Instructions for use

DHEA-S Saliva ELISASA E-6500R

Free

1. INTENDED USE
Competitive immunoenzymatic colorimetric method for quantitative determination of DHEA-S concentration insaliva. DHEA-S ELISA (Saliva) is intended for research use only.
1.1

SUMMARY AND EXPLANATION

Dehydroepiandrosterone sulfate (DHEA-S) is a endogenous natural steroid hormone with 19 carbon atoms. It is
the principal steroid hormone produced by the secretion of the adrenal glands, but it is also produced in thegonads and brain. DHEA is the most abundant circulating steroid in human beings.
DHEA-S is a natural steroid hormone found primarily in the kidneys and it is derived from the enzymaticconversion of DHEA in the adrenal and extra-adrenal tissues. It is the most abundant hormone in the humanbody and is a precursor of all sex steroids. As most DHEA-S is produced by the zona reticularis of the adrenal, it
is argued that there is a role in the immune and stress response. DHEA-S may have more biologic roles: forexample its production in the brain suggests a role as neurosteroid.
The majority of DHEA-S in saliva is non-protein bound and enters the saliva via intracellular mechanisms.
Salivary DHEA-S levels are unaffected by salivary flow rate or salivary enzymes.
Measurement of saliva DHEA-S is a useful marker of adrenal androgen synthesis. Abnormally low levels may
occur in have been reported in hypoadrenalism, while elevated levels occur in several conditions, e.g. virilizing
adrenal adenoma and carcinoma, 21-hydroxylase and 3β-hydroxysteroid dehydrogenase deficiencies and in somecases of female hirsutism. Women with polycystic ovary syndrome tend to have normal or mildly elevated levelsof DHEAS.
2. PRINCIPLEThe DHEA-S (antigen) in the sample competes with the antigenic DHEA-S conjugated with horseradish
peroxidase (HRP) for binding to the limited number of antibodies anti DHEA-S coated on the microplate (solidphase).
After incubation, the bound/free separation is performed by a simple solid-phase washing.
Then, the enzyme HRP in the bound-fraction reacts with the Substrate (H2O2) and the TMB Substrate anddevelops a blue colour that changes into yellow when the Stop Solution (H2SO4) is added.
The colour intensity is inversely proportional to the DHEA-S concentration of in the sample.
DHEA-S concentration in the sample is calculated through a standard curve.
3. REAGENTS, MATERIALS AND INSTRUMENTATION
3.1

Reagents and materials supplied in the kit

Standards and Controls
Cat. no.

SymbolStandards

Concentration

Volume/Vial

Standard 00 ng/mL

1 ml

Standard 1

0.2 ng/mL

1 ml

Standard 2

1 ng/mL

1 ml

Standard 3

3 ng/mL

1 ml

SA E-6551

Standard 4
Control low

12 ng/mL

1 ml
1 ml

SA E-6552

Control highSA E-6501
SA E-6502
SA E-6503
SA E-6504
SA E-6505

Refer to vial labels for expectedvalue and acceptable range!

1 ml

SA E-6513 Incubation Buffer(1 vial) 30 mL; Phosphate buffer pH 7.5, BSA 1 g/L
SA E-6540 Conjugate(1 vial) 1 mL; DHEA-S conjugated with horseradish peroxidase (HRP)SA E-6531 Microtiterwells(1 microplate breakable); Anti-DHEA-S antibody adsorbed on microplate
SA E-0030 Conc. Wash Solution 50X(1 vial) 20 mL; NaCl 45 g/L; Tween 20, 55 g/L
MS E-0055 TMB-Substrate(1 vial) 15 mL; H2O2-TMB 0.26 g/L, (avoid any skin contact)

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MS E-0080 Stop Solution(1 vial) 15 mL; Sulphuric acid 0.15 mol/L (avoid any skin contact)
Hazards
identification:
H290 May be corrosive to metals.
H314 Causes severe skin burns and eye damage.
3.2

Reagents necessary not supplied

Distilled water.
3.3

Auxiliary materials and instrumentation

Automatic dispenser.
Microplates reader (450 nm, 620-630).
Saliva Collection Device e.g. SALI SET 100 REF SA D-6100 or Salivette Sarstedt REF 51.1534.500
NoteStore all reagents at 2- 8 °C in the dark.
Open the bag of the Coated Microplate only when it is at room temperature and close immediately after use;
once opened the microplate is stable until the expiry date of the kit.

