Instructions for use

Free Testosterone ELISAAA E-1800R

2nd Generation

Free Testosterone ELISA 2nd Generation

EnglishINTENDED USE
For the direct quantitative determination of Free Testosterone 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 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 is added. The enzymatic reaction is terminated by addition of the stopping solution. Theabsorbance is measured on a microtiter plate reader. The intensity of the colour formed is inversely
proportional to the concentration of free testosterone in the sample. A set of standards is used to plot astandard curve from which the amount of free testosterone in samples and controls can be directly read.
The Free Testosterone ELISA 2nd Generation kit utilizes a highly specific rabbit anti-testosterone polyclonal
antibody at a low binding capacity (Keq x concentration) to keep minimum disturbances of the testosteroneprotein equilibrium. The other components in the test system are also optimized in order to not alter theoriginal free testosterone concentration.
CLINICAL APPLICATIONS
Testosterone is a C-19 steroid secreted from the testis and the adrenal cortex in men and from the adrenal
cortex and ovaries in women. Testosterone is also produced by peripheral tissues from androstenedione,
which is of little physiological significance in men; in women however, about half of the circulating
testosterone is derived from this origin. Testosterone measurements are used mainly for clinical evaluationof hypogonadism in males and hyperandrogenic states in females.
Testosterone circulates in the blood bound to three proteins: sex hormone binding globulin (60 - 80%),albumin and cortisol binding globulin. Only about 1 - 2 % of the total circulating testosterone remains
unbound or free. Even though it is still under investigation, most researchers accept the free testosterone
determination as a measure of the biologically active fraction. Free testosterone determinations arerecommended to overcome the influences caused by variations in transport proteins on the total
testosterone concentration.
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
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 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 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 itshould not be used.
11. When dispensing the substrate and stopping solution, do not use pipettes in which these liquids will
come into 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.

Version: 8.0-r

Effective: 2018-10-10

2/8

LIMITATIONS
1. All the reagents within the kit are calibrated for the direct determination of free testosterone in humanserum. The kit is not calibrated for the determination of free testosterone in 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. Samples reading higher than 60 pg/ml should not be diluted. Dilution will alter the equilibrium between
free testosterone and serum proteins.
SAFETY CAUTIONS AND WARNINGS
POTENTIAL BIOHAZARDOUS MATERIAL
Human serum that may be used in the preparation of the standards and control has been tested and foundto be non-reactive for Hepatitis B surface antigen and has also been tested for the presence of antibodies toHCV and Human Immunodeficiency Virus (HIV) and found to be negative. No test method however, canoffer complete assurance that HIV, HCV and Hepatitis B virus or any infectious agents are absent. Thereagents should be considered as potential biohazard and handled with the same precautions as applied toany blood specimen.
CHEMICAL HAZARDS
Avoid contact with reagents containing TMB, hydrogen peroxide and sulfuric acid. If contacted with any ofthese reagents, wash with plenty of water. TMB is a suspected carcinogen.
SPECIMEN COLLECTION AND STORAGE
Approximately 0.1 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 at4°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
human specimens 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 25, 50, 100, 150 and 350 μl
2. Disposable pipette tips
3. Distilled or deionized water
4. A 37 °C incubator
5. Microplate reader with a filter set at 450nm and an upper OD limit of 3.0 or greater* (see assayprocedure step 11).
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.

Version: 8.0-r

Effective: 2018-10-10

3/8

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.
4. Standards and Controls- Ready To Use.
* Listed below are approximate concentrations, please refer to vial labels for exact concentrations:Cat. no.

SymbolStandard

Concentration*
0 pg/mlStandard A

AA E-1801

Volume/Vial
0.5 mlAA E-1802

Standard B

0.1 pg/ml

AA E-1803

Standard C

1 pg/ml

0.5 mlAA E-1804

Standard D

5 pg/ml

0.5 mlAA E-1805

Standard E

20 pg/ml

0.5 mlAA E-1806

Standard F

60 pg/ml

AA E-1851

Control 1

0.5 ml
0.5 mlAA E-1852

Control 2

Refer to vial labels for expectedvalue and acceptable range!

0.5 ml

0.5 mlContents:

Vials containing testosterone in a human serum-based buffer with a non-mercury
preservative. Prepared by spiking serum with a precise quantity of testosterone equivalent
to approximately 0, 0.1, 1, 5, 20 and 60 pg/ml * of free testosterone.

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 freezing and thawing
cycles.

