Instructions for use

Pregnenolone ELISAFR E-2700R

Pregnenolone ELISAINTENDED USE
For the direct quantitative determination of Pregnenolone 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 ofpregnenolone in the sample. A set of standards is used to plot a standard curve from which the concentration ofpregnenolone in 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. It is recommended to all customers to prepare their own control materials or serum pools that should beincluded in every run at a high and low level for assessing the reliability 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 provided in the kit should be included in every run and fall within established confidence
limits.
8. Improper procedural techniques, imprecise pipetting, in- complete 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 pregnenolone in humanserum. The kit is not calibrated for the determination of pregnenolone in saliva, plasma or otherspecimens 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 lead to false results.
SAFETY CAUTIONS AND WARNINGS
POTENTIAL BIOHAZARDOUS MATERIAL
Human serum that maybe used in the preparation of the standards and controls has been tested and found to
be non-reactive 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 – X10

Contents:

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

Volume:50 ml/vial

Storage:

Refrigerate at 2 - 8 °CStability:

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/vial

Storage:

Refrigerate at 2 - 8 °C

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 °C

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.

SymbolStandard

FR E-2701

Standard A

Concentration
0 ng/ml

2.0 mlFR E-2702

Standard B

0.1 ng/ml

0.5 mlFR E-2703

Standard C0.4 ng/ml

0.5 mlFR E-2704

Standard D

1.6 ng/ml

0.5 mlFR E-2705

Standard E

6.4 ng/ml

0.5 mlFR E-2706

Standard F

25.6 ng/ml

FR E-2751

Control 1

0.5 ml
0.5 mlFR E-2752

Control 2

Refer to vial labels for
acceptable range!

Volume/Vial

0.5 mlContents:

Pregnenolone in a protein-based buffer with a non-mercury preservative. Prepared byspiking buffer with a defined quantity of pregnenolone.

Storage:

Refrigerate at 2 - 8 °C

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 freezing andthawing cycles.

5. FR E-2713
Contents:

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

Volume:15 ml/vial

Storage:

Refrigerate at 2 - 8 °C

Stability:

12 months or as indicated on label.

6. FR E-2731

Rabbit Anti-Pregnenolone Antibody Coated Microwell Plate-BreakApart Wells - Ready To Use

Contents:

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

Storage:

Refrigerate at 2 - 8 °C

Stability:

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

7. FR E-2740
Contents:

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

Volume:300 µl/vial

Storage:

Refrigerate at 2 - 8 °C

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 12ml 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 pregnenolone-HRP conjugate and wash buffer.

2.

Remove the required number of well 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 1 hour 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 wel.

8.

Incubate 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 ofspecimens/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 25.6 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
Sample data only. Do not use to calculate results.
StandardOD 1

OD 2

Mean OD

Value(ng/ml)

A

2.891

2.808

2.850

0

B

2.613

2.651

2.632

0.1

C

2.350

2.343

2.347

0.4

D

1.823

1.879

1.851

1.6

E

1.237

1.197

1.217

6.4

F

0.589

0.594

0.591

25.6

Unknown

1.431

1.451

1.441

4.0

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TYPICAL STANDARD CURVE
Sample curve only. Do not use to calculate results.
3OD 450nm

2.5
2
1.5
1
0.5
0

0.1

0.4

1.6

6.4

25.6

Pregnenolone (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 thePregnenolone ELISA kit is 0.05 ng/ml.
SPECIFICITY (CROSS REACTIVITY)
The following compounds were tested for cross-reactivity with the Pregnenolone ELISA kit with pregnenolone
cross-reacting at 100%.
Steroid% Cross Reactivity

