Instructions for use

Testosterone ELISAAA E-1300R

Testosterone ELISAINTRODUCTION
Intended Use
The Testosterone ELISA is an enzyme immunoassay for the quantitative measurement of Testosterone inserum and plasma.
PRINCIPLE OF THE TESTThe Testosterone ELISA Kit is a solid phase enzyme-linked immunosorbent assay (ELISA), based on theprinciple of competitive binding.
The microtiter wells are coated with a monoclonal [mouse] antibody directed towards an unique antigenic siteon the Testosterone molecule.
Endogenous Testosterone of a sample competes with a Testosterone
horseradish peroxidase conjugate for binding to the coated antibody. After incubation the unbound conjugate iswashed off.
The amount of bound peroxidase conjugate is reverse proportional to the concentration of Testosterone in thesample. After addition of the substrate solution, the intensity of colour developed is reverse proportional to theconcentration of Testosterone in the sample.

WARNINGS AND PRECAUTIONS
1. This kit is for research use only.
2. All reagents of this test kit which contain human serum or plasma have been tested and confirmed
negative for HIV I/II, HBsAg and HCV by FDA approved procedures. All reagents, however, should betreated as potential biohazards in use and for disposal.
3. Before starting the assay, read the instructions completely and carefully. Use the valid version of thepackage insert provided with the kit. Be sure that everything is understood.
4. 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.
5. Pipetting of samples and reagents must be done as quickly as possible and in the same sequence for eachstep.
6. 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.
7. Mix the contents of the microplate wells thoroughly to ensure good test results. Do not reuse microwells.
8. Do not let wells dry during assay; add reagents immediately after completing the rinsing steps.
9. 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.
10. Never pipet by mouth and avoid contact of reagents and specimens with skin and mucous membranes.
11. Do not smoke, eat, drink or apply cosmetics in areas where specimens or kit reagents are handled.
12. Wear disposable latex gloves when handling specimens and reagents. Microbial contamination of reagentsor specimens may give false results.
13. Handling should be done in accordance with the procedures defined by an appropriate national biohazard
safety guideline or regulation.
14. Do not use reagents beyond expiry date as shown on the kit labels.
15. All indicated volumes have to be performed according to the protocol. Optimal test results are only
obtained when using calibrated pipettes and microtiterplate readers.
16. 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.
17. Avoid contact with Stop Solution containing 0.5 M H2SO4. It may cause skin irritation and burns.
18. Some reagents contain Proclin 300, BND and/or MIT as preservatives. In case of contact with eyes or skin,flush immediately with water.
19. TMB substrate has an irritant effect on skin and mucosa. In case of possible contact, wash eyes with anabundant volume of water and skin with soap and abundant water. Wash contaminated objects before
reusing them. If inhaled, take the person to open air.
20. Chemicals and prepared or used reagents have to be treated as hazardous waste according to the national
biohazard safety guideline or regulation.
21. For information on hazardous substances included in the kit please refer to Material Safety Data Sheets.
Material Safety Data Sheets for this product are available upon request directly from the manufacturer.

Version: 8.0-b-r

Effective: 2018-06-27

2/8

REAGENTS
Reagents provided
AA E-1331 Microtiterwells
12x8 (break apart) strips, 96 wells; Wells coated with a mouse monoclonal anti-Testosterone antibody.
Standards
ready to useCat. no.
AA E-1301

Standard
Standard A (0)Concentration
0 ng/ml

Volume/Vial
1 ml

AA E-1302

Standard B (1)0.2 ng/ml

1 ml

AA E-1303

Standard C (2)0.5 ng/ml

1 ml

AA E-1304

Standard D (3)1 ng/ml

1 ml

AA E-1305

Standard E (4)2 ng/ml

1 ml

AA E-1306

Standard F (5)6 ng/ml

1 ml

AA E-1307
Standard G (6)16 ng/ml
Conversion: 1 ng/mL = 3.467 nmol/Lcontain 0.03% Proclin 300 + 0.005% gentamicin sulfate as a preservative.

1 ml

AA E-1340 Enzyme Conjugate
1 vial, 25 ml, ready to use; Testosterone conjugated to horseradish peroxidase;* contain 0.03% Proclin 300, 0.015% BND and 0.010% MIT as a preservative.
SA E-0055 Substrate Solution
1 vial, 25 ml, ready to use; Tetramethylbenzidine (TMB).
FR E-0080 Stop Solution
1 vial, 14 ml, ready to use; contains 0.5 M H2SO4.
Avoid contact with the stop solution. It may cause skin irritations and burns.
Hazards
identification:
H290 May be corrosive to metals.
H314 Causes severe skin burns and eye damage.
FR E-0030 Wash Solution
1 vial, 30 ml (40X concentrated); see „Preparation of Reagents“.
* BND = 5-bromo-5-nitro-1,3-dioxane
MIT = 2-methyl-2H-isothiazol-3-one
Note: Additional Standard A for sample dilution is available upon request.

