Instructions for use

DHEA-S ELISAAA E-1100R

DHEA-S ELISAINTENDED USE
For the direct quantitative determination of DHEAS by an enzyme immunoassay in human serum.
PRINCIPLE OF THE TESTThe principle of the following enzyme immunoassay test follows the typical competitive binding scenario.
Competition occurs between an unlabelled antigen (present in standards, controls and samples) and an
enzyme-labelled antigen (conjugate) for a limited number of antibody binding sites on the microplate. The
washing and decanting procedures remove unbound materials. After the washing step, the enzyme substrate isadded. The enzymatic reaction is terminated by addition of the stopping solution. The absorbance is measured
on a microtiter plate reader. The intensity of the colour formed is inversely proportional to the concentration ofDHEAS in the sample. A set of standards is used to plot a standard curve from which the amount of DHEAS insamples and controls can be directly read.
PROCEDURAL CAUTIONS AND WARNINGS
1. Users should have a thorough understanding of this protocol for the successful use of this kit. Reliable
performance will only be attained by strict and careful adherence to the instructions provided.
2. Control materials or serum pools should be included in every run at a high and low level for assessing thereliability of results.
3. When the use of water is specified for dilution or reconstitution, use deionized or distilled water.
4. In order to reduce exposure to potentially harmful substances, gloves should be worn when handling kit
reagents and human specimens.
5. All kit reagents and specimens should be brought to room temperature and mixed gently but thoroughly
before use. Avoid repeated freezing and thawing of reagents and specimens.
6. A standard curve must be established for every run.
7. The controls should be included in every run and fall within established confidence limits.
8. Improper procedural techniques, imprecise pipetting, incomplete washing as well as improper reagent
storage may be indicated when assay values for the controls do not reflect established ranges.
9. When reading the microplate, the presence of bubbles in the wells will affect the optical densities (ODs).
Carefully remove any bubbles before performing the reading step.
10. The substrate solution (TMB) is sensitive to light and should remain colourless if properly stored.
Instability or contamination may be indicated by the development of a blue colour, in which case it should
not be used.
11. When dispensing the substrate and stopping solution, do not use pipettes in which these liquids will comeinto contact with any metal parts.
12. To prevent contamination of reagents, use a new disposable pipette tip for dispensing each reagent,
sample, standard and control.
13. Do not mix various lot numbers of kit components within a test and do not use any component beyond
the expiration date printed on the label.
14. Kit reagents must be regarded as hazardous waste and disposed of according to national regulations.
LIMITATIONS
1. All the reagents within the kit are calibrated for the direct determination of DHEAS in human serum. The
kit is not calibrated for the determination of DHEAS in saliva, plasma or other specimens of human or
animal origin.
2. Do not use grossly hemolyzed, grossly lipemic, icteric or improperly stored serum.
3. Any samples or control sera containing azide or thimerosal are not compatible with this kit, as they may
lead to false results.
4. Only standard A may be used to dilute any high serum samples. The use of any other reagent may leadto false results.
SAFETY CAUTIONS AND WARNINGS
POTENTIAL BIOHAZARDOUS MATERIAL
Human serum that may be used in the preparation of the standards and controls has been tested and found tobe nonreactive for Hepatitis B surface antigen and has also been tested for the presence of antibodies to HCV
and Human Immunodefi ciency Virus (HIV) and found to be negative. No test method however, can offer
complete assurance that HIV, HCV and Hepatitis B virus or any infectious agents are absent. The reagentsshould be considered a potential biohazard and handled with the same precautions as applied to any blood
specimen.
CHEMICAL HAZARDS
Avoid contact with reagents containing TMB, hydrogen peroxide and sulfuric acid. If contacted with any of thesereagents, wash with plenty of water. TMB is a suspected carcinogen.