Pregnenolone

100Progesterone

6.0

Dehydroisoandrosterone

5.25α-Androstandiol

4.7

Epiandrosterone

1.0

Pregnenolone Sulfate

0.4

Androstandione

0.35α-Androsterone

0.3

DHEAS

0.2

Etiocholanolone

0.1

The following steroids were tested but cross-reacted at less than 0.1%: Adrenosterone, Aldosterone,Androstenedione, Cholesterol, Corticosterone, 5α-DHT, 17β-Estradiol, Estriol and Testosterone.
INTRA-ASSAY PRECISION
Three samples were assayed ten times each on the same standard curve. The results (in ng/ml) are tabulatedbelow:
SampleMeanSD

CV %

1

0.19

0.02

10.6

2

1.04

0.85

8.2

3

4.77

0.37

7.8

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

CV %

1

0.22

0.03

14.5

2

1.14

0.14

12.3

3

4.56

0.44

9.6

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RECOVERY
Spiked samples were prepared by adding defined amounts of pregnenolone to four serum samples. The results(in ng/ml) are tabulated below:
SampleObs. Result

Exp. Result

Recovery %1 Unspiked+ 4.14

0.37
5.31

4.51

117.7

2 Unspiked+ 4.01

0.77
5.69

4.78

119.0

3 Unspiked+ 3.98

0.85
5.18

4.83

107.2

4 Unspiked+ 3.78

1.47
6.31

5.25

120.2

LINEARITY
Three serum samples were diluted with Standard A. The results (in ng/ml) are tabulated below:
SampleObs. Result

Exp. Result

Recovery %

5.31
2.89
1.26
0.71
6.51
2.75
1.54
0.80
8.34
3.78
2.15
1.05

2.66
1.33
0.66
3.26
1.63
0.81
4.17
2.09
1.04

108.6
94.7
107.6
84.4
94.598.8
90.6
102.9
101.0

1
1:2
1:4
1:8
2
1:2
1:4
1:8
3
1:2
1:4
1:8

EXPECTED NORMAL VALUESEach laboratory should collect data and establish their own range of expected normal values.
GroupN

Mean (ng/ml)

Abs. Range (ng/ml)

Males

30

0.50

0.1 - 3.4Females

50

0.55

0.1 - 3.8

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REFERENCES
1. Abraham GE, et al. Radioimmunoassay of Plasma Pregnenolone, 17-hydroxy-pregnenolone and Dehydroepiandrosterone Under Various Physiological Conditions. J Clin Endocrinol Metab. 1973; 37(1):140–4.
2. Hill M, et al. Age Relationships and Sex Differences in Serum Levels of Pregnenolone and 17hydroxypregnenolone in Healthy Subjects; Clin Chem Lab Med. 1999; 37(4):439–47.
3. Robel P, et al. Biosynthesis and Assay of Neurosteroids in Rats and Mice. J Steroid Biochem Mol Biol.
1995; 53:355–60.
4. Weusten JJ, et al. Early Time Sequence in Pregnenolone Metabolism to Testosterone in Homogenates of
Human and Rat Testis. Endocrinlogy. 1987; 120(5):1909–13.
5. Akwa Y, et al. Neurosteroids: Biosynthesis, Metabolism and Function of Pregnenolone andDehydroepiandrosterone in the brain. J Steroid Biochem Mol Biol. 1991; 40(1-3):71–81.
6. McKenna TJ, Brown RD. Pregnenolone in Man: Plasma Levels in States of Normal and Abnormal
Steroidogenesis. J Clin Endocrinol Metab. 1974; 38(3):480–5.
7. McKenna TJ, et al. Pregnenolone, 17-OH-Pregnenolone, and Testosterone in Plasma of Patients withCongenital Adrenal Hyperplasia. J Clin Endocrinol Metab. 1976; 42(5):918–25.
8. McKenna TJ, et al. Plasma Pregnenolone in Patients with Adrenal Tumors, ACTH Excess, or IdiopathicHirsutism. J Clin Endocrinol Metab. 1977; 44(2):231–6.
9. Wilson JD, Foster, DW, eds. Williams Textbook of Endocrinology 8th Edition. London: W.B. Saunders
Company; 1992:495-7.
10. 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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