Materials required but not provided

A microtiter plate calibrated reader (450±10 nm)
Calibrated variable precision micropipettes.
Absorbent paper.
Distilled or deionized waterTimer
Semi logarithmic graph paper or software for data reduction

Storage Conditions
When stored at 2 °C to 8 °C unopened reagents will retain reactivity until expiration date. Do not use reagents
beyond this date.
Opened reagents must be stored at 2 °C - 8 °C. Microtiter wells must be stored at 2 °C - 8 °C. Once the foil
bag has been opened, care should be taken to close it tightly again.
Reagent Preparation
Allow all reagents and required number of strips to reach room temperature prior to use.

Version: 8.0-b-r

Effective: 2018-06-27

3/8

Wash Solution
Add deionized water to the 40X concentrated Wash Solution.
Dilute 30 mL of concentrated Wash Solution with 1170 mL deionized water to a final volume of 1200 mL.
The diluted Wash Solution is stable for 2 weeks at room temperature.
Disposal of the Kit
The disposal of the kit must be made according to the national regulations. Special information for this productis given in the Material Safety Data Sheets (see chapter 13).
Damaged Test Kits
In case of any severe damage to the test kit or components, the manufacturer has to be informed in writing, atthe latest, one week after receiving the kit. Severely damaged single components should not be used for a testrun. They have to be stored until a final solution has been found. After this, they should be disposed according
to the official regulations.
SPECIMEN COLLECTION AND PREPARATION
Serum or plasma (EDTA-, Heparin- or citrate plasma) can be used in this assay.
Do not use haemolytic, icteric or lipaemic specimens.
Please note: Samples containing sodium azide should not be used in the assay.
Specimen Collection
Serum:
Collect blood by venipuncture (e.g. Sarstedt Monovette # 02.1388.001), allow to clot, and separate serum bycentrifugation at room temperature. Do not centrifuge before complete clotting has occurred.
Plasma:Whole blood should be collected into centrifuge tubes containing anti coagulant and centrifuged immediately
after collection.
(E.g. for EDTA plasma Sarstedt Monovette – red cap - # 02.166.001; for Heparin plasma Sarstedt Monovette –orange cap - # 02.165.001; for Citrate plasma Sarstedt Monovette – green cap - # 02.167.001.)
Specimen Storage and Preparation
Specimens should be capped and may be stored for up to 5 days at 2-8°C prior to assaying.
Specimens held for a longer time should be frozen only once at -20°C prior to assay. Thawed samples should
be inverted several times prior to testing.
Specimen Dilution
If in an initial assay, a specimen is found to contain more than the highest standard, the specimens can be
diluted with Standard A and reassayed as described in Assay Procedure.
For the calculation of the concentrations this dilution factor has to be taken into account.
Example:a) Dilution 1:10: 10 µL Serum + 90 µL Standard A (mix thoroughly)b) Dilution 1:100: 10 µL dilution a) 1:10 + 90 µL Standard A (mix thoroughly).
ASSAY PROCEDUREGeneral Remarks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.

Version: 8.0-b-r

Effective: 2018-06-27

4/8

Test Procedure
Each run must include a standard curve.
1.

Secure the desired number of Microtiter wells in the holder.

2.

Dispense 25 µL of each Standard, Control and samples with new disposable tips into appropriatewells.

3.

Dispense 200 µL Enzyme Conjugate into each well.
Thoroughly mix for 10 seconds. It is important to have a complete mixing in this step.

4.

Incubate for 60 minutes at room temperature (without covering the plate).

5.

Briskly shake out the contents of the wells.
Rinse the wells 3 times with diluted Wash Solution (400 µL per well). Strike the wells sharply on
absorbent paper to remove residual droplets.
Important note:
The sensitivity and precision of this assay is markedly influenced by the correct performance of thewashing procedure!

6.

Add 200 µL of Substrate Solution to each well.

7.

Incubate for 15 minutes at room temperature.

8.

Stop the enzymatic reaction by adding 100 µL of Stop Solution to each well.

9.

Determine the absorbance (OD) of each well at 450 ± 10 nm with a microtiter plate reader.
It is recommended that the wells be read within 10 minutes after adding the Stop Solution.

Calculation of Results
1. Calculate the average absorbance values for each set of standards, controls and 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 the IFU have been calculated automatically using a 4 PL (4 ParameterLogistics) curve fit. 4 Parameter Logistics is the preferred method. Other data reduction functions may giveslightly different results.
5. The concentration of the samples can be read directly from this standard curve. Samples with concentrations
higher than that of the highest standard have to be further diluted or reported as > 16 Ing/mL. For thecalculation of the concentrations this dilution factor has to be taken into account.
Example of Typical Standard Curve
The following data is for demonstration only and cannot be used in place of data generations at the time ofassay.
Standard

Optical Units (450 nm)

Standard A (0 ng/mL)

2.1

Standard B (0.2 ng/mL)

1.71

Standard C (0.5 ng/mL)