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SPECIMEN COLLECTION AND STORAGE
Approximately 0.1 mL of serum is required per duplicate determination. Collect 4–5 mL of blood into anappropriately labelled tube and allow it to clot. Centrifuge and carefully remove the serum layer. Store at 4°C
for up to 24 hours or at -10°C or lower if the analyses are to be done at a later date. Consider all humanspecimens as possible biohazardous materials and take appropriate precautions when handling.
SPECIMEN PRETREATMENT
This assay is a direct system; no specimen pretreatment is necessary.
REAGENTS AND EQUIPMENT NEEDED BUT NOT PROVIDED
1. Precision pipettes to dispense 25, 50, 150, 200 and 300 μL
2. Disposable pipette tips
3. Distilled or deionized water
4. Plate shaker
5. Microplate reader with a filter set at 450 nm and an upper OD limit of 3.0 or greater*(see assay procedure step 10)
REAGENTS PROVIDED
1. AA E-0030

Wash Buffer Concentrate – requires preparation X10

Contents:

One bottle containing buffer with a non-ionic detergent and a non-mercury preservative.

Volume:50 ml/bottle

Storage:

Refrigerate at 2 – 8 oC

Stability:

12 months or as indicated on label.

Preparation:

Dilute 1:10 in distilled or deionized water before use. If the whole plate is to be used dilute
50 ml of the wash buffer concentrate in 450 ml of water.
TMB Substrate - Ready To Use.

2. AA E-0055
Contents:

One bottle containing tetramethylbenzidine and hydrogen peroxide in a non-DMF or DMSO
containing buffer.

Volume:16 ml/bottle

Storage:

Refrigerate at 2 – 8 oC

Stability:

12 months or as indicated on label.
Stopping Solution - Ready To Use.

3. AA E-0080
Contents:

One vial containing 1M sulfuric acid.

Volume:6 ml/vial

Storage:

Refrigerate at 2 – 8 oC

Stability:

12 months or as indicated on label.

Hazards
identification:
H290 May be corrosive to metals.
H314 Causes severe skin burns and eye damage.

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4. Standards and Controls- Ready To Use.
Listed below are approximate concentrations, please refer to vial labels for exact concentrations:Cat. no.

SymbolAA E-1101

Standard
Standard A

Concentration

AA E-1102

Standard B

0.005 µg/ml

0.5 mlAA E-1103

Standard C

0.02 µg/ml

0.5 mlAA E-1104

Standard D

0.1 µg/ml

0.5 mlAA E-1105

Standard E

0.5 µg/ml

0.5 mlAA E-1106

Standard F

2.5 µg/ml

0.5 mlAA E-1107

Standard G

10 µg/ml

AA E-1151

Control 1

AA E-1152

Control 20 µg/ml

Refer to vial labels for expectedvalue and acceptable range!

Volume/Vial
2.0 ml

0.5 ml
0.5 ml
0.5 mlContents:

DHEAS in a protein-based buffer with a non-mercury preservative. Prepared by spiking
buffer with a defined quantity of DHEAS.

Storage:

Refrigerate at 2 – 8 oC

Stability:

12 months in unopened vials or as indicated on label. Once opened the standards andcontrols should be used within 14 days or aliquoted and stored frozen. Avoid multiple
freezing and thawing cycles.

5. AA E-1113
Contents:

Assay Buffer - Ready To Use.
One vial containing a protein-based buffer with a non-mercury preservative.

Volume:30 ml/vial

Storage:

Refrigerate at 2 – 8 oC

Stability:

12 months or as indicated on label.

6. AA E-1131

Rabbit Anti-DHEAS Antibody Coated Break Apart Well Microplate Ready To Use.

Contents:

One 96 well (12x8) polyclonal antibody-coated microplate in a resealable pouch withdesiccant.

Storage:

Refrigerate at 2 – 8 oC

Stability:

12 months or as indicated on label.
DHEAS-Horseradish Peroxidase (HRP) Conjugate Concentrate –requires preparation X50

7. AA E-1140
Contents:

DHEAS-HRP conjugate in a protein-based buffer with a non-mercury preservative.