5. AA E-1413
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 oC

Stability:

12 months or as indicated on label.
Rabbit Anti-Free Testosterone Antibody-Coated Break-Apart Well
Microplate - Ready To Use.

6. AA E-1431
Contents:

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

Storage:

Refrigerate at 2 - 8 oC

Stability:

12 months or as indicated on label.

7. AA E-1840

Free Testosterone-Horseradish Peroxidase (HRP) Conjugate
Concentrate – Requires Preparation x50

Contents:

Free Testosterone-HRP
preservative.

Volume:300 µl/vialStorage:

Refrigerate at 2 - 8 oC

conjugate

ina

protein-basedbufferwitha

non-mercury

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.

Version: 8.0-r

Effective: 2018-10-10

4/8

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 free testosterone-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 25 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.

Gently shake the plate for 10 seconds.

6.

Incubate the plate at 37 oC for 1 hour.

7.

Wash the wells 3 times with 350 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).

8.

Pipette 150 µl of TMB substrate into each well at timed intervals.

9.

Incubate the plate at 37 oC for 10-15 minutes.
(or until Standard A attains dark blue colour for desired OD).

10.

Pipette 50 µl of stopping solution into each well at the same timed intervals as in step 8.

11.

Read the plate on a microplate reader at 450 nm within 20 minutes after addition of the stopping solution.
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 thestandard concentrations on the X-axis. If immunoassay software is being used, a 4-parameter or 5parameter 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.
TYPICAL TABULATED DATA
Sample data only. Do not use to calculate results.
Standard

Mean OD (450 nm)

Value (pg/ml)

A

2.292

0B

1.680

0.1

C

1.181

1

D

0.780

5

E

0.426

20F

0.214

60

Unknown

1.066

1.59

Unknown

0.441

19.6

Version: 8.0-r

Effective: 2018-10-10

5/8

OD (450 nm)

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

Free Testosterone (pg/ml)
PERFORMANCE CHARACTERISTICSSENSITIVITY
The limit of detection (LoD) was determined from the analysis of 64 replicates of a low value sample andfrom the LoB.
LoD = LoB + 1.645σS,
where σS is the standard deviation of the low value sample. σS was determined to be 0.0093 based on 64
measurements of a low value sample.
LoD = 0.0025 + (1.645*0.0093) = 0.018 pg/ml.
SPECIFICITY (CROSS REACTIVITY)
The following compounds were tested for cross-reactivity with the Free Testosterone ELISA 2nd Generation
kit with testosterone cross-reacting at 100%.
Steroid
Testosterone
5α-DHT
Androstenedione
Progesterone
Androsterone
Aldosterone
Cholesterol
Cortisone
DHEA
DHEAS
17β-Estradiol
Estriol
Pregnenolone% Cross Reactivity
100
3.5
0.17
0.007
0.075
<0.008<0.0001
0.00250.071
0.00140.15<0.0080.028

INTRA-ASSAY PRECISION
Five samples were assayed 24 times each on the same standard curve. The results (in pg/ml) are tabulated
below:Sample
1234
5Version: 8.0-rMean
2.24
3.81
13.6
13.7
23.7

CV %
6.7
6.4
6.0
5.9
4.8

Effective: 2018-10-10

6/8

INTER-ASSAY PRECISION
Three samples were assayed twenty times in duplicate over a period of greater than ten days.
The results (in pg/ml) are tabulated below:
Sample
123

Mean
3.53
13.8
23.3

CV %
8.1
11.5
6.9

COMPARATIVE STUDIESThe Free Testosterone ELISA 2nd Generation Kit (y) was compared with a competitors Free Testosterone Coated
Tube RIA Kit (x).The comparison of 60 serum samples yielded the following linear regression results:
y = 0.9362x (competitor) + 3.8794, r = 0.97
EFFECT OF SEX HORMONE BINDING GLOBULIN (SHBG)
The purpose of this study was to investigate a possible interference caused by the binding of SHBG to thefree testosterone-HRP conjugate. A charcoal-stripped human serum pool was spiked precisely with SHBG atconcentrations ranging from 6.25 - 200 μg/ml and was assayed with the Free Testosterone ELISA 2nd Generation
Kit. Results tabulated below (in pg/ml):
SHBG Added
06.25
12.550200OD 450nm
2.37
2.37
2.34
2.362.27Percent B/Bo (%)
100.0
99.9
98.7
99.5
95.6