4. WARNINGS
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This kit is intended for research use by professional persons only. Not for internal or external use in Humansor Animals.
Use appropriate personal protective equipment while working with the reagents provided.
Follow Good Laboratory Practice (GLP) for handling blood products.
Some reagents contain small amounts of Proclin 300 as preservative. Avoid the contact with skin or mucosa.
The TMB Substrate contains an irritant, which may be harmful if inhaled, ingested or absorbed through theskin. To prevent injury, avoid inhalation, ingestion or contact with skin and eyes.
The Stop Solution consists of a diluted sulphuric acid solution. Sulphuric acid is poisonous and corrosive andcan be toxic if ingested. To prevent chemical burns, avoid contact with skin and eyes.
Avoid the exposure of reagent TMB/H2O2 to directed sunlight, metals or oxidants. Do not freeze the solution.
This method allows the determination of DHEA-S from 0.2 ng/mL to 12 ng/mL.

5. PRECAUTIONS
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Please adhere strictly to the sequence of pipetting steps provided in this protocol. The performance data
represented here were obtained using specific reagents listed in this Instruction for Use.
All reagents should be stored refrigerated at 2 °C - 8 °C in their original container. Any exceptions are clearlyindicated. The reagents are stable until the expiry date when stored and handled as indicated.
Allow all kit components and specimens to reach room temperature (22 °C - 28 °C) and mix well prior to use.
Do not interchange kit components from different lots. The expiry date printed on box and vials labels must
be observed. Do not use any kit component beyond their expiry date.
If you use automated equipment, the user has the responsibility to make sure that the kit has been
appropriately tested.
The incomplete or inaccurate liquid removal from the wells could influence the assay precision and/or increase
the background.
To improve the performance of the kit on automatic systems is recommended to increase the number ofwashes.
It is important that the time of reaction in each well is held constant for reproducible results. Pipetting ofsamples should not extend beyond ten minutes to avoid assay drift. If more than 10 minutes are needed,follow the same order of dispensation. If more than one plate is used, it is recommended to repeat the dose
response curve in each plate
Addition of the TMB Substrate solution initiates a kinetic reaction, which is terminated by the addition of theStop Solution. Therefore, the TMB Substrate and the Stop Solution should be added in the same sequence toeliminate any time deviation during the reaction.
Observe the guidelines for performing quality control in medical laboratories by assaying controls and/orpooled saliva samples.
Maximum precision is required for reconstitution and dispensation of reagents.
Plate readers measure vertically. Do not touch the bottom of the wells.

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6. PROCEDURE
6.1

Preparation of the standard (standard A-E)

Before use, mix for 5 minutes with a rotating mixer.
The Standards are ready to use and have the following concentrations of DHEA-S:ng/mL

standard A0

standard B
0.2

standard C
1.0

standard D
3.0

standard E
12.0

Samples with concentration greater than 12.0 ng/mL have to be diluted 1:2 with standard A.
Once open, the standards are stable at 2 °C – 8 °C for 6 months.
For SI UNITS: ng/mL x 2.71 = nmol/L
6.2

Preparation of Diluted Conjugate

Prepare immediately before use.
Add 10 µl of Conjugate to 1.0 mL of Incubation Buffer. Mix gently.
Stable 3 hours at room temperature (22 °C – 28 °C).
6.3

Preparation of Wash Solution

Dilute the content of each vial of Concentrate Wash Solution with distilled water to a final volume of 1000 mLprior to use. For smaller volumes respect the 1:50 dilution ratio.
The diluted wash solution is stable for 30 days at 2 °C - 8 °C.
6.4

Preparation of the Sample

The determination of DHEA-S with this kit should be performed in saliva.
It is recommended to collect saliva samples with a centrifuge glass tube and a plastic straw, with the Sali Set(catalogue no. SA D-6100) or with the “Salivette” (Sarstedt, Ref. 51.1534.500).
Other commercially available sample collector devices have not been tested.
6.4.1

Method and Limitations

Collect saliva samples at the times indicated.
If no specific instructions have been given, saliva samples may be collected at any time, paying attention to thefollowing indications:a. If saliva collection is carried out in the morning ensure that this is carried out prior to brushing teeth
b. During the day allow 1 hour after a meal, oral intake of pharmaceutical drugs or tooth cleaning.
c. It is very important that a good clear sample is received – i.e. no contamination with food, lipstick, blood(bleeding gums) or other extraneous materials.
6.4.2

Saliva Processing Instructions with glass tubes

Let the saliva flow down through the straw into the centrifuge glass tube.
1. Centrifuge the sample for 15 minutes at 3000 rpm
2.

