Instructions for use

DHEA Saliva ELISASA E-7000R

DHEA Saliva ELISA
1. INTRODUCTION
1.1 Intended use and principle of the test
An enzyme immunoassay for the measurement of active free DHEA in saliva.
2. PRINCIPLEThe DHEA Saliva ELISA Kit is a solid phase enzyme-linked immunosorbent assay (ELISA), based on theprinciple of competitive binding. The microtiter wells are coated with an antibody directed against the DHEA
molecule. Endogenous DHEA of a patient sample competes with a DHEA-horseradish peroxidase conjugate
for binding to the coated antibody. After incubation the unbound conjugate is washed off.
The amount of bound peroxidase conjugate is inversely proportional to the concentration of DHEA in thesample. After addition of the substrate solution, the intensity of color developed is inversely proportional tothe concentration of DHEA in the patient sample.
3. WARNINGS and PRECAUTIONS
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This kit is for research use only.
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.
The microplate contains snap-off strips. Unused wells must be stored at 2 °C to 8 °C in the sealed foil
pouch and used in the frame provided.
Pipetting of samples and reagents must be done as quickly as possible and in the same sequence foreach step.
Use reservoirs only for single reagents. This especially applies to the substrate reservoirs. Using areservoir for dispensing a substrate solution that had previously been used for the conjugate solution
may turn solution colored. Do not pour reagents back into vials as reagent contamination may occur.
Mix the contents of the microplate wells thoroughly to ensure good test results. Do not reusemicrowells.
Do not let wells dry during assay; add reagents immediately after completing the rinsing steps.
Allow the reagents to reach room temperature (21 - 26 °C) before starting the test. Temperature willaffect the absorbance readings of the assay. However, values for the patient samples will not beaffected.
Never pipet by mouth and avoid contact of reagents and specimens with skin and mucous membranes.
Do not smoke, eat, drink or apply cosmetics in areas where specimens or kit reagents are handled.
Wear disposable latex gloves when handling specimens and reagents. Microbial contamination ofreagents or specimens may give false results.
Handling should be done in accordance with the procedures defined by an appropriate national
biohazard safety guideline or regulation.
Do not use reagents beyond expiry date as shown on the kit labels.
All indicated volumes have to be performed according to the protocol. Optimal test results are only
obtained when using calibrated pipettes and microtiterplate readers.
Do not mix or use components from kits with different lot numbers. It is advised not to exchange wellsof 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 result slightly different.
Avoid contact with Stop Solution. It may cause skin irritation and burns.
Chemicals and prepared or used reagents have to be treated as hazardous waste according to thenational biohazard safety guideline or regulation.
For information please refer to Safety Data Sheets. Safety Data Sheets for this product are available
upon request directly from the manufacturer.

4. REAGENTS
4.1 Reagents provided
SA E-7031

Microtiterwells

Content:

Wells coated with an anti-DHEA antiserum (polyclonal).

Volume:12 x 8 (break apart) strips, 96 wells.

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Standards and Controls -Ready to use
Cat.-no.
Component
Concentration

Volume / Vial

SA E-7001

0 pg/ml

3.0 mlSA E-7002

10 pg/ml

1.0 mlSA E-7003

40 pg/ml

1.0 mlSA E-7004

160 pg/ml

1.0 mlSA E-7005

640 pg/ml

1.0 mlSA E-7006

2560 pg/ml

1.0 mlSA E-7051

1.0 mlSA E-7052

For control values andranges please refer to
QC-Datasheet.

SA E-7040

Enzyme Conjugate - Ready to use

Content:

DHEA conjugated to horseradish peroxidase.

Volume:1 x 11 ml/vial

1.0 mlSubstrate Solution - Ready to use

AR E-0055
Content:

Tetramethylbenzidine (TMB).

Volume:1 x 22 ml/vial

Hazards
identification:
H360D May damage the unborn child.
Stop Solution - Ready to use

AR E-0080
Content:

contains 2 N hydrochlorid acid solution.

