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Instructions for use

Testosterone rat/mouse ELISAAR E-8000

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Testosterone rat/mouse ELISA
1. INTRODUCTION
1.1 INTENDED USE
The Testosterone rat/mouse ELISA is a competitive immunoassay for the measurement of testosterone
in rat and mouse serum or plasma. For research use only. Not for use in diagnostic procedures.
1.2 SUMMARY AND EXPLANATION

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Testosterone is a steroid hormone from the androgen group synthesized by the Leydig cells in the testes inmales, the ovaries in females, and adrenal glands in both sexes. It exerts a wide-ranging influence oversexual behaviour, muscle mass and strength, energy, cardiovascular health and bone integrity.
Testosterone biosynthesis coincides with the spermatogenesis and fetal Leydig cell differentiation in the malerat. Several in vivo models including hormone-suppression, hormone-restoration and hypophysectomy were
established for the study of the hormonal regulation of spermatogenesis by testosterone (1-3).
In the Brown Norway rat, serum testosterone levels decrease with aging, accompanied by increases inserum FSH. The capacity of Leydig cells to produce testosterone is higher in young than in old rats (4).
Testosterone secreted during late gestational and neonatal periods causes significant brain sexual
dimorphism in the rat. This results in both sex-specific behaviour and endocrinology in adults (5).
Analyses concerning the regulation of synthesis reveal that testosterone is able to regulate its own synthesis
and indicate that this autoregulation is the result of rapid, specific inhibition by testosterone of 17 alphahydroxylase activity (6).

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2. PRINCIPLE

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The Testosterone rat/mouse ELISA Kit is a solid phase enzyme-linked immunosorbent assay (ELISA),
based on the principle of competitive binding. An unknown amount of testosterone present in the sample
and a defined amount of testosterone conjugated to horseradish peroxidase compete for the binding sites of
testosterone antiserum coated to the wells of a microplate. After one-hour incubation on a shaker the
microplate is washed four times. After addition of the substrate solution the concentration of testosterone isinversely proportional to the optical density measured.

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3. WARNINGS AND PRECAUTIONSThis kit is strictly intended for research use only. Use by staff, who is specially informed and trained inmethods which are carried out by use of immunoassays.

2.

Before starting the assay, read the instructions completely and carefully. Use the valid version of the
package 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 each
step.

5.

Use reservoirs only for single reagents. This especially applies to the substrate reservoirs. Using a reservoir
for 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.

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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 and burns.
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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 manufacturer.
4. REAGENTS
4.1 Reagents providedkiStandard - ready to use.
Listed below are approximate concentrations, please refer to vial labels for exact concentrations:t

AR E-8031
Microtiter Plate, 12 x 8 (break apart) strips with 96 wells;
Wells coated with anti-testosterone antibody.

AR E-8003

Standard C

0.4 ng/ml

AR E-8004

Standard D

1.5 ng/ml

AR E-8005

Standard E

6.0 ng/ml

Standard F

25 ng/ml

0.3 ml0.3 ml0.3 ml0.3 ml0.3 mlse

AR E-8006e

0.1 ng/mlthStandard Bw
ithStandard A

AR E-8002

Volume/Vial
0.3 mld

AR E-8001

Concentration
0 ng/mlideStandardov

SymbolprCat. no.

AR E-8013

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Incubation Buffer, 1 vial, 11 ml, ready to use;

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AR E-8040
Enzyme Conjugate, 1 vial, 7 ml, ready to use;
Testosterone conjugated to horseradish peroxidise.

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AR E-0055
Substrate Solution, 1 vial, 22 ml, ready to use;
contains tetramethylbenzidine (TMB) and hydrogen peroxide in a buffered matrix.

Inst

AR E-0080
Stop Solution, 1 vial, 7 ml, ready to use;
contains 2 N Hydrochloric Acid solution.

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Hazards
identification:

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H290 May be corrosive to metals.
H314 Causes severe skin burns and eye damage.
H335 May cause respiratory irritation.

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AR E-0030
Wash Solution, 1 vial, 50 ml (10x concentrated);see „Preparation of Reagents“.

theNote: Additional Standard A for sample dilution is available upon request.

