrifuged at least once in order to separate the mucins. Upon
arrival of the samples in the lab the samples have to be stored frozen at least overnight. In the next morning
the frozen samples are thawed and brought to room temperature and mixed carefully. Then the samples haveto be centrifuged for 5 to 10 minutes. Now the clear colorless supernatant is easy to pipette. If the sample
should show even a slight reddish tinge it should be discarded. Otherwise the concentration value mostprobably will be falsely elevated. Due to the episodic variations of the cortisol secretion we highly recommend
the strategy of multiple sampling. If such a set of multiple samples has to be tested the lab (after at least one
freezing, thawing, and centrifugation cycle) has to mix the aliquots of the five single samples in a separate
sampling device and perform the testing from this mixture. If the shape of the morning peak has to be
determined all five morning samples have to be tested separately.
5.3

Specimen Dilution

If in an initial assay, a specimen is found to contain more cortisol than the highest standard, the specimens canbe diluted with Standard A and re-assayed as described in Assay Procedure.
For the calculation of the concentrations this dilution factor has to be taken into account.
6

ASSAY PROCEDURE

6.1

General remarksAll reagents and specimens must be allowed to come to room temperature (18-25 °C) before use. All
reagents must be mixed without foaming.
Once the test has been started, all steps should be completed without interruption.
Use new disposal plastic pipette tips for each standard, control or sample in order to avoid crosscontamination.
Absorbance is a function of the incubation time and temperature. Before starting the assay, it isrecommended that all reagents are ready, caps removed, all needed wells secured in holder, etc. This will
ensure equally elapsed time for each pipetting step without interruption.
As a general rule the enzymatic reaction is linearly proportional to time and temperature.
Respect the incubation times as stated in this instructions for use.
Duplicate determination of standards, controls and samples is recommended in order to identify potential
pipetting errors.

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6.2

Assay Procedure

Each run must include a standard curve.
1.

Secure the desired number of coated strips in the frame holder.

2.

Dispense 50 µl of each Standard, Control and Samples in duplicates with new disposable tips into
appropriate wells.

3.

Dispense 50 µl of Enzyme Conjugate into each well.
Thoroughly mix for 10 seconds. It is important to have a complete mixing in this step.

4.

Incubate for 60 minutes at room temperature (18-25 °C). Shaking on a horizontal shaker during
incubation is not necessary, but it improves the sensitivity of the test.

5.

Briskly empty the contents of the wells by aspiration or by decanting.
Rinse the wells 4 times with diluted Wash Solution (300 µl per well). Strike the wells sharply on
absorbent paper to remove residual droplets.
Important note: The sensitivity and precision of this assay is markedly influenced by the correct
performance of the washing procedure!

6.

Add 200 µl of Substrate Solution to each well.

7.

Incubate for 30 minutes in the dark at room temperature (18-25 °C).

8.

Stop the enzymatic reaction by adding 50 µl of Stop Solution to each well.

9.

Determine the absorbance of each well at 450±10 nm within 15 minutes after adding the Stop
Solution.

6.3 Calculation of results
1. Calculate the average absorbance values for each set of standards, controls and samples.
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2. The obtained OD of the standards (y-axis, linear) are plotted against their concentration (x-axis,
logarithmic) either on semi-logarithmic paper or using an automated method.
3. Using the mean absorbance value for each sample determine the corresponding concentration from thestandard curve.
4. Automated method: The results in this instruction for use have been calculated automatically using a 4 PL
(4 Parameter Logistics) curve fit. 4 Parameter Logistics is the preferred calculation method. Other data
reduction functions may give slightly different results.
5. The concentration of the samples can be read directly from this standard curve. Samples with concentrations
higher than that of the highest standard have to be further diluted. For the calculation of the concentrations
this dilution factor has to be taken into account.
6.3.1 Example of typical standard curve
The following data is for demonstration only and cannot be used in place of data generations at the time of
assay.
Standard
Optical Density (450 nm)

7Standard A

0.0 ng/ml

3.099

Standard B

0.1 ng/ml

2.647

Standard C

0.4 ng/ml

1.932

Standard D

1.7 ng/ml

0.856Standard E

7.0 ng/ml

0.357

Standard F

30 ng/ml

0.172

EXPECTED NORMAL VALUES

In order to determine the normal range of Cortisol free in Saliva samples from adult male and female
apparently healthy subjects, were collected and analyzed using the Cortisol free in Saliva ELISA kit.
The following range was calculated from this study.
Time of day
Morning
MiddayAfternoon
EveningMidnight

5-95% percentile(ng/ml)1.6 – 9.2
0.9 – 6.9
0.6 – 3.6
0.4 – 3.9
< 1.2

n
234
427
129
419
26

Since Cortisol levels show diurnal cycles, we recommend to always collecting a series of samples in the morningand another one in the evening. The difference between morning and evening is an important parameter.
Furthermore, we recommend that each laboratory determines its own range for the population tested.

