Instructions for use

DHEA ELISAAA E-1600R

DHEA ELISAINTRODUCTION

Intended Use
The DHEA ELISA is a competitive immunoassay for the quantitative measurement of Dehydroepiandrosterone(DHEA) in serum and plasma.
PRINCIPLE
The DHEA ELISA Kit is a solid phase enzyme-linked immunosorbent assay (ELISA), based on the principle ofcompetitive binding.
The microtiter wells are coated with an anti-DHEA antibody. An unknown amount of DHEA present in thesample competes with an DHEA-horseradish peroxidase conjugate for binding to the coated antibody. Afterincubation the unbound conjugate is washed off. The amount of bound peroxidase conjugate is inversely
proportional to the concentration of DHEA in the sample. After addition of the substrate solution, the intensityof color developed is inversely proportional to the concentration of DHEA in the sample.
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 thepackage 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 °C to 8 °C in the sealed foil
pouch and used in the frame provided.

4.

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

5.

Use reservoirs only for single reagents. This especially applies to the substrate reservoirs. Using a reservoirfor dispensing a substrate solution that had previously been used for the conjugate solution may turnsolution 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 (21-26°C) before starting the test. Temperature will affectthe absorbance readings of the assay. However, values for the samples will not be affected.

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.
14. All indicated volumes have to be performed according to the protocol. Optimal test results are only obtained
when using calibrated pipettes and microtiterplate readers.
15. Do not mix or use components from kits with different lot numbers. It is advised not to exchange wells ofdifferent 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.
16. Avoid contact with Stop Solution. It may cause skin irritation.
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.

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REAGENTS

Reagents provided
AA E-1631 Microtiterplate
12x8 (break apart) strips with 96 wells; Wells coated with anti-DHEA antibody.
Standards
ready to use
Cat. no.

Standard

AA E-1601

Standard A (0)0 ng/ml

0.6 mlAA E-1602

Standard B (1)0.3 ng/ml

0.6 mlAA E-1603

Standard C (2)1 ng/ml

0.6 mlAA E-1604

Standard D (3)3 ng/ml

0.6 mlAA E-1605

Standard E (4)10 ng/ml

0.6 mlAA E-1606Standard F (5)30 ng/ml

0.6 ml+Concentration

Volume/Vial

AA E-1651 + AA E-1652 Control low / Control high2 vials, 0.6 ml each, ready to use; containing DHEA in serum.
For control values and ranges please refer QC-Datasheet
AA E-1640 Enzyme Conjugate
1 vial, 13 ml, ready to use; horseradish peroxidase labeled DHEA in buffered matrix.
AA E-1655 Substrate Solution
1 vial, 26 ml each, ready to use; contains Tetramethylbenzidine (TMB)
Hazards
identification:
H360D May damage the unborn child.
AA E-1680 Stop Solution
1 vial, 9 ml, ready to use; contains 2 N hydrochloric acid solution
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
2 vials, each 50 ml (10X concentrated);see “Reagent Preparation”.
Note: Additional Standard A for sample dilution is available upon request.
Materials required but not provided







A microtiter plate reader capable for endpoint measurement at 450 nm
Calibrated variable precision micropipettes (25 µL, 100 µL, 200 µL, 300 µL).
Microplate mixer operating more than 600 rpm
Absorbent paper
Distilled or deionized waterTimer
Semi logarithmic graph paper or software for data reduction

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°C to 8°C. After first opening the reagents are stable for
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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Reagent preparation
Allow the reagents and the required number of wells to reach room temperature (21-26°C) before starting thetest.
Wash Solution:
Dilute 50 mL of 10X concentrated Wash Solution with 450 mL deionized water to a final volume of 500 mL.
The diluted Wash Solution is stable for at least 12 weeks at room temperature (21-26°C).
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.
Damaged test kitsIn case of any severe damage of the test kit or components, the manufacturer have 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.
SPECIMENFor determination of DHEA serum or plasma (EDTA) can be used. The procedure calls for 25 µL sample perwell. The samples should be assayed immediately or aliquoted and stored at ≤ -20°C. Avoid repeated freezethaw cycles. Samples expected to contain DHEA concentrations higher than the highest standard (30 ng/mL)
should be diluted with Standard A before assay. The additional dilution step has to be taken into account for thecalculation of the results. Do not use grossly haemolytic, icteric or grossly lipaemic specimens.
ASSAY PROCEDURE
General RemarksAll reagents and specimens must be allowed to come to room temperature before use. All reagents must bemixed 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.

Assay procedure
Each run must include a standard curve.
1.

Prepare a sufficient number of microplate wells to accommodate standards and samples in duplicates.

2.

Dispense 25 µL of each Standard, Sample and Control with new disposable tips into appropriate
wells.

3.

Dispense 100 µL of Enzyme Conjugate into each well.

4.

Incubate for 60 minutes at room temperature on a plate shaker (> 600 rpm).
Important Note:
Optimal reaction in this assay is markedly dependent on shaking of the microplate!

5.

Discard the content of the wells and rinse the wells 4 times with diluted Wash Solution (300 µL perwell). Remove as much Wash Solution as possible by beating the microplate on absorbent paper.

6.

Add 200 µL of Substrate Solution to each well.

7.

Incubate without shaking for 30 minutes in the dark.

8.

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

9.

Determine the absorbance of each well at 450 nm. It is recommended to read the wells within
15 minutes.