1.44

Standard D (1 ng/mL)

1.18

Standard E (2 ng/mL)

0.89Standard F (6 ng/mL)

0.46Standard G (16 ng/mL)

0.24

Version: 8.0-b-r

Effective: 2018-06-27

5/8

EXPECTED NORMAL VALUES
It is strongly recommended that each laboratory should determine its own normal and abnormal values.
In a study conducted with apparently normal healthy adults, using the Testosterone ELISA the following values
are observed:Population

5% Percentile

95% Percentile

MalesFemales

2.0 ng/mL
0.26 ng/mL

6.9 ng/mL
1.22 ng/mL

QUALITY CONTROLGood laboratory practice requires that controls be run with each standard curve. A statistically significant
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 state and federal regulations. The use of control
samples is advised to assure the day to day validity of results. Use controls at both normal and pathologicallevels.
The controls and the corresponding results of the QC-Laboratory are stated in the QC certificate added to thekit. The values and ranges stated on 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 analysing 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.
After checking the above mentioned items without finding any error contact your distributor or themanufacturer directly.
PERFORMANCE CHARACTERISTICS
Assay Dynamic RangeThe range of the assay is between 0.083 – 16 ng/mL.
Specificity of Antibodies (Cross Reactivity)
The following substances were tested for cross reactivity of the assay:
AnalyteCross Reactivity

Testosterone
5α-Dihydrotestosterone
Androstenedione
11β-Hydroxyestosterone
17α-Methyltestosterone
19-Nortestosterone
Epitestosterone
Oestradiol
Progesterone
Cortisol
Oestrone
Danazol

100.0
0.8
0.9
3.3
0.1
3.3
< 0.1
< 0.1
< 0.1
< 0.1
< 0.1
< 0.1

Sensitivity
The analytical sensitivity of the Testosterone ELISA was calculated by subtracting 2 standard deviations fromthe mean of 20 replicate analyses of the Standard A (S0) and was found to be 0.083 ng/mL.
Reproducibility

Version: 8.0-b-r

Effective: 2018-06-27

6/8

Intra Assay Variation
The within assay variability is shown below:
SamplenMean (ng/mL)

CV (%)

12
3

20
20
20

0.73
4.88
11.26

4.16
3.28
3.34

Inter Assay Variation
The between assay variability is shown below:
SamplenMean (ng/mL)

CV (%)

12320
20200.82
5.20
11.38

9.94
6.71
4.73

Recovery
Samples have been spiked by adding Testosterone solutions with known concentrations in a 1:1 ratio.
The expected values were calculated by addition of half of the values determined for the undiluted samples andhalf of the values of the known solutions. The % Recovery has been calculated by multiplication of the ratio of
the measurements and the expected values with 100.
SampleAdded Concentration

Measured Conc.

Expected Conc.

Recovery

1:1 (v/v) (ng/mL)(ng/mL)(ng/mL)

(%)

0.0
16.0
6.0
2.0
1.0
0.0
16.0
6.0
2.0
1.0
0.0
16.0
6.0
2.0
1.0

1.10
9.31
3.93
1.67
0.91
6.07
11.81
6.65
3.73
3.26
11.62
14.76
9.33
7.29
6.75

8.55
3.55
1.55
1.05

109.0
110.7
107.9
86.9

11.03
6.03
4.03
3.53

107.1
110.1
92.5
92.2

13.63
8.63
6.63
6.13

108.3
108.1
110.0
110.1

12

3

Linearity
SampleDilution

123

Version: 8.0-b-r

None
1:2
1:4
1:8
None
1:2
1:4
1:8
1:16
None
1:2
1:4
1:8
1:16

Measured Conc

Expected Conc.

Recovery(ng/mL)(ng/mL)

(%)

1.10
0.51
0.24
0.15
6.07
3.36
1.66
0.68
0.37
11.26
5.76
2.76
1.55
0.77

1.10
0.55
0.27
0.14
6.07
3.03
1.52
0.76
0.38
11.26
5.63
2.81
1.41
0.70

Effective: 2018-06-27

93.8
86.1
106.6
110.6
109.2
89.0
97.0
102.4
97.9
110.0
109.5

7/8

LIMITATIONS OF USE
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.
Interfering Substances
Haemoglobin (up to 4 mg/mL), Bilirubin (up to 0.25 mg/mL) and Triglyceride (up to 7.5 mg/mL) have noinfluence on the assay results.
Drug Interferences
Until today no substances (drugs) are known to us, which have an influence to the measurement ofTestosterone in a sample.
High-Dose-Hook EffectNo hook effect was observed in this test.
REFERENCES / LITERATURE
1. Tietz, N.W. Textbook of Clinical Chemistry. Saunders, 1986.

Symbols:

Version: 8.0-b-r

Storagetemperature

Manufacturer

Expiry date

Batch code

Consult instructions
for useContentCaution

Cataloguenumber
Effective: 2018-06-27

Contains sufficient for<n> tests

For research use
only!
8/8