8

QUALITY CONTROLGood laboratory practice requires that controls be run with each standard curve. A statistically significant
number of controls should be assayed to establish mean values and acceptable ranges to assure proper
performance.
It is recommended to use control samples according to state and federal regulations. The use of control
samples is advised to assure the day to day validity of results. Use controls at both normal and pathologicallevels.
The controls and the corresponding results of the QC-Laboratory are stated in the QC certificate included in thekit. The values and ranges stated on the QC sheet always refer to the current kit lot and should be used for
direct comparison of the results.
It is also recommended to make use of national or international Quality Assessment programs in order toensure the accuracy of the results.
Employ appropriate statistical methods for analysing control values and trends. If the results of the assay do
not fit to the established acceptable ranges of control materials results should be considered invalid.
In this case, please check the following technical areas: Pipetting and timing devices, microtiter plate reader,
expiration dates of reagents, storage and incubation conditions, aspiration and washing methods. Afterchecking the above mentioned items without finding any error contact your distributor or the manufacturer
directly.

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9
9.1

PERFORMANCE CHARACTERISTICS
Analytical Sensitivity

The analytical sensitivity of the Cortisol free in Saliva ELISA was calculated by subtracting two standard
deviations from the mean of twenty (20) replicate analyses of Standard A. The analytical sensitivity of theassay is 0.019 ng/ml.
9.2

Specificity (Cross Reactivity)

The following materials have been evaluated for cross reactivity.
Steroids
Testosterone
Corticosterone
Cortisone
11-Deoxycorticosterone
11-Deoxycortisol
Dexamethasone
Estriol
EstronePrednisolone
Prednisone
Progesterone
17-Hydroxyprogesterone
Danazole
Pregnenolone
Estradiol
Androstenedione

9.3% Crossreactivity
< 0.1
6.2
0.8
2.6
50
< 0.1
< 0.1
< 0.1
100
0.9
< 0.1
1.3
< 0.1
< 0.1
< 0.1
< 0.1

Assay Dynamic Range

The range of the assay is between 0.1 – 30 ng/ml.
9.4

Reproducibility

9.4.1 Intra-Assay
The intra-assay variation was determined by replicate measurements of three saliva samples within one runusing the Cortisol free in Saliva ELISA. The intra-assay variation is shown below:

Mean (ng/ml)
SD (ng/ml)
CV (%)
n=

Sample 1

Sample 2

Sample 3

0.64
0.05
7.1
20

2.01
0.08
4.1
20

4.87
0.21
4.3
20

9.4.2 Inter-Assay
The inter-assay variation was determined by duplicate measurements of three saliva samples in ten different
runs using the Cortisol free in Saliva ELISA. The inter-assay variation is shown below:

Mean (ng/ml)
SD (ng/ml)
CV (%)
n=

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Sample 1

Sample 2

Sample 3

0.65
0.03
4.2
10

2.05
0.15
7.5
10

5.31
0.48
9.1
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9.5

RecoveryRecovery of the Cortisol free in Saliva ELISA was determined by adding increasing amounts of the analyte tothree different saliva samples containing different amounts of endogenous analyte. Each sample (non-spiked
and spiked) was assayed and analyte concentrations of the samples were calculated from the standard curve.
The percentage recoveries were determined by comparing expected and measured values of the samples.