Store at -20 °C for at least 1 hour

3.

Centrifuge again for 15 minutes at 3000 rpm

4.

The saliva sample is now ready to be tested.

5.

Store the sample at 2 °C - 8 °C for one week or at -20 °C for longer time.

6.4.3 Saliva Processing Instructions with Salivette Sardstedt
1. Remove the swab from the suspended insert of the Salivette
2. Gently chewing the swab for 1 minute produces a sufficient quantity of saliva.
3. Replace the swab into the Salivette and firmly close the tube using the stopper.
4. Centrifuge the Salivette for 2 minutes at 1000g (rcf) for saliva generation.
5. Remove the insert complete with the swab from the centrifuge vessel and discard. The clear saliva is now
ready for analysis (at least 1 mL of saliva should be recovered with this method).

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6.5
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Procedure

Allow all reagents to reach room temperature (22 °C - 28 °C). At the end of the assay, store
immediately the reagents at 2 °C - 8 °C: avoid long exposure to room temperature.
Unused coated microwell strips should be released securely in the foil pouch containing desiccant and stored
at 2 °C - 8 °C.
To avoid potential microbial and/or chemical contamination, unused reagents should never be transferred into
the original vials.
As it is necessary to perform the determination in duplicate in order to improve accuracy of the test results,
prepare two wells for each point of the standard curve (standard A-E), two for each Control, two for eachsample, one for Blank.

Reagent

Standard

Sample/Controls

Sample/Controls

Blank

50 µL

Standards (A-E)

50 µL

Diluted conjugate

150 µL150 µL

Incubate at 37 °C for 15 minutes.
Remove the contents from each well; wash the wells 3 times with 0.3 mL of diluted wash solution.
Important note: During each washing step, gently shake the plate for 5 seconds and remove excess solution bytapping the inverted plate on an absorbent paper towel.
Automatic washer: if you use automated equipment, wash the wells at least 5 times.
TMB Substrate

100 µL100 µL100 µL

Incubate at room temperature (22 °C - 28 °C) for 15 minutes in the dark.
Stop Solution

100 µL100 µL100 µL

Shake the microplate gently.
Read the absorbance (E) at 450 nm against a reference wavelength of 620-630 nm or against Blank within
5 minutes.

7. QUALITY CONTROL
Each laboratory should assay controls at normal, high and low levels range of DHEA-S for monitoring assayperformance. These controls should be treated as unknowns and values determined in every test procedure
performed. Quality control charts should be maintained to follow the performance of the supplied reagents.
Pertinent statistical methods should be employed to ascertain trends. The individual laboratory should set
acceptable assay performance limits. Other parameters that should be monitored include the 80, 50 and 20%
intercepts of the standard curve for run-to-run reproducibility. In addition, maximum absorbance should beconsistent with past experience. Significant deviation from established performance can indicate unnoticed
change in experimental conditions or degradation of kit reagents. Fresh reagents should be used to determine
the reason for the variations.

8. RESULTS
8.1

Mean AbsorbanceCalculate the mean of the absorbance (Em) for each point of the standard curve (standard A-E) and of eachsample8.2

Standard Curve

Plot the mean value of absorbance (Em) of the standards (standards A-E) against concentration. Draw the bestfit curve through the plotted points. (es: Four Parameter Logistic).
8.3

Calculation of ResultsInterpolate the values of the samples on the standard curve to obtain the corresponding values of theconcentrations expressed in ng/mL.

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9. REFERENCE VALUESAs the values of salivary DHEA-S have a cicardian pattern we suggest to collect the samples at the same hour
(8 A.M.).
The following values can be used as preliminary guideline until each laboratory established its own normal range.