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Centrifuge
A microtiter plate reader capable for endpoint measurement at 450 nm
Microplate mixer operating more than 600 rpm
Vortex mixer
Calibrated variable precision micropipettes (10 µl, 50 µl, 100 µl, 200 µl).
Absorbent paper
Distilled or deionized water
Timer
Semi logarithmic graph paper or software for data reduction

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4.2 Materials required but not provided

4.3 Reagent preparation
All reagents should be at room temperature before use.
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 3 months at room temperature.

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4.4 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 - 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.
Protect TMB-Substrate Solution from light.
4.5 Disposal of the kits
The disposal of the kit must be made according to the national regulations. Special information for thisproduct is given in the Safety Data Sheet.
4.6 Damaged test kits

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In 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 to the official regulations.

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5. SAMPLE

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For determination of Testosterone rat/mouse serum and plasma can be used. The procedure calls for
10 µl matrix per well. The samples should assay immediately or aliquot and stored at -20 °C. Avoid repeatedfreeze-thaw cycles. Samples expected to contain rat/mouse Testosterone concentrations higher than thehighest standard (25 ng/ml) should be diluted with Standard A before assay. The additional dilution step has
to be taken into account for the calculation of the results.

foPlease note: The use of plasma as sample can result in a diminished precision of this assay.

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6. ASSAY PROCEDURE

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6.1 General Remarks

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 All reagents and specimens must be allowed to come to room temperature before use. All reagents must
be mixed 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.
 For internal quality control we suggest to use Rat Control Set coded AR K-8000. For more
information please contact the manufacturer directly.

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6.2 Assay ProceduretheEach run must include a standard curve.
Prepare a sufficient number of microplate wells to accommodate Standards and Samples in duplicates2.

Dispense 10 µl of each Standard, Control and Sample with new disposable tips into appropriate wells.

3.

Dispense 100 µl of Incubation Buffer into each well.

4.

Add 50 µl Enzyme Conjugate into each well.

5.

Incubate for 60 minutes at room temperature on a Microplate mixer.
Important Note:
Optimal reaction in this assay is markedly dependent on shaking of the microplate!
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.

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1.

6.
7.

Add 200 µl of Substrate Solution to each well.

8.

Incubate without shaking for 30 minutes in the dark.

9.

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

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

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6.3 Calculation of resultskit

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 absorbanceobtained 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 thestandard curve.
4. Automated method: The results in the IFU 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
the concentrations, this dilution factor has to be taken into account.

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Conversion to SI units:
Testosterone (pg/ml) x 3.47 = pmol/l

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6.3.1 Example of Typical Standard Curve
Following data are intended for illustration only and should not be used to calculate results from another
run.

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7. EXPECTED NORMAL VALUES

Absorbance Units
2.478
2.078
1.668
1.170
0.645
0.330

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Standard
Standard A (0 ng/ml)
Standard B (0.1 ng/ml)
Standard C (0.4 ng/ml)
Standard D (1.5 ng/ml)
Standard E (6.0 ng/ml)
Standard F (25.0 ng/ml)

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Male
♂
Female ♀

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In order to determine the normal range of serum testosterone in rat, samples from 35 male rats and 20
female rats were collected and analyzed using the Testosterone rat/mouse ELISA kit. The following ranges
are calculated with the results of this study.
Range (ng/ml)
0.66 – 5.4
0.11 – 0.31

Mean (ng/ml)
3.06
0.21

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In further studies serum samples of 10 mice were collected between 11.00 am and 3.00 pm und analyzed ina similar manner.

♂

Range (ng/ml)
1.7 – 14.4

Mean (ng/ml)
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It is recommended that each laboratory establish its own normal range since testosterone levels can vary
due to handling and sampling techniques.

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8. PERFORMANCE CHARACTERISTICS

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8.1 Analytical sensitivity
The lowest analytical detectable level of testosterone that can be distinguished from the Standard A is
0.066 ng/ml at the 2SD confidence limit.

