Instructions for use

Androstanediol-Glucuronide ELISAAA E-1500R

5α-ANDROSTANE-3α, 17β-DIOL GLUCURONIDE (3α DIOL G) ELISAINTENDED USE
For the direct quantitative determination of 3α Diol G by 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 anenzyme-labelled antigen (conjugate) for a limited number of antibody binding sites on the microplate. Thewashing 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 of
3α-Diol G in the sample. A set of standards is used to plot a standard curve from which the amount of 3α-Diol Gin samples 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 microwells 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.
The assay buffer is sensitive to light and should be stored in the original dark bottle away from direct
sunlight.
12.
When dispensing the substrate and stopping solution, do not use pipettes in which these liquids will comeinto contact with any metal parts.
13.
To prevent contamination of reagents, use a new disposable pipette tip for dispensing each reagent,
sample, standard and control.
14.
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.
15.
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 3α-Diol G in human serum.
The kit is not calibrated for the determination of 3α-Diol G in saliva, plasma or other specimens of humanor 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 HCVand Human Immunodeficiency 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 0.2 ml of serum is required per duplicate determination. Collect 4–5 ml of blood into an
appropriately 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 50, 100, 150 and 300 µl
2. Disposable pipette tips
3. Distilled or deionized water
4. Plate shaker
5. Microplate reader with a filter set at 450nm 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.

SymbolStandardAA E-1501Standard A

Concentration
0 ng/ml

Volume/Vial
2.0 mlAA E-1502

Standard B

0.25 ng/ml

0.6 mlAA E-1503

Standard C

1 ng/ml

0.6 mlAA E-1504

Standard D

3 ng/ml

0.6 mlAA E-1505

Standard E

10 ng/ml

0.6 mlAA E-1506

Standard F

50 ng/ml

0.6 mlAA E-1551

Control 1

AA E-1552

Control 2

Refer to vial labels for
expected value andacceptable range!

0.6 ml

0.6 mlContents:

3α Diol G in a protein-based buffer with a non-mercury preservative.
spiking buffer with a defined quantity of 3α Diol G.

Storage:

Refrigerate at 2 - 8 oC

Stability:

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

5. AA E-1513

Assay Buffer - Ready To Use.

Warm to completely dissolve before use.

Contents:

One vial containing a protein-based buffer with a non-mercury preservative.

Volume:15 ml/bottle

Storage:

Refrigerate at 2 - 8 oC

Stability:

12 months or as indicated on label.

6. AA E-1531

Prepared byRabbit Anti-3α Diol G Antibody Coated Microwell Plate-Break Apart
Wells - Ready To Use.

Contents:

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

Storage:

Refrigerate at 2 - 8 oC

Stability:

12 months or as indicated on label.
3α Diol G-Horseradish Peroxidase (HRP) Conjugate Concentrate –requires preparation X50

7. AA E-1540
Contents:

3α Diol G -HRP conjugate in a protein-based buffer with a non-mercury preservative.

Volume:300 µl/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 240 µl of HRP in 12 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 3α Diol G-HRP conjugate and wash buffer.

2.

Remove the required number of microwell strips. Reseal the bag and return any unused strips to therefrigerator.

3.

Pipette 50 µl of each standard, control and specimen sample into correspondingly labelled wells induplicate.

4.

Pipette 100 µl of the conjugate working solution into each well.
(We recommend using a multichannel pipette).

5.

Incubate on a plate shaker (approximately 200 rpm) for 30 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 the plate on a plate shaker for 10-15 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 microwell plate reader at 450 nm within 20 minutes after addition of the stoppingsolution.
If the OD exceeds the upper limit of detection or if a 450nm filter is unavailable, a 405 or 415nm filter may
be substituted. The optical densities will be lower, however, this will not affect the results ofspecimen/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 50 ng/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:StandardOD 1

OD 2

Mean OD

A

2.480

2.474

2.477

Value(ng/ml)
0B

2.102

2.106

2.104

0.25

C

1.428

1.413

1.421

1D

0.877

0.883

0.880

3E

0.360

0.368

0.364

10F

0.147

0.143

0.145

50Unknown

0.598

0.596

0.597

5.4

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TYPICAL STANDARD CURVE
Sample curve only. Do not use to calculate results:
2.5

OD 450nm

21.5
10.5
00.25

3110503 Diol G (ng/ml)

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 theDirect 3α-Diol G ELISA kit is 0.1 ng/ml.
SPECIFICITY (CROSS REACTIVITY)
The following compounds were tested for cross-reactivity with the Direct 3α-Diol G ELISA kit with 3α-Diol G
cross-reacting at 100%.
Steroid
3α Diol G
Testosterone
Progesterone
Androstenedione
Cortisol%Cross Reactivity
100
0.2
0.16
0.14
0.05

The following steroids were tested but cross-reacted at less than 0.01%:
Dehydroepiandrosterone, Dihydrotestosterone, Epiandrosterone, 17β-Estradiol and Estrone.