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Calculation of results
1. Calculate the average absorbance values for each set of standards, controls and samples.
2. Using semi logarithmic graph paper, construct a standard curve by plotting the mean absorbance obtained
from each standard against its concentration with absorbance value on the vertical (Y) axis andconcentration on the horizontal (X) axis.
3. Using the mean absorbance value for each sample, determine the corresponding concentration from thecalibration 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 determined directly from this standard curve. Samples withconcentrations higher than that of the highest standard have to be further diluted. For the calculation of theconcentrations, this dilution factor has to be taken into account.
Example of typical standard curve
Following data are intended for illustration only and should not be used to calculate results from another run.
Standard
Standard A
Standard B
Standard C
Standard DStandard E
Standard F(0 ng/mL)(0.3 ng/mL)(1 ng/mL)(3 ng/mL)(10 ng/mL)(30 ng/mL)

Optical Units(450nm)
3.003
2.501
1.912
1.220
0.647
0.341

EXPECTED VALUES
It is strongly recommended that each laboratory should determine its own normal and abnormal values.
In a study conducted with apparently normal healthy adults, using the DHEA ELISA the following values areobserved:
Population

Range

Adult Males

1.8 – 12.5 ng/mL

Adult Woman

1.3 –9.8 ng/mL

PERFORMANCE CHARACTERISTICS

Analytical Sensitivity
The lowest analytical detectable level of DHEA that can be distinguished from Standard A is 0.07 ng/mL at the2SD confidence limit.

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Specificity (Cross Reactivity)
The following materials have been evaluated for cross reactivity. The percentage indicates cross reactivity at
50% displacement compared to DHEA.
Steroid
DHEA-S% Cross reaction< 0,01

Testosterone< 0,015α-Dihydrotestosterone< 0,01Androstendione

0,06

Progesterone

0,2317α-Hydroxyprogesterone< 0,01Pregnenolone

0,01

17-Hydroxy-Pregnenolone

0,07

Desoxycorticosterone

0,05

Corticosterone< 0,01

Cortisol< 0,01

Assay dynamic range
The range of the assay is between 0.3 – 30 ng/mL.
Reproducibility
Intra-Assay
The intra-assay variation was determined by 20 replicate measurements of three serum samples within one
run. The within-assay variability is shown below:
Serum 1

Serum 2

Serum 3

Mean (ng/mL)

2.08

5.34

20.84

SD

0.16

0.39

1.511

CV (%)

7.9

7.3

7.3

n=202020

Inter-Assay
The inter-assay (between-run) variation was determined by duplicate measurements of three serum samples in11 different tests.
Serum 1

Serum 2

Serum 3

Mean (ng/mL)

2.14

5.26

20.63

SD

0.15

0.27

1.03

CV (%)

6.9

5.1

5.0

n=

111111

RecoveryUsing the standard matrix a spiking solution of 1000 ng DHEA/mL was prepared. 500 µL of three sera werespiked with 1.5, 3 and 5 µL of the spiking solution leaving the serum matrices relatively intact. All samples weremeasured by the DHEA ELISA procedure.

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Sample1Spiking(ng/mL)-Measured(ng/mL)
0Expected(ng/mL)-Recovery
(%)
-

3

2.63

388%

236

5.52

692%

10

10.04

10100%

-

0.96

-

-

3

3.36

3.96

85%

65.67

6.96

81%

108.73

10.96

80%

-

1.69

--

3

4.05

4.69

86%

6

7.11

7.69

92%

10

10.24

11.69

88%

LinearityThree serum samples were assayed undiluted and diluted with Standard A.
Serum
12

3

Measured

Expected(ng/mL)(ng/mL)1 in 2
1 in 41 in 8

11.34
5.91
3.24
1.55./.
5.67
2.84
1.48./.
104%
114%
105%

1 in 2
1 in 41 in 8

4.78
2.57
1.23
0.49./.
2.39
1.2
0.6./.
108%
103%
82%

1 in 2
1 in 4

12.08
6.57
3.57./.
6.04
3.02./.
109%
118%

1 in 8

1.82

1.51

120%

Dilution

Linearity (%)

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.
Drug Interferences
Any medication (cream, oil, pill etc.) containing DHEA will significantly influence the measurement of thisanalyte.

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REFERENCES
1. Labrie F, Luu-The V, Belanger A, Lin SX, Simard J, Pelletier G, Labrie C.
hormone?

Is dehydroepiandrosterone aJ Endocrinol. 2005 Nov;187(2):169-96.
2. De Pergola G, Giagulli VA, Garruti G, Cospite MR, Giorgino F, Cignarelli M, Giorgino R. Low
dehydroepiandrosterone circulating levels in premenopausal obese women with very high body mass index.
Metabolism. 1991 Feb;40(2):187-90
3. Zumoff B, Rosenfeld RS, Strain GW, Levin J, Fukushima DK. Sex differences in the twenty-four-hour mean
plasma concentrations of dehydroisoandrosterone (DHA) and dehydroisoandrosterone sulfate (DHAS) andthe DHA to DHAS ratio in normal adults.
J Clin Endocrinol Metab. 1980 Aug;51(2):330-3
4. Carlstrom K, Brody S, Lunell NO, Lagrelius A, Mollerstrom G, Pousette A, Rannevik G, Stege R, von Schoultz
B.
Dehydroepiandrosterone sulphate and dehydroepiandrosterone in serum: differences related to ageand sex.
Maturitas. 1988 Dec;10(4):297-306
5. Lee PD, Winter RJ, Green OC. Virilizing adrenocortical tumors in childhood: eight cases and a review of theliterature.
Pediatrics. 1985 Sep;76(3):437-44.
6. Belanger A, Candas B, Dupont A, Cusan L, Diamond P, Gomez JL, Labrie F. Changes in serum concentrationsof conjugated and unconjugated steroids in 40- to 80-year-old men.
J Clin Endocrinol Metab. 1994 Oct;79(4):1086-90.

Symbols:
Storagetemperature

Manufacturer

Expiry date

Batch code

Consult instructions
for useContentCaution

Cataloguenumber

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For research use
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