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8.2 Specificity
The following materials have been evaluated for cross reactivity. The percentage indicates cross reactivity at
50% displacement compared to Testosterone.
Steroid% Cross reaction

Dihydroxyandrosterone

7.4

Estron< 0.1

Estradiol< 0.1

Estriol< 0.1

Cortisol< 0.1

11-Deoxycortisol

< 0.1
< 0.1

17OH-Progesterone

< 0.1

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Progesterone

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< 0.1

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< 0.1

DihydroandrosteronethEpiandrosterone

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< 0.1

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< 0.1

Androsterone

ideAndrostenedione

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69.6

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Dihydrotestoteronese

8.3 ReproducibilityrU

8.3.1 Intra-Assay

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The intra-assay variation was determined by 20 replicate measurements of 3 serum samples within one run.
The within-assay variability is shown below:
3.23

1.44

SD

0.21

0.12

0.09

CV (%)

6.50

8.06

11.07

20

20

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Mean (ng/ml)

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0.84

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The inter-assay (between-run) variation was determined by duplicate measurements of 3 serum samples
over 10 days.
0.29

1.23

9.50

SD

0.03

0.11

0.88

CV (%)

11.3

9.3

9.3

n=

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Mean (ng/ml)

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8.4 Recovery
Using the Standard Matrix three spiking solutions were prepared (A = 50 ng/ml, B = 100 ng/ml and C = 150
ng/ml). A 25 µl aliquot of each solution was spiked into 475 µl of six different rat sera with low testosterone
concentrations for a spiking ratio of 1 to 20, leaving the serum matrix of the spiked samples relatively intact.
All samples were then measured by the Testosterone rat/mouse ELISA procedure. To calculate expectedvalues 95% of the unspiked values were added to 5% of the spiking solution concentrations (2,5, 5 and 7,5
ng/ml, respectively).

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112%
96%
93%
118%
109%
100%
101%
103%
95%
96%
89%
91%
110%
89%
93%
100%
86%
93%

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O/E %

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Expected
(E)
2,81
5,31
7,81
2,90
5,40
7,90
2,86
5,36
7,86
2,75
5,25
7,75
2,88
5,38
7,88
2,78
5,38
7,78

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Observed
(0)
0,31
3,15
5,11
7,27
0,40
3,42
5,88
7,90
0,36
2,88
5,50
7,50
0,25
2,65
4,65
7,08
0,38
3,17
4,79
7,36
0,28
2,78
4,61
7,22

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Spiking
Solution
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C

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Serum

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8.5 Linearity

ionFive native serum samples were assayed undiluted and diluted with the standard matrix.
Dilution

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Serum

native1 in 2
1 in 41 in 8native1 in 2
1 in 41 in 8native1 in 2
1 in 41 in 8native1 in 2
1 in 41 in 8native1 in 2
1 in 41 in 8

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Version: 7.0c

Observed (0)(ng/ml)
2.54
1.22
0.61
0.34
1.85
0.86
0.43
0.21
1.94
1.04
0.52
0.26
0.75
0.45
0.18
0.09
1.35
0.75
0.30
0.13

Effective: 2020-08-10

Expected(E)(ng/ml)
1.27
0.64
0.32
0.93
0.46
0.23
0.97
0.49
0.24
0.38
0.19
0.09
0.68
0.34
0.16

O/E %
96%
95%
106%
92%
93%
91%
107%
106%
108%
118%
95%
100%
110%
88%
81%

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9. 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.
9.1 Drug Interferences
Until now no substances (drugs) are known influencing the measurement of rat or mouse testosterone inserum and plasma. Lipemic and haemolysed samples can cause false results.
10. LEGAL ASPECTS

kit

10.1 Reliability of results

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The test must be performed exactly as per the manufacturer’s instructions for use. Moreover the user muststrictly adhere to the rules of GLP (Good Laboratory Practice) or other applicable national standards and/or
laws. This is especially relevant for the use of control reagents. It is important to always include, within thetest procedure, a sufficient number of controls for validating the accuracy and precision of the test.
The test results are valid only if all controls are within the specified ranges and if all other test parameters
are also within the given assay specifications. In case of any doubt or concern please contact the
manufacturer directly.

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Any modification of the test kit and/or exchange or mixture of any components of different lots from one
test kit to another could negatively affect the intended results and validity of the overall test. Suchmodification and/or exchanges invalidate any claim for replacement.
Regardless, in the event of any claim, the manufacturer’s liability is not to exceed the value of the test kit.
Any damage caused to the test kit during transportation is not subject to the liability of the manufacturer.