Volume:1 x 7 ml/vial
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.
Wash Solution - Concentrated 10x

AR E-0030
Volume:1 x 50 ml/vial
see “Preparation of Reagents”

All reagents contain azide-free and mercury-free preservatives.
Note: Additional Standard A for sample dilution is available upon request.
4.2 Material required but not provided
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microcentrifuge
A microtiterplate reader capable for endpoint measurement at 450 ±10nm
Microplate mixer operating at about 600 - 900 rpm
Vortex 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 calculation

4.3 Storage conditions
When stored at 2°C to 8°C unopened reagents will be stable until expiration date. Do not use reagents
beyond this date. Opened reagents must be stored at 2° - 8 °C. After first opening the reagents are stablefor 30 days if used and stored properly.
Microtiter wells must be stored at 2°C to 8°C. Take care that the foil bag is sealed tightly.
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4.4 Reagents preparation
Allow the reagents and the required number of wells to reach room temperature (21 - 26 °C) before starting
the test.
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 3 months at room temperature.
4.5 Disposal of the kits
The disposal of the kit must be made according to the national regulations. Special information for thisproduct is given in the 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 to the official regulations.
5. SPECIMEN COLLECTION AND PREPARATION
Samples containing sodium azide should 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 give falsely
elevated concentration values. In case of visible blood contamination the patient should discard the sample,
rinse the collection device with water, also rinse the mouth with (preferably) cold water, wait for 10 minutes
and take a new sample. Do not chew anything during the sampling period. 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 use only appropriate devices made from ultrapure polypropylene. Do not use any PE devices or cotton based Salivettes for sampling. False readings will
result. Glass tubes can be used as well, but in this case special attention is necessary for excluding any
interference caused by the stopper. Please contact the manufacturer for more details.
As food might contain significant amounts of steroid hormones samples preferably should be taken while
fasting. If fasting should be a problem at least any food of animal origin (meat or dairy products) should be
avoided prior to finalizing the collection. In the morning breakfast should be done only after finalizing thecollection procedure. During the day the collection period should be timed just before an anticipated meal.
As the steroid hormone secretion in saliva as well as in serum shows an obvious dynamic secretion pattern
throughout the day it is important to always collect 5 samples during a 2 hour period; this means every 30
minutes one sample. If possible the volume of each single sample should be a minimum of 0.5 ml (better 1
ml). Saliva flow may be stimulated by drinking water. This is allowed and even recommended before andduring the collection period. Drinking of water is not allowed during the last 5 minutes before taking thesingle samples.
5.2 Specimen Storage and Preparation
Saliva samples in general are stable at ambient temperature for several days. Therefore mailing of such
samples by ordinary mail without cooling will not create a problem. Storage at 4 °C can be done for a periodof up to one month. Whenever possible samples preferable should be kept at a temperature of -20 °C. Even
repeated thawing and freezing is no problem. Each sample has to be frozen, thawed, and centrifuged atleast once anyhow in order to separate the mucins by centrifugation. Upon arrival of the samples in the lab
the samples have to be kept frozen at least overnight. Next morning the frozen samples are warmed up to
room temperature and mixed carefully. Then the samples have to be centrifuged for 5 to 10 minutes. Now
the clear colorless supernatant is easy to pipette. If the sample should show even a slightly red color it
should be discarded. Otherwise the concentration value most probably will be falsely elevated. Due to theepisodic variations of the steroid 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 5 single samples in a separate sampling device and perform the testingfrom this mixture.

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5.3 Specimen Dilution
If in an initial assay, a specimen is found to contain more than the highest standard, the specimens can be
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.
Example:a) Dilution 1:10:b) Dilution 1:100:

10 µl saliva + 90 µl Standard A (mix thoroughly)
10 µl of dilution a) + 90 µl Standard A (mix thoroughly).

6. ASSAY PROCEDURE
6.1 General remarks
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All reagents and specimens must be allowed to come to room temperature 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 equal 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

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 100 µl of each Standard, Control and sample with new disposable tips into appropriate
wells.

3.

Dispense 100 µl of Enzyme Conjugate into each well.

4.
5.

Incubate for 60 minutes at room temperature on a Microplate mixer (≥ 600 rpm).
Important note: Optimal reaction in this assay is markedly dependent on shaking of the microplate!
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.

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.
It is recommended that the wells are read within 15 minutes.