Corticosterone,INTRA-ASSAY PRECISION
Three samples were assayed ten times each on the same standard curve. The results (in ng/ml) are tabulatedbelow:
Sample
12
3

Mean
0.87
6.86
21.26

SD
0.07
0.49
1.29

CV%
7.8
7.2
6.0

INTER-ASSAY PRECISION
Three samples were assayed ten times over a period of four weeks. The results (in ng/ml) are tabulated below:
Sample123

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Mean
0.98
7.05
20.92

SD
0.10
0.46
2.26

CV%
10.4
6.5
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RECOVERY
Spiked samples were prepared by adding defined amounts of 3α Diol G to three serum samples. The results (inng/ml) are tabulated below:
Sample1 Unspiked+0.5+5.0+15.0
2 Unspiked+0.5+5.0+15.0
3 Unspiked+0.5+5.0+15.0

Obs. Result
0.67
1.07
4.99
12.66
1.83
2.07
6.18
17.64
12.76
15.32
19.22
22.68

Exp. Result
1.17
5.67
15.67
2.33
6.83
16.83
13.26
17.76
27.76

Recovery %
91.4
88.0
80.8
88.8
90.5
104.8
115.5
108.2
81.7

LINEARITY
Three serum samples were diluted with Standard A. The results (in ng/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
6.24
2.83
1.550.74
13.55
6.00
2.71
1.70
17.05
6.93
4.09
2.34

Exp. Result
3.12
1.56
0.78
6.77
3.39
1.64
8.53
4.26
2.13

Recovery %
90.7
99.4
94.9
88.6
80.0
103.6
81.2
96.0
109.8

EXPECTED NORMAL VALUESEach laboratory should collect data and establish their own range of expected normal values.
GroupMales
Premenopausal
Postmenopausal
Puberty (Female)

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Range (ng/ml)
1.53 - 14.82
0.22 - 4.64
0.61 - 3.71
0.51 - 4.03

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REFERENCES
1. Jacobi GH, Wilson JD. Formation of 5α-Androstane-3α, 17β-Diol by Normal and Hypertrophic Human
Prostate. J Clin Endocrinol Metab. 1977; 44(1):107–15.
2. Deslypere JP, et al. Plasma 5α-Androstane-3α,17β-Diol and Urinary 5α-Androstane-3α,17β-Diol
Glucuronide, Parameters of Peripheral Androgen Action: A comparative study. J Clin Endocrinol Metab.
1982; 54(2):386–91.
3. Moghissi E, et al. Origin of Plasma Androstanediol Glucuronide in Men. J Clin Endocrinol Metab. 1984;
59(3):417–21.
4. Scanlon MJ, et al. Serum A ndrostanediol G lucuronide Concentrations in Normal and Hirsute Women andPatients with Thyroid Dysfunction. Clin Endocinol (Oxf). 1988; 29(5):529–38.
5. Reiner BJ, et al. Serum 3α-Androstanediol Glucuronide Measurements in Sexually Mature Women withCongenital Adrenal Hyperplasia During Therapy. J Clin Endocrinol Metab. 1989; 69(1):105–9.
6. Vexiau P, et al. Increase in plasma 5α-Androstane-3α,17β- Diol G lucuronide a s a M arker o f Pe ripheral A
ndrogen Action in Hirsutism: A Side-effect Induced by Cyclosporine A. J Steroid Biochem. 1990;
35(1):133–7.
7. Pang S, et al. 3α-Androstanediol Glucuronide in Virilizing Congenital Adrenal Hyperplasia: A Useful Serum
Metabolic Marker of Integrated Adrenal Androgen Secretion. J Clin Endocrinol Metab. 1991; 73(1):166–74.
8. Riddick L, et al. 3α-Androstanediol Glucuronide in Premature and Normal Pubarche. J Clin Endocrinol
Metab. 1991; 72(1): 46–50.
9. 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.

Symbols:

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for useContentCaution

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numberEffective: 2018-11-20

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