The results showed % binding values between 95 - 100 %(Bo = unspiked serum) even at higher thannormal SHBG levels. In conclusion, the results showed that there was no significant binding by SHBG on thefree testosterone-HRP conjugate.
EXPECTED NORMAL VALUESEach laboratory should collect data and establish their own range of expected normal values. The results ofan expected range study with apparently normal healthy subjects yielded the following results (all values arereported in pg/ml):
Cohort Group;
Gender/Age
Males / < 13
Males / 13 - 19
Males /20 - 39
Males / 40 - 59
Males / ≥ 60
Females / < 13Females / 13 - 19
Females / 20 - 39
Females / 40 - 59
Females / ≥ 60

Version: 8.0-r

N

95% Confidence Range

Absolute Range

4437120120120631712012060

9.1 - 32.2
5.7 - 30.7
5.9 - 27.0
0.1 - 6.3
0.2 - 4.1
0.5 - 3.9

ND - 1.6
ND - 22.3
ND - 1.3
0.2 - 2.0-Effective: 2018-10-10

7/8

REFERENCES
1. Winter SJ, et al. The Analog Free Testosterone Assay: Are the Results in Men Clinically Useful. Clin
Chem. 1998; 44(10):2178–82.
2. Ooi DS, et al. Establishing Reference Interval for DPC’s Free Testosterone Radioimmunoassay. Clin
Biochem. 1998; 31(1):15–21.
3. Marcus GJ, Dumford R. A Simple Enzyme-Linked Immunosorbent Assay for Testosterone. Steroids.
1985; 46:975–86.
4. Joshi UM, et al. A Sensitive Specific Enzyme Immunoassay for Serum Testosterone. Steroids. 1979;
34(1):35–46.
5. Swinkels LM, et al. Salivary and Plasma Free Testosterone and Androstenedione Levels in Women. AmClin Biochem. 1988; 25(Pt 4):354–9.
6. Swinkels LM, et al. A Symetric Dialysis Method for the Determination of Free Testosterone in HumanPlasma. Clin Chem Acta. 1987; 165(2–3):341–9.
7. Ekins R. Hirsutism: Free and Bound Testosterone. Ann Clin Biochem. 1990; 27(Pt 1):91–4.
8. Manni A, et al. Bioavailability of Albumin-Bound Testosterone.J Clin Endo Metab. 1985; 61(4):705–10.
9. Ekins R. The Science of Free Testosterone Measurement.Proc UK NEQAS Meeting. 1998; 3:35–9.
10. Longcope C, et al. Free Estradiol, Free Testosterone, and Sex Hormone-Binding Globulin in
Perimenopausal Women. J Clin Endo Metab. 1987; 64(3):513–8.
11. Vermeulen A, et al. The Apparent Free Testosterone Concentration: An Index of Androgenicity. J Clin
Endo Metab. 1971; 33(5):759–67.
12. Paulson JD, et al. Free Testosterone Concentration in Serum. Elevation is the Hallmark of Hirsutism. AmJ Obs Gyneco.1977; 128(8):851–7.
13. Cumming DC, Wall SR. Non-Sex Hormone-Binding Globulin-Bound Testosterone as a Marker forHyperandrogenism. JClin Endo Metab. 1985; 61(5):873–6.
14. Baxendale PM, et al. Salivary Testosterone: Relationship to Unbound Plasma Testosterone in Normaland Hyperandrogenic Women. Clin Endocrinol (Oxf). 1982; 16(6):595–603.
15. Biffignandi P, et al. Female Hirsutism: Pathoplysiological Considerations and Therapeutics Implications.
Endocrinol Rev.1984; 5(4):498–513.
16. Wu CH. Plasma Free and Protein-Bound Testosterone inHirsutism. Obstet Gynecol. 1982; 60(2):188–94.
17. Bamman BL, et al. Total and Free Testosterone During Pregnancy. Am J Obsetet Gynecol. 1980;
137(3):293–8.
18. Halpern EP, et al. Production of Anti-Testosterone Antisera in Rabbits. Clin Chem. 1980; 26:68.
19. Wheeler MJ. The Determination of Bio-Available Testosterone. Ann Clin Biochem. 1995; 32(Pt 4):345–57.
20. 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:

Version: 8.0-r

Storagetemperature

Manufacturer

Expiry date

Batch code

Consult instructions
for useContentCaution

Catalogue
numberEffective: 2018-10-10

Contains sufficient for<n> tests

For research use
only!
8/8