Volume:0.8 ml/vial

Storage:

Refrigerate at 2 – 8 oC

Stability:

12 months or as indicated on label.

Preparation:

Dilute 1:50 in assay buffer before use (eg. 40 µl of HRP in 2 ml of assay buffer). If thewhole plate is to be used dilute 0.5 ml of HRP in 25 ml of assay buffer. Discard any that isleft over.

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ASSAY PROCEDURE
Specimen Pretreatment: None.
All reagents must reach room temperature before use. Standards, controls and specimen samples should beassayed in duplicate. Once the procedure has been started, all steps should be completed without interruption.
1.

Prepare working solutions of the DHEAS-HRP conjugate and wash buffer.

2.

Remove the required number of well strips. Reseal the bag and return any unused strips to therefrigerator.
Pipette 25 μL of each standard, control and specimen sample into correspondingly labelled wells induplicate.
Pipette 200 μL of the conjugate working solution into each well.
(We recommend using a multichannel pipette.)

3.
4.
5.

Incubate on a plate shaker (approximately 200 rpm) for 45 minutes at room temperature

6.

Wash the wells 3 times with 300 μL of diluted wash buffer per well and tap the plate firmly against
absorbent paper to ensure that it is dry.
(The use of a washer is recommended.)7.

Pipette 150 μL of TMB substrate into each well at timed intervals..

8.

Incubate on a plate shaker for 15–20 minutes at room temperature(or until standard A attains dark blue colour for desired OD).

9.

Pipette 50 μL of stopping solution into each well at the same timed intervals as in step 7.

10.

Read the plate on a microplate reader at 450 nm within minutes after addition of the stopping solution.
If the OD exceeds the upper limit of detection or if a 450 nm filter is unavailable, a 405 or 415 nm filter
may be substituted. The optical densities will be lower, however, this will not affect the results ofdonor/control samples.

CALCULATIONS
1. Calculate the mean optical density of each standard duplicate.
2. Draw a standard curve on semi-log paper with the mean optical densities on the Y-axis and the standard
concentrations on the X-axis. If immunoassay software is being used, a 4-parameter or 5-parameter
curve is recommended.
3. Calculate the mean optical density of each unknown duplicate.
4. Read the values of the unknowns directly off the standard curve.
5. If a sample reads more than 10 μg/mL then dilute it with standard A at a dilution of no more than 1:8.
The result obtained should be multiplied by the dilution factor.
TYPICAL TABULATED DATA
Sample data only. Do not use to calculate results.
StandardABCDEFG
Unknown

OD 1
2.106
1.910
1.636
1.398
0.966
0.496
0.250
0.690

OD 2
2.060
1.863
1.769
1.382
0.938
0.479
0.252
0.688

Mean OD
2.083
1.887
1.703
1.390
0.952
0.488
0.251
0.689

Value (µg/ml)
00.005
0.02
0.1
0.5
2.5
10
1.20

TYPICAL STANDARD CURVE
Sample curve only. Do not use to calculate results.
21.8
1.6

OD 450nm1.4
1.2
1
0.8
0.6
0.4
0.2
00.0010.01

0.1110100DHEAS (ug/mL)

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PERFORMANCE CHARACTERISTICSSENSITIVITY
The lower detection limit is calculated from the standard curve by determining the resulting concentration ofthe mean OD of standard A (based on 10 replicate analyses) minus 2 SD.
Therefore, the sensitivity of the Direct DHEAS ELISA kit is 0.005 μg/mL.
SPECIFICITY (CROSS REACTIVITY)
The following compounds were tested for cross-reactivity with the Direct DHEAS ELISA kit with DHEAS crossreacting at 100%.
Steroid%Cross
Reactivity
DHEAS
100
Androsterone
16.0
Androstenedione
1.7
Testosterone
0.9Progesterone
0.6
DHT
0.6
Cortisol
0.5
The following steroids were tested but cross-reacted at less than 0.001%: 17β-Estradiol, Estrone, EstroneSulfate and Pregnenolone.
INTRA-ASSAY PRECISION
Three samples were assayed ten times each on the same standard curve. The results (in µg/ml) are tabulated
below:Sample
MeanSD
CV%
1
0.24
0.02
7.5
22.02
0.18
8.9
3
9.54
0.11
11.5
INTER-ASSAY PRECISION
Three samples were assayed ten times over a period of four weeks. The results (in µg/ml) are tabulated below:
Sample
123