6.3 Calculation of results
1. Calculate the average absorbance values for each set of standards, controls and patient samples.
2. Construct a standard curve by plotting the mean absorbance obtained from each standard against its
concentration with absorbance value on the vertical (Y) axis and concentration on the horizontal (X) axis.
3. Using the mean absorbance value for each sample determine the corresponding concentration from thestandard curve.
4. Automated method: The results in the package insert 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 withconcentrations 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.

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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
Standard A

Optical Units (450 nm)

0 pg/ml

2.940

Standard B

10 pg/ml

2.701

Standard C

40 pg/ml

2.290

Standard D

160 pg/ml

1.657

Standard E

640 pg/ml

0.890

Standard F

2560 pg/ml

0.426

7. EXPECTED NORMAL VALUES
In order to determine the normal range of DHEA free in Saliva samples from adult male and female
apparently healthy subjects were collected and analyzed using the DHEA Saliva ELISA kit. The following
range was calculated from this study.
Men
Age Group(years)Women

5% - 95% Percentile Median(pg/ml)(pg/ml)n

5% - 95% Percentile(pg/ml)Median(pg/ml)n< 2130.4 - 537.7

200.7

7

27.2 - 565.4

215.7

24

21 - 30

291.4 - 826.7

464.4

10

73.5 - 780.7

605.2

50

31 - 40306.7 - 892.3

514.2

10

124.5 - 745.1

335.0

50

41 - 50

86.8 - 713.7

285.2

25

85.7 - 480.8

222.3

50

51 - 60

79.1 - 525.3

228.4

23

76.7 - 620.2

217.7

50

> 60

39.4 - 694.9

171.2

28

34.7 - 467.1

170.8

50

8. QUALITY CONTROLGood laboratory practice requires that controls need to be run with each Standard curve. A statisticallysignificant 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.
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 patient results should be considered invalid.
In this case, please check the following technical areas: Pipetting and timing devices; photometer, expiration
dates of reagents, storage and incubation conditions, aspiration and washing methods.
After checking the above mentioned items without finding any error contact your distributor or the
manufacturer directly.
9. PERFROMANCE CHARACTERISTICS
9.1 Analytical Sensitivity
The analytical sensitivity of the DHEA Saliva ELISA was calculated by subtracting 2 standard deviations fromthe mean of twenty-two (22) replicate analyses of Standard A.
The analytical sensitivity of the assay is 3.7 pg/ml.

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9.2 Specificity (Cross Reactivity)
The following materials have been evaluated for cross reactivity.
Steroids% Crossreactivity at 50% Binding

DHEA-S< 0.01

Testosterone< 0.015α-Dihydrotestosterone< 0.01Androstendione

0.06

Progesterone

0.23

17α-Hydroxyprogesterone< 0.01Pregnenolone

0.01

17-Hydroxy-Pregnenolone

0.07

Deoxycorticosterone

0.05

Corticosterone< 0.01

Cortisol< 0.01

11-Desoxycortisol

0.01

Estradiol-17β< 0.01

Estradiol-17α< 0.01

Estrone< 0.01

Estriol< 0.01

9.3 Assay Dynamic Range
The range of the assay is between 10 - 2560 pg/ml.
9.4 Reproducibility
9.4.1 Intra-Assay
The intra-assay variation was determined by replicate measurements of 3 saliva samples within one runusing the DHEA Saliva ELISA. The within-assay variation is shown below:
Sample 1

Sample 2

Sample 3

Mean (pg/ml)

255.1

672.8

822.8

SD (pg/ml)

13.3

60.7

39.9

CV (%)

5.2

9.0

4.9

n=

19

19

19

9.4.2 Inter-Assay
The inter-assay variation was determined by duplicate measurements of 3 saliva samples in 10 different
runs using the DHEA Saliva ELISA. The inter-assay variation is shown below:
Sample 1

Sample 2

Sample 3

Mean (pg/ml)

238.6

648.8

797.5

SD (pg/ml)

10.6

44.2

51.2

CV (%)

4.5

6.8

6.4

n=

10

10

10

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9.5 Recovery
Using the standard matrix a spiking solution of 20 ng DHEA/ml was prepared. 500 µl of three saliva werespiked with 5, 10 and 15 µl of the spiking solution leaving the saliva matrices relatively intact. All samples
were measured by the DHEA Saliva ELISA procedure.
Saliva1

