Instructions for use

Testosterone Saliva ELISASA E-6100R

Free

TESTOSTERONE FREE IN SALIVA ELISA
1. INTRODUCTION
1.1 Intended Use
An Enzyme Immunoassay for the quantitative measurement of free active testosterone in saliva.
Measurement of testosterone is used in research of disorders involving the male sex hormones (androgens),
including primary and secondary hypogonadism, delayed or precocious puberty, impotence in males and infemales hirsutism (excessive hair) and virilization (masculinization) due to tumors, polycystic ovaries, andadrenogenital syndromes. It is intended only for research use.
1.2 Summary and explanation
At present, the majority of steroid hormone determinations are conducted from serum samples, even if
results in the low or very low concentration range are expected, for example, in elderly patients. This is areal challenge for any laboratory as shown by Taieb et al in 2003 (10) and others (9). Recently there has been
an official position statement of the Endocrine Society(14) stating that reliable Testosterone measurements inserum either need an extraction step or have to be done by chromatographic methods like Tandem MS or
GCMS. There now is sufficient evidence that the commercial Testosterone assays are unable to quantify low
concentrations in a reliable way.
Another major problem associated with the measurement of free hormone levels from serum is the episodic
secretion pattern of steroid hormones. Even in 1973(1) it could be shown that steroid secretion shows asignificant episodic pattern. Nevertheless, the majority of the determinations are still made from just one
serum sample, resulting in non-reproducible values due to the biological variation. In general, serum
measurements can only give the total steroid hormone concentration, whereas saliva testing results in themeasurement of the free active hormone fraction (3,5).
So far, all attempts for a direct quantification of free Testosterone in serum or plasma samples bycommercial immunoassays have failed (7).
Taking into consideration the above mentioned drawbacks of the current analytical procedures, salivary
testing seems to be a reliable alternative. It has been shown in the literature (3,5,13,15) that the measurementof free salivary Testosterone gives valid results even in the low concentration range. In salivary testing it iseasy to compensate for the episodic secretion pattern provided multiple sampling is done (preferably 5
samples within 2 hours). The measurement of free Testosterone is done with a mixture of these 5 samples.
In contrast to this, measurements from just one single saliva sample always will give arbitrary results (likein serum).
2. PRINCIPLEThe Testosterone free in Saliva ELISA Kit is a solid phase enzyme-linked immunosorbent assay (ELISA),
based on the principle of competitive binding. An unknown amount of free testosterone present in thesample and a defined amount of testosterone conjugated to horseradish peroxidase compete for the bindingsites of rabbit polyclonal 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 theconcentration of testosterone is inversely proportional to the optical density measured.
3. 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 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 areservoir for dispensing a substrate solution that had previously been used for the conjugate solution may
turn solution 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.

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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.
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.
4. REAGENTS PROVIDED
4.1 Reagents provided
Standards and Controls
Cat. no.
SA E-6101
SA E-6102
SA E-6103
SA E-6104
SA E-6105
SA E-6106

Standard
Standard A (0)Standard B (1)Standard C (2)Standard D (3)Standard E (4)Standard F (5)Concentration
0 pg/ml
10 pg/ml
30 pg/ml
100 pg/ml
300 pg/ml
1000 pg/ml

SA E-6151

Control low

SA E-6152

Control highPlease refer to
QC-Datasheet

Volume/Vial
3 ml
1 ml
1 ml
1 ml
1 ml
1 ml
1 ml
1 ml

Conversion: Testosterone (pg/ml) x 3.47 = pmol/l
SA E-6131 Coated Microplate
12 x 8 (break apart) strips with 96 wells;
Wells coated with an anti-Testosterone antibody (rabbit polyclonal antibody).
SA E-6140 Enzyme Conjugate
1 vial, 12 ml, ready to use;
Testosterone conjugated to horseradish peroxidase
AR E-0055 Substrate Solution
1 vial, 22 ml, ready to use;
contains tetramethylbenzidine (TMB)
Hazards
identification:
H360D May damage the unborn child.
AR E-0080 Stop Solution
1 vial, 7 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.

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AR E-0030 Wash Solution
1 vial, 50 ml (10X concentrated);see „Preparation of Reagents“.
Note: Additional Standard A for sample dilution is available upon request.
4.2 Materials required but not provided
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Microcentrifuge
A microtiter plate reader capable for endpoint measurement at 450 nm
Microplate mixer operating more than 600 rpm
Vortex mixer
Calibrated variable precision micropipettes (50 µl, 100 µl, 200 µl).
Absorbent paper.
Distilled or deionized water
Timer
Semi logarithmic graph paper or software for data reduction