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11. REFERENCES

Huang HF, Marshall GR, Rosenberg R & Nieschlag E (1987): Restoration of spermatogenesis by highlevels of testosterone in hypophysectomised rats after long-term regression. Acta Endocrinologica 116,
433–444.

2.

Sun YT, Irby DC, Robertson DM & de Kretser DM (1989): The effects of exogenously administered
testosterone on spermatogenesis in intact and hypophysectomized rats. Endocrinology 125, 1000–1010.

3.

O’Donnell L, McLachlan RI, Wreford NG & Robertson DM (1994): Testosterone promotes the conversion ofround spermatids between stages VII and VIII of the rat spermatogenic cycle. Endocrinology 135 2608–
2614.

4.

Zirkin BR & Chen H. (2000): Regulation of Leydig cell steroidogenic function during aging. Biol. Reprod.
63(4): 977-81

5.

Sakuma Y (2009): Gonadal steroid action and brain sex differentiation in the rat. J. Neuroendocrinol. 21(4): 410-4

6.

Darney KJ Jr, Zirkin BR, Ewing LL (1996): Testosterone autoregulation of its biosynthesis in the rat testis:
inhibition of 17 alpha-hydroxylase activity. J. Androl. 17 (2): 137-42

7.

Moore AM, Prescott M, Campbell RE (2013): Estradiol negative and positive feedback in a prenatal
androgen-induced mouse model of polycystic ovarian syndrome. Endocrinology, February 2013, 154(2):796-806

8.

Niakani A, Farrokhi F. and Hasanzadeh S (2013): Decapeptyl ameliorates cyclophosphamide-induced
reproductive toxicity in male Balb/C mice: histomorphometric, stereologic and hormonal evidences. Iran J
Reprod Med Vol.11 No.10. pp: 791-800, October 2013

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1.

9.

Clarkson J, Busby ER, Kirilov M, Schütz G, Sherwood NM and Herbison AE (2014): Sexual differentiationof the brain requires perinatal Kisspeptin-GnRH Neuron Signaling. The Journal of Neuroscience,
November 12, 2014, 34(46): 15297-15305

10. Slimen S, Saloua EF, Najoua G (2014): Oxidative stress and cytotoxic potential of anticholinesterase
insecticide, malathion in reproductive toxicology of male adolescent mice after acute exposure. Iranian J
Basic Med Sci, Vol 17, No 7, Jul 2014
11. Zhu W, Liu P, Yu L, Chen Q, Liu Z, Yan K, Lee WM, Cheng CY and Han D (2014): p204-Initiated innate
antiviral response in mouse Leydig cells. Biology of Reproduction (2014) 91(1):8, 1-9
12. O´Hara L, McInnes K, Simitsidellis I, Morgan S, Atanassova N., Slowikowska-Hilczer J, Kula K, SzarrasCzapnik M, Milne L, Mitchell RT and Smith LB (2015):Autocrine androgen action is essential for Leydig cell
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maturation and function, and protects against late-onset Lexdig cell apoptosis in both mice and men. The
FASEB Journal, Vol.29, March 201513. Schellino R, Trova S, Cimino I, Farinetti A, Jongbloets BC, Pasterkamp RJ, Panzica G, Giacobini P,
DeMarchis S and Peretto P (2016). Opposite-sex attraction in male mice requires testosterone-dependent
regulation of adult olfactory bulb neurogenesis. Scientific Reports / 6:36063/DOI:10.1038/srep36063

Storagetemperature

Manufacturer

Expiry date

Batch code

Consult instructions
for useContent

CautionCatalogue
numberPlease

Symbols:

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14. Soylu-Kucharz R, Baldo B and Petersén A (2016): Metabolic and behavioural effects of mutant huntingtin
deletion in Sim1 neurons in the BACHD mouse model of Huntington´s disease. Scientific Reports /6:28322/DOI: 10.1038/srep28322

Version: 7.0c

Effective: 2020-08-10

Contains sufficient for<n> tests

For research use
only!

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