WOMENMENng/mL
0.2 – 2.5
0.2 – 2.7

Please pay attention to the fact that the determination of a range of expected values for a “normal” population ina given method is dependent on many factors, such as specificity and sensitivity of the method used and type of
population under investigation.
Therefore each laboratory should consider the range given by the manufacturer as a general indication andproduce their own range of expected values based on the indigenous population where the laboratory works.

10. PERFORMANCE AND CHARACTERISTICS
10.1 Precision
10.1.1 Intra Assay Variation
Within run variation was determined by replicate measurements (14x) of two different control sera in one assay.
The within assay variability is ≤ 7.8%.
10.1.2 Inter Assay Variation
Between run variation was determined by replicate measurements (9x) of three different control sera withdifferent lots of kit. The between assay variability is ≤ 14.9%.
10.2 Accuracy
The recovery of 0.5 – 1.5 – 6.0 ng/mL of DHEA-S added to sample gave an average value (±SD)
of 108.86% ± 3.27% with reference to the original concentrations.
10.3 Sensitivity
The lowest detectable concentration of DHEA-S that can be distinguished from the standard A is 0.05 ng/ml at
the 95 % confidence limit.
10.4 Specificity
The cross reaction of the antibody calculated at 50% according to Abraham are shown in the table:
DHEA-S
90 %
DHEA

100 %

Androsterone-S-Na

48 %Androstendione

20 %Etiocolanone-S-Na

0.2 %5-Androstendione

0.01 %

Testosterone

0.01 %

Progesterone

0.01 %

17 OH Progesterone

0.01 %

Estrone0.01 %

Cortisol0.001 %

Cholesterol

0.001 %

10.5 Correlation
The DHEA-S Saliva ELISA was compared to an analogous commercially available kit. 31 saliva samples wereanalysed according in both test systems.
The linear regression curve was calculated:
y = 0.37x + 1.10
r2 = 0.826
y = DHEA-S Saliva ELISA
x = DHEA-S Saliva Salimetrics Kit

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11. WASTE MANAGEMENT
Reagents must be disposed off in accordance with local regulations.

12. BIBLIOGRAPHY
1.

Joshi, U. M., et al. Steroids 34 (1) 35 (1979)

2.

Turkes, A., et al. J Endocrinol. 81 (2) P165 (1979)3.

Ismail A.A, et al. J.Clin.Endocr.Metab. 34,177-184 (1972)

4.

Rajkowski,K.M, et al. Steroids 29 no 5 (1977)

5.

Widsdom G.B. Clin. Chem. 22/8, 1243 - 1255 (1976)

6.

Abraham G.E, et al. Obstet.Gynecol.,47(4),395 (1976)

7.

Abraham G.E, et al. Obstet. Gynecol.,53(1),111 (1979)8.

Hopper B.R, et al. J.Clin.Endoc.Metab.40(3),458 (1975)

9.

Winter J.S.D,et al. Clin.Obste.and Gynec.,21(1),67 (1978)

10. D. Riad et al. Endocr. Reviews, 3 (4) 304 367 (1978)

13. TROUBLESHOOTINGERRORS / POSSIBLE CAUSES / SUGGESTIONS
No colorimetric reaction
 no conjugate pipetted
 contamination of conjugates and/or of substrate errors in performing the assay procedure (e.g. accidental pipetting of reagents in a wrong sequence or fromthe wrong vial, etc.)
Too low reaction (too low ODs)
 incorrect conjugate (e.g. not from original kit) incubation time too short, incubation temperature too low
Too high reaction (too high ODs)
 incorrect conjugate (e.g. not from original kit) incubation time too long, incubation temperature too high water quality for wash buffer insufficient (low grade of deionization)
 insufficient washing (conjugates not properly removed)
Unexplainable outliers
 contamination of pipettes, tips or containers
 insufficient washing (conjugates not properly removed)
too high within-run CV% reagents and/or strips not pre-warmed to room temperature prior to use plate washer is not washing correctly (suggestion: clean washer head)
too high between-run CV %
 incubation conditions not constant (time, temperature) controls and samples not dispensed at the same time (with the same intervals) (check pipetting order) person-related variation

Symbols:
Storagetemperature

Manufacturer

Expiry date

Batch code

Consult instructions
for useContentCaution

Catalogue
numberVersion 8.0-b-r

Effective: 2018-06-20

Contains sufficient for<n> tests

For research use
only!
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