4.3 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.
4.4 Reagent preparation
Allow the reagents and the required number of wells to reach room temperature (21-26°C) before starting
the test.
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.
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 Material Safety Data Sheet.
4.6 Damaged test kits
In case of any severe damage of the test kit or components, the manufacturer have to be informed inwriting within one week after receiving the kit. Severely damaged single components should not be used fora 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.
5. SPECIMEN
Samples containing sodium azide should not be used in the assay. The saliva samples should be completely
colorless. Even the slightest red color shows blood contamination. Blood contamination will give falsely
elevated concentration values. In case of visible blood contamination one should discard the sample, rinse
the sampling device with water, wait for 10 minutes and take a new sample.
5.1 Specimen Collection
For the correct collection of saliva we are recommending to use appropriate devices made from ultra-purepolypropylene. Do not use any PE devices for sampling to avoid significant interferences. Glass tubes can beused as well, but in this case special attention is necessary for excluding any interference caused by the
stoppers. For more details please contact the manufacturer. As the Testosterone secretion in saliva as wellas in serum shows an obvious episodic secretion pattern it is important to care for a proper timing of thesampling. In order to avoid arbitrary results we are recommending to collect 5 samples within a period of 2hours (multiple sampling) preferably in the early morning of a normal day directly after waking up. As foodmight contain significant amounts of steroid hormones samples preferably should be taken while fasting. If
fasting should be a problem the collection period should be timed just before lunch or before dinner. In theearly morning Testosterone levels of males are significantly higher compared to those ones during the day.
The Testosterone concentration in the morning is roughly twice as high compared to the evening
concentration.
Do not chew anything during the sampling period. Any pressure to the teeth may result in falsely elevatedmeasurements due to an elevated content of gingival liquid in the saliva sample.

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5.2 Specimen Storage and Preparation
Saliva samples in general are stable at ambient temperature for up to seven days. Therefore mailing of such
samples by ordinary mail without cooling will not create any problem. Storage at 4°C can be done for aperiod of up to one month. Whenever possible, samples should preferably be kept at a temperature of 20°C. Even repeated thawing and freezing is not a problem. Each sample has to be frozen, thawed, andcentrifuged at least once anyhow in order to separate the mucins by centrifugation. Upon arrival of thesamples at the lab the samples have to kept frozen at least overnight. Next morning the samples are thawed
and mixed carefully. The samples have to be centrifuged for 5 to 10 minutes. The clear colorless
supernatant is easy to pipette. If the sample should show even a slighty red color it should be discarded.
Otherwise the value most probably will be falsely elevated. Due to the episodic variations of the steroid
secretion we highly recommend the strategy of multiple sampling. If such a set of multiple samples has tobe tested the staff of lab (after at least one freezing, thawing, and centrifugation cycle) should mix aliquotsof the 5 single samples and perform the determination using the mixture.
5.3 Specimen Dilution
Samples expected to contain testosterone concentrations higher than the highest standard (1000 pg/ml)
should be diluted with the standard A before assay. The additional dilution step has to be taken into accountfor the calculation of the result.
Example:a) Dilution 1:10: 10 µl saliva + 90 µl Standard A (mix thoroughly)b) Dilution 1:100: 10 µl of dilution a) + 90 µl Standard A (mix thoroughly).
6. ASSAY PROCEDURE
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.
Respect the incubation times as stated in this instructions for use.

6.2 Assay procedure
Each run must include a standard curve.

1.
2.
3.
4.
5.
6.
7.
8.
9.

Prepare a sufficient number of microplate wells to accommodate standards, controls and samples.
Dispense 100 µl of each Standard, Control and sample with new disposable tips into appropriate wells
Dispense 100 µl of Enzyme Conjugate into each well.
Incubate for 60 minutes at room temperature on a Microplate mixer (≥ 600 rpm).
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 per well).
Remove as much Wash Solution as possible by beating the microplate on absorbent paper.
Add 200 µl of Substrate Solution to each well.
Incubate for 30 minutes in the dark.
Stop the reaction by adding 50 µl of Stop Solution to each well.
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 results
1. Calculate the average absorbance values for each set of standards, controls and patient samples.
2. Construct a standard curve by plotting the mean absorbance obtained from each standard against its
concentration with absorbance value on the vertical (Y) axis and concentration 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 this IFU have been calculated automatically using a 4 PL (4parameters Logistics) curve fit. 4 Parameters 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.
Conversion to SI units:
Testosterone (pg/ml) x 3.47 = pmol/l
6.3.1 Example of Typical Standard Curve
Following data are intended for illustration only and should not be used to calculate results from anotherrun.
Standard
Standard A (0 pg/ml)
Standard B (10 pg/ml)
Standard C (30 pg/ml)
Standard D (100 pg/ml)
Standard E (300 pg/ml)
Standard F (1000 pg/ml)