Mean
0.13
1.11
6.38

SD
0.02
0.09
0.27

CV%
15.3
8.1
4.2

RECOVERY
Spiked samples were prepared by adding defi ned amounts of DHEAS to three serum samples. The results (inμg/mL) are tabulated below:
Sample1 Unspiked+0.1+1.0+5.0
2 Unspiked+0.1+1.0+5.0
3 Unspiked+0.1+1.0+5.0

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Obs.Result0.67
0.84
1.97
5.80
1.06
1.40
1.82
5.26
1.73
1.90
2.21
5.79

Exp.Result
0.77
1.67
5.67
1.16
2.06
6.06
1.83
2.73
6.73

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Recovery%
109.1
117.9
102.3
120.7
88.3
86.8
103.8
80.9
86.0

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LINEARITY
Three serum samples were diluted with standard A. The results (in μg/ml) are tabulated below:
Sample
1
1:2
1:4
1:8
2
1:2
1:4
1:8
3
1:2
1:4
1:8

Obs.Result
2.84
1.48
0.81
0.46
6.32
3.17
1.63
0.78
7.12
3.09
1.78
0.80

Exp.Result
1.42
0.71
0.36
3.16
1.58
0.79
3.56
1.78
0.89

Recovery%
104.2
87.8
104.2
100.3
103.2
98.7
86.8
86.5
89.9

EXPECTED NORMAL VALUESEach laboratory should collect data and establish their own range of expected normal values.
GroupRange (μg/ml)
Males
0.39-4.63
Females0.46-2.75
Postmenopausal
0.48-2.08
Females
REFERENCES
1. Chasalow FI, Blethen SL, Bradlow HL. Dehydroepiandrosterone Sulfate (DHEAS) and DHEASlike
Compounds in Fibrocystic Disease of the Breast. Steroids. 1988; 52(3):205–15.
2.

Chasalow FI, et al. Serum Levels of Dehydroepiandrosterone Sulfate as Determined by Commercial Kitsand Reagents. Steroids. 1989; 54(4):373–81.

3.

de Peretti E, Forest MG. Pattern of Plasma Dehydroepiandrosterone Sulfate Levels in Humans from Birthto Adulthood: Evidence for Testicular Production. J Clin Endocrinol Metab. 1978; 47(3):572–77.

4.

Holtzclaw WD, Gordon GB. Measurement of Serum Levels of Dehydroepiandrosterone Sulfate: AComparison of Radioimmunoassay and Enzymatic Analysis. Steroids,1989; 54(4): 355–71.

5.

Koritnik DR, et al. A Radioimmunoassay for Dehydroepiandrosterone Sulfate in the Circulation of RhesusMonkeys. Steroids. 1983; 42(6):653–67.

6.

Orentreich N, et al. Age Changes and Sex Differences in Serum Dehydroepiandrosterone Sulfate
Concentrations Throughout Adulthood. J Clin Endocrol Metab. 1984; 59(3):551–55.

7.

Smith MR, et al. A Radioimmunoassay for the Estimation of Serum Dehydroepiandrosterone Sulphate inNormal and Pathological Sera. Clin Chim Acta. 1975; 65(1): 5–13.

8.

Check JH, et al. Falsely Elevated Steroidal Assay Levels Related to Heterophile Antibodies Against Various
Animal Species. Gynecol Obstet Invest. 1995; 40(2):139–40.

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Manufacturer

Expiry date

Batch code

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for useContentCaution

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numberEffective: 2018-11-20

Contains sufficient for<n> tests

For research use
only!

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