2

3

Spiking(pg/ml)-Observed(pg/ml)291

Expected(pg/ml)-Recovery
(%)
-

200

517

491

105

400

668

691

97

600

859

891

96

-

64

-

-

200

287

264

109

400

520

464

112

600

661

664

100

-

247

-

-

200

490

447

110

400

668

647

103

600

847

847

100

9.6 Linearity
Four saliva samples containing different amounts of analyte were serially diluted with Standard A andassayed with the DHEA Saliva ELISA. The percentage recovery was calculated by comparing the expected
and observed values for DHEA.
Saliva12

3

4

nativeObserved(pg/ml)569

Expected(pg/ml)-Linearity(%)-

1 in 2

284

285

100

1 in 4

127

142

89

Dilution

1 in 8

77

71

108

native384--

1 in 2

167

192

87

1 in 478

96

81

1 in 840

48

83

native238--

1 in 2

86

119

72

1 in 4

63

60

105

1 in 8

30

30

100

native292--

1 in 2

135

146

92

1 in 4

73

73

100

1 in 8

40

37

108

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 good laboratory practice.
Any improper handling of samples or modification of this test might influence the results.
10.1 High-Dose-Hook EffectNo hook effect was observed in this test.
10.2 Drug Interference
Any medication (cream, oil, pill etc) containing DHEA of course will significantly influence the measurementof this analyte in saliva.
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11. REFERENCES
1. Assessing cortisol and dehydroepiandrosterone (DHEA) in saliva: effects of collection method.
Gallagher P, Leitch MM, Massey AE, McAllister-Williams RH, Young AH
J Psychopharmacol, Sep 2006 (Vol. 20, Issue 5, Pages 643-9)
2. Effects of DHEA administration on episodic memory, cortisol and mood in healthy young men: a doubleblind, placebo-controlled study.
Alhaj HA, Massey AE, McAllister-Williams RH Psychopharmacology (Berl), Nov 2006 (Vol. 188, Issue 4,
Pages 541-51)
3. Bacteria in the oral mucosa and its effects on the measurement of cortisol, dehydroepiandrosterone, andtestosterone in saliva.
Whembolua GL, Granger DA, Singer S, Kivlighan KT, Marguin JA Horm Behav, Apr 2006 (Vol. 49, Issue 4,
Pages 478-83)
4. Anthropometry and body composition do not predict bioavailable androgen or progesterone concentrationin adolescent girls.
Bond LJ, Vella ET, Kiparissis Y, Wynne-Edwards KE Am J Hum Biol, Sep 2006 (Vol. 18, Issue 5, Pages
639-53)
5. The influence of 10 min of the Johrei healing method on laboratory stress.
Laidlaw TM, Naito A, Dwivedi P, Hansi NK, Henderson DC, Gruzelier JH Complement Ther Med, Jun 2006
(Vol. 14, Issue 2, Pages 127-32)
6. Salivary cortisol, dehydroepiandrosterone-sulphate (DHEA-S) and testosterone in women with chronic
migraine.
Patacchioli FR, Monnazzi P, Simeoni S, De Filippis S, Salvatori E, Coloprisco G, Martelletti P J HeadachePain, Apr 2006 (Vol. 7, Issue 2, Pages 90-4)
7. Aggression, dominance, and affiliation: Their relationships with androgen levels and intelligence in 5year-old children.
Azurmendi A, Braza F, Garcia A, Braza P, Munoz JM, Sanchez-Martin JR Horm Behav, Jun 2006 (Vol. 50,
Issue 1, Pages 132-40)
8. Cognitive abilities, androgen levels, and body mass index in 5-year-old children.
Azurmendi A, Braza F, Sorozabal A, Garcia A, Braza P, Carreras MR, Munoz JM, Cardas J, SanchezMartin JR Horm Behav, Aug 2005 (Vol. 48, Issue 2, Pages 187-95)
9. Effect of prolonged stress on the adrenal hormones of individuals with irritable bowel syndrome.
Sugaya N., Izawa S., Saito K., Shirotsuki K., Nomura S. and Shimada H.
BioPsychoSocial Medicine 2015, 9:4

For updated literature or any other information please contact your local supplier.
Symbols:

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Storagetemperature

Manufacturer

Expiry date

Batch code

Consult instructions
for useContent

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Contains sufficient for<n> tests

For research use
only!

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