Optical Units (450 nm)
2.267
2.040
1.721
1.019
0.592
0.299

7. EXPECTED NORMAL VALUES
In order to determine the normal range of salivary Testosterone, saliva samples from children, adult maleand adult female apparently healthy subjects were collected in the morning and analyzed using the
Testosterone free in Saliva ELISA kit. The following ranges are calculated with the results of this study.
The concentrations are given in pg/ml.
Men ♂
Age Group 5th-95th Percentile

Women ♀
Mediann

5th-95th Percentile Median

n

Years

[pg/ml][pg/ml][pg/ml][pg/ml]15 – 55

33.6 – 205.0

90.0

83

11.6 – 88.1

33.8

538

>55

25.1 – 140.7

68.3

42

9.3 – 83.0

27.2

137

Children
Age Group 5th-95th Percentile

MedianYears[pg/ml][pg/ml]≤ 11

5.8 – 45.3

11.5

n
8

8. QUALITY CONTROLGood laboratory practice requires that controls should be run with each standard curve. A statisticallysignificant number of controls should be assayed to establish mean values and acceptable ranges to assure
proper performance.
It is recommended to use control samples according to national regulations. The use of control samples isadvised to assure the day to day validity of results. Use controls at both normal and pathological levels.
The controls and the corresponding results of the QC-Laboratory are stated in the QC certificate added to
the kit. The values and ranges stated at the QC sheet always refer to the current kit lot and should be used
for direct comparison of the results.
It is also recommended to make use of national or international Quality Assessment programs in order toensure the accuracy of the results.
Employ appropriate statistical methods for analyzing control values and trends. If the results of the assay do
not fit to the established acceptable ranges of control materials results should be considered invalid.
In this case, please check the following technical areas: Pipetting and timing devices; photometer, expiration
dates of reagents, storage and incubation conditions, aspiration and washing methods.
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9. PERFORMANCE CHARACTERISTICS
9.1 Analytical Sensitivity
The lowest analytical detectable level of testosterone that can be distinguished from the Standard A is
2.2 pg/ml at the 2SD confidence limit.
9.2 Specificity (Cross Reactivity)
The following materials have been evaluated for cross reactivity. The percentage indicates cross reactivity at
50% displacement compared to Testosterone.
Steroid% Cross reaction

Testosterone

100%5α-Dihydrotestosterone

23.3%

Androstenedione

32.2%

Androsteron< 0.1%

5α-Androstane< 0.1%

5ß-Androstane-3α,17ß-diole< 0.1%

Corticosterone< 0.1%

11-Desoxycorticosterone< 0.1%

Dexamethasone< 0.1%

Estradiol< 0.1%

Progesterone< 0.1%

17α-Hydroxyprogesterone< 0.1%

Cortisol< 0.1%

11-Desoxycortisol< 0.1%

Cortison< 0.1%

Estrone< 0.1%

Pregenolone< 0.1%

Prednisone< 0.1%

Prednisolon< 0.1%

Prednisone< 0.1%

Danazol< 0.1%

9.3 Assay Dynamic range
The range of the assay is between 10 – 1000 pg/ml.
9.4 Reproducibility
9.4.1 Intra-Assay
The intra-assay variation was determined by 20 replicate measurements of 3 saliva samples within one run.
The within-assay variability is shown below:

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Sample 1

Sample 2

Sample 3

Mean (pg/ml)

61.0

90.7

216.3

SD

5.92

6.57

11.92

CV (%)

9.7

7.2

5.5

n=

20

20

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9.4.2 Inter-Assay
The inter-assay (between-run) variation was determined by duplicate measurements of 3 saliva samples
over 10 days.
Sample 1
Sample 2
Sample 3
Mean (pg/ml)

53.4

74.6

288.3

SD
CV (%)
n=

10.42
9.9
11

5.90
7.9
11

27.74
9.6
11

9.5 Recovery
Using the Standard Matrix six spiking solutions were prepared (A = 2000 pg/ml, B = 4000 pg/ml,
C = 6000 pg/ml, D = 500 pg/ml, E = 1000 pg/ml and F = 2000 pg/ml). A 25 µl aliquot of each solution was
spiked into 475 µl of different salivas, for a spiking ratio of 1 to 20, leaving the saliva matrix of the spiked
samples relatively intact. All samples were then measured by Salivary Testosterone procedure. To calculate
expected values 95% of the unspiked values were added to 5% of the spiking solution concentrations.

