Instructions for use

DHT ELISAAA E-1700R

Dihydrotestosterone (DHT) ELISAINTENDED USE
For the direct quantitative determination of Dihydrotestosterone 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 ofDHT in the sample. A set of standards is used to plot a standard curve from which the amount of DHT insamples 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. Control materials or serum pools should be included in every run at a high and low level for assessing thereliability 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 thoroughlybefore 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 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 theexpiration 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 DHT in human serum. The kit is
not calibrated for the determination of DHT in saliva, plasma or other specimens 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 leadto false results.
4. Only standard A may be used to dilute any high serum samples. The use of any other reagent may lead tofalse results.
SAFETY CAUTIONS AND WARNINGS
POTENTIAL BIOHAZARDOUS MATERIAL
Human serum that may be used in the preparation of the standards and controls has been tested and found tobe nonreactive for Hepatitis B surface antigen and has also been tested for the presence of antibodies to HCV
and 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 reagents should be considered a 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 of thesereagents, wash with plenty of water. TMB is a suspected carcinogen.
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
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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. Microplate reader with a fi lter set at 450 nm and an upper OD limit of 3.0 or greater*(see assay procedure step 10)
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.
Stopping Solution - Ready To Use.

3. AA E-0080
Contents:

One bottle containing 1M sulfuric acid.

Volume:6 ml/bottle

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.

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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

Concentration

Volume/Vial
2.0 mlAA E-1701

Standard A

0 pg/ml

AA E-1702

Standard B

25 pg/ml

0.6 mlAA E-1703

Standard C

100 pg/ml

0.6 mlAA E-1704

Standard D

500 pg/ml

0.6 mlAA E-1705

Standard E1000 pg/ml

0.6 mlAA E-1706

2500 pg/ml

AA E-1751

Standard F
Control 1

AA E-1752

Control 2

Refer to vial labels for expectedvalue and acceptable range!

0.6 ml
0.6 ml
0.6 mlContents:

DHT in a protein-based buffer with a non-mercury preservative. Prepared by spiking buffer
with a defined quantity of DHT.

Storage:

Refrigerate at 2 - 8 oC

Stability:

12 months in unopened vial or as indicated on label. Once opened, the standards andcontrols should be used within 14 days or aliquoted and stored frozen.
Avoid multiple freezing and thawing cycles.

5. AA E-1713
Contents:

Assay Buffer - Ready To Use.
One bottle 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-DHT Antibody-Coated Break-Apart Well Microplate

6. AA E-1731- Ready To Use.
Contents:

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

Storage:

Refrigerate at 2 - 8 oC

Stability:

12 months or as indicated on label.
Dihydrotestosterone-Horseradish Peroxidase (HRP)ConjugateConcentrate – Requires Preparation X100

7. AA E-1740
Contents:

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

Volume:200 µl/vial

Storage:

Refrigerate at 2 - 8 oC

Stability:

12 months or as indicated on label.

Preparation:

Dilute 1:100 in assay buffer before use (eg. 20 μl of conjugate concentrate in 2 ml of assaybuffer). If the whole plate is to be used dilute 120 µl of conjugate concentrate in 12 ml of
assay buffer.
Discard any that is left 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 DHT-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.

Gently shake the plate for 10 seconds and incubate for 1 hour at room temperature (no shaking).

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 well at timed intervals.

8.

Gently shake the plate for 10 seconds and incubate for 10 – 15 minutes at room temperature (noshaking) 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 stopping
solution.
If the OD exceeds the upper limit of detection or if a 450 nm filter is unavailable, a 405 or 415nm filter may
be substituted. The optical densities will be lower, however, this will not affect the results of 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 curveis 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 2500 pg/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:

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Standard

OD 1

OD 2

Mean OD

A

2.320

2.279

2.300

Value(pg/ml)
0B

1.976

1.928

1.952

25C

1.058

1.077

1.068

100

D

0.359

0.354

0.357

500

E

0.222

0.205

0.214

1000

F

0.131

0.128

0.130

2500

Unknown

0.515

0.507

0.511

300

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TYPICAL STANDARD CURVE
Sample curve only. Do not use to calculate results.
2.5

OD 450nm

21.5
10.5
025100500

1000

2500

DHT (pg/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 theDirect Dihydrotestosterone ELISA kit is 6.0 pg/ml.
SPECIFICITY (CROSS REACTIVITY)
The following compounds were tested for cross-reactivity with the Direct Dihydrotestosterone ELISA kit withdihydrotestosterone cross-reacting at 100%.
Steroid% Cross
Reactivity
1008.72.00.2Dihydrotestosterone
Testosterone
5ß Dihydrotestosterone
Androstenedione