Saliva Spiking

1

2

3

9.6

native3 ng/ml
5 ng/ml
7 ng/mlnative3 ng/ml
5 ng/ml
7 ng/mlnative3 ng/ml
5 ng/ml
7 ng/ml

MeasuredConcentration(ng/ml)
0.53
3.35
6.57
8.31
0.54
3.52
7.02
8.61
0.82
3.51
6.32
9.15

Expected
Cencentration(ng/ml)
3.53
5.53
7.53
3.54
5.54
7.54
3.82
5.82
7.82

Recovery %
95%
119%
110%
99%
126%
114%
92%
108%
117%

LinearityThree saliva samples containing different amounts of analyte were serially diluted with Standard A and assayedwith the Cortisol free in Saliva ELISA. The percentage linearity was calculated by comparing the expected andmeasured values for cortisol.
Expected
Measured
Cencentration Linearity %SalivaDilution
Concentration(ng/ml)(ng/ml)native4.13
1 in 2
2.04
2.07
99%
1
1 in 4
1.07
1.03
104%
1 in 8
0.60
0.52
115%
native4.13
1 in 2
2.26
2.07
109%
2
1 in 4
1.24
1.03
120%
1 in 8
0.66
0.52
127%
native4.48
1 in 2
2.32
2.24
104%
3
1 in 4
1.33
1.12
119%
1 in 8
0.65
0.56
116%

10 LIMITATIONS OF PROCEDURE
Reliable and reproducible results will be obtained when the assay procedure is performed with a complete
understanding of the package insert instruction and with adherence to GLP (Good Laboratory Practice).
Any improper handling of samples or modification of this test might influence the results.
10.1 High-Dose-Hook Effect
A High-Dose-Hook Effect is not known for competitive assays.
10.2 Drug Interferences
Any medication (cream, oil, pill etc) containing Cortisol of course will significantly influence the measurement of
this analyte in saliva. The same is true for any medication containing Prednisolone.

11 REFERENCES
1.

Fleiner T., Zijlstra W, Dauth H., Haussermann P.
Evaluation of a hospital-based day-structuring exercise programme on exacerbated behavioural andpsychological symptoms in dementia – the exercise carrousel: study protocol for a randomised controlled
trial, BioMedCentral 2015 16:228

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2.

De Steenwinkel F.D.O, Hokken-Koelega A.C.S, Hazes J.M.W, Dolhain R.J.E.M.
The influence of foetal prednisone exposure on the cortisol level in the offspring
Clinical Endocrinology 2014, 80, 804-810
3. Balsalobre-Fernández C., Tjero-González C.M., del Campo-Vecino J.
Relationships between Training Load, Salivary Cortisol Responses and Performance during Season Training
in Middle and Long Distance Runners; PLOS ONE 2014, Vol. 9, Issue 8
4. LK Nieman, BMK Biller, JW Findling, J Newell-Price, VM Montori
The diagnosis of Cushing's syndrome: An Endocrine Society Clinical Practice Guideline
J. Clin. Endocrin. & Metabol. 2008, Vol.93, No.5, pages 1526-1540
5. Kirschbaum C., Hellhammer DH: Salivary cortisol in psychobiological Research: An overview,
Neuropsychobiology, 1989, 22, 150-169
6. Kirschbaum C, Hellhammer Dh: Salivary cortisol in psychoneuroendocrine Research:
Recent developments and applications, Psychoneuroendocrinology 1994, 19, pp 313-333
7. Vining RF, et al., Salivary cortisol: a better measure of adrenal cortical function than serum cortisol, Ann
Clin Biochem, 1983, 20, 329-335
8. L.D. Dorn, J.F. Lucke, T.L. Loucks, S.L. Berga
Salivary Cortisol reflects serum Cortisol: Analysis of circadian profiles.
Ann Clin Biochem 2007 Vol. 44, pages 281-284
9. Geburt K, Friedrich M, Piechotta M, Gauly M, König von Borstel U
Validity of physiological biomarkers for maternal behavior in cows – A comparison of beef and dairy cattle;
Physiology & Behavior 139 (2015) 361-368
10. Aurich J, Wulf M, Ille N, Erber R, von Lewinski M, Palme R, Aurich C
Effects of season, age, sex and housing on salivary cortisol concentrations in horses
Domestic Animal Endocrinology 52 (2015) 11-16
11. Davies NA, Gramotnev G, McAlpine C, Seabrook L, Baxter G, Lunney D, Rhodes JR, Bradley A
Physiological Stress in Koala Populations near the Arid Edge of Their Distribution
PLOS ONE 2013, Volume 8, Issue 11
12. Aardal E. and Holm AC
Cortisol in Saliva – Reference Ranges and Relation to Cortisol in Serum
Eur. J. Clin. Chem. Clin. Biochem. (1995), 33:927-932
13. Garde A.H. and Hansen A.M.
Long-term stability of salivary cortisol
Scand J Clin Lab Invest 2005; 65:433-436

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For research use
only!
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