SampleMeasured(pg/ml)
61.8
153.3
259.0
365.0

Expected(pg/ml)
158.7
258.7
358.7

Recovery
(%)
96.6
100.1
101.8

Sample

2

74.0
164.6
270.4
345.4

170.3
270.3
370.3

96.6
100.0
93.3

3

30.9
49.4
76.6
109.0

54.3
79.3
129.3

91.0
96.6
84.3

1

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Measured(pg/ml)
87.3
105.8
125.8
195.6

Expected(pg/ml)
107.9
132.9
182.9

Recovery
(%)
98.0
94.7
106.9

5

71.1
85.6
94.5
154.9

92.5
117.5
167.5

92.5
80.4
92.5

6

74.7
87.9
140.3
168.4

95.9
120.9
170.9

91.6
116.0
98.5

4

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9.6 Linearity
Six saliva samples containing different amounts of analyte were serially diluted with zero standard (A) andassayed with the ELISA. Four native samples were serially diluted, and two samples were spiked withtestosterone and then serially diluted up to 1:8. The percentage recovery was calculated by comparing the
expected and measured values for testosterone.
Saliva

1

2

3

4

5

6

10.

Observed
0
224
118
51
27
205
110
50
27
106.7
55.6
27,7
11,9
66.6
28,9
12,3
6,9
91.1
51,1
26,3
12,9
133.7
62.6
33.0
19,5

Dilution
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 8native1 in 2
1 in 41 in 8

Expected
(E)
112
56
28
103
51
26
53.4
26,7
13,3
33.3
16,70
8,30
45.6
22,8
11,4
66,9
33,4
16,7

O/E %
105%
91%
96%
107%
98%
104%
104%
104%
89%
87%
74%
83%
112%
115%
113%
94%
99%
117%

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.
10.1 Interfering Substances
Blood contamination in saliva samples will affect results, and usually can be seen by eye.
10.2 Drug Interferences
Any medication (cream, oil, pill, etc.) containing testosterone of course will significantly influence themeasurement of this analyte.
11.

REFERENCES
1.
2.
3.
4.
5.

Charles D. West et.al. (1973): Simultaneous Measurement of Multiple Plasma Steroids byRadioimmunoassay Demonstrating Episodic Secretion.
J Clin Endocrin Metabol Vol. 36 (No.6), pages 1230 – 1236.
G.E.Butler et. al. (1989): Salivary Testosterone levels and the progress of puberty in the normal boy.
Clin Endocrin Vol 30, pages 587 – 596
Josko Osredkar et. al. (1989): Salivary free testosterone in hirsutism
Ann. Clin. Biochem. Vol. 26, pages 522 – 526
G. Lac, N. Lac, A. Robert (1993): Steroid assays in saliva: A method to detect plasmatic
contaminations.
Arch Int de Physiol Bioch Biophys Vol 101, pages 257-262
James M. Dabbs et. al. 1995: Reliability of salivary testosterone measurements: A Multicenter
Evaluation
Clin. Chem. Vol. 41 (11), pages 1581 – 1584

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6.

J. Valero-Politi, X. Fuentes-Arderiu 1996: Daily rhythmic and non-rhythmic variations of LH, FSH,
SHBG, and Testosterone in men
Eur J Clin Chem Clin Biochem. Vol. 34, pages 455 – 462
7. William Rosner 2001: An extraordinarily inaccurate assay for free Testosterone is still with us. J Clin
Endocrin Metabol Vol.86 (6), page 2903
8. Karen K. Miller et. al. 2004: Measurement of free Testosterone in normal women and women withandrogen deficiency: Comparison of methods
J Clin Endocrin Metabol Vol. 89 (2), pages 525 – 533
9. Frank Z. Stanczyk et. al. 2003: Limitations of direct Estradiol and Testosterone immunoassay kits.
Steroids Vol. 68, pages 1173 – 1178
10. Joelle Taieb et. al. 2003: Testosterone Measured by 10 Immunoassays and by Isotope-Dilution Gas
Chromatography-Mass Spectrometry in sera from 116 men, women, and children. Clin Chem Vol. 49(8), pages 1381 – 1250
11. P Celec et. al. 2003: Circatrigintan cycle of salivary Testosterone in human male. Biological Rhythm
Research Vol. 34 (3), pages 305-315.
12. John G. Lewis 2006: Steroid analysis in saliva: An overview (Review article).
Clin Biochem Rev. Vol.27, pages 139-146
13. J.E. Morley et. al. 2006: Validation of salivary Testosterone as a screening test for male hypogonadism.
Aging Male Vol.9, pages 165-169
14. W. Rosner 2007: Utility, Limitations, and pitfalls in measuring Testosterone: An Endocrine Society
Position Statement
J Clin Endocrin Metabol Vol. 92 (2), pages 405-413
15. M. Yasuda et. al. 2007: Low testosterone level of middle-aged Japanese men – the association between
low testosterone levels and quality of life.
Journal of Men’s Health and Gender Vol. 4 (2), pages 149-155
16. Reimers L. & Diekhof EK (2015): Testosterone is associated with cooperation during intergroupcompetition by enhancing parochial altruism.
Frontiers in Neuroscience, June 2015, Volume 9, Article 183

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Contains sufficient for<n> tests

For research use
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