The following steroids were tested but cross-reacted at less than 0.01%: Dehydroepiandrosterone Sulfate, 17βEstradiol, Estriol, Estrone, Progesterone, 17-OH Progesterone, Cortisol, and Pregnenolone.
INTRA-ASSAY PRECISION
Three samples were assayed ten times each on the same standard curve. The results (in pg/ml) are tabulated
below:
Sample123Mean
236.74869.031008.14

SD
26.89
47.41
39.36

CV%
11.4
5.46
3.90

INTER-ASSAY PRECISION
Three samples were assayed ten times over a period of four weeks. The results (in pg/ml) are tabulated below:
Sample123

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Mean
280.88721.401025.41

SD
34.07
54.20
60.45

Effective: 2018-10-09

CV%
12.1
7.5
5.9

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RECOVERY
Spiked samples were prepared by adding defined amounts of DHT to three serum samples.
The results (in pg/ml) are tabulated below:
Sample1 Unspiked+117.53+235.06+470.13
2 Unspiked+117.53+235.06+470.13
3 Unspiked+117.53+235.06+470.13

Obs.Result
290.54361.51501.66744.81324.75389.43505.23712.44720.11
758.13856.461013.61

Exp.Result
408.07525.60760.67442.29559.81794.88837.64955.171190.24

Recovery%
88.6
95.4
97.9
88.0
90.3
89.6
90.5
89.7
85.1

LINEARITY
Three serum samples were diluted with Standard A. The results (in pg/ml) are tabulated below:
Sample
1
1:2
1:4
1:8
2
1:2
1:4
1:8
3
1:2
1:4
1:8

Obs.Result
340.67165.3595.39
48.47
1086.01508.58232.11114.951313.21612.98318.63134.98Exp.Result
170.3485.17
42.58
543.00271.50135.75656.61
328.30164.15

Recovery%
97.1
112.0
113.8
93.7
85.5
84.7
93.4
97.182.2

COMPARATIVE STUDIESThe Direct Dihydrotestosterone ELISA kit (Kit A) was compared with a competitors coated tube RIA kit (Kit B).
The results (in pg/ml) are tabulated below:
GroupNFemales
Males

1010Kit A
Mean
95.5
280.0

Kit B
Mean
99.0
252.0

EXPECTED NORMAL VALUESAs for all clinical assays each laboratory should collect data and establish their own range of expected normalvalues.
GroupFemales:
Premenopausal
Postmenopausal
Males

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Effective: 2018-10-09

24-368
10-181
250-990

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REFERENCES
1. Bassett RM. A Simple Chromatographic Method for the Radioimmunoassay of Four Androgenic Steroids.
Med Lab Sci. 1980; 37(1):31–8.
2. Baxendale PM, et al. Plasma and Salivary Androstenedione and Dihydrotestosterone in Women withHyperandrogenism. Clin Endocrinol (Oxf). 1983; 18(5):447–57.
3. Brooks RV, Androgens. Physiology and Pathology. In: Makin HL J, ed., Biochemistry of Steroid Hormones,
2nd ed., Oxford Blackwell Scientifi c Publications, 1984; 565.
4. Cameron EHD, et al., eds. Steroid Immunoassay: Proceedings of the Fifth Tenovus Workshop, Cardiff,April 1974. Cardiff: Alpha Omega Publishing; 1975.
5. Dunn JF, et al. Transport of Steroid Hormones: Binding of 21 Endogenous Steroids to TestosteroneBinding Globulin and Corticosteroid-Binding Globulin in Human Plasma. J Clin Endocrinol Metab. 1981;
53(1):58–68.
6. Hammond GL, et al. The Simultaneous Radioimmunoassay of Seven Steroids in Human Spermatic andPeripheral Venous Blood. J Clin Endocrinol Metab. 1977; 45(1):16–24.
7. Ito T, Horton R. Dihydrotestosterone in Human Peripheral Plasma. J Clin Endocrinol Metab. 1970;31(4):362–8.
8. Mean F, et al. Study of the Binding of Dihydrotestosterone, Testosterone and Oestradiol with SexHormone Binding Globulin. Clin Chim Acta. 1977; 80(1):171–80.
9. Mooradian AD, et al. The Biological Actions of Androgens. Endocr Rev. 1987; 8(1):1–28.
10. Pazzagli M, et al. Radioimmunoassay of Plasma Dihydrotestosterone in Normal and Hypogonadal Men.
Clin Endocrinol (Oxf). 1975; 4(5):513–20.
11. Wakelin K, et al. Relationship of 5ßdihydrotestosterone and 5αdihydrotestosterone to testosetrone inhealth and disease. J Endocrinol. 1980; 87:450.
12. Wang C, et al. Salivary Androgens in Hirsutism: Are They of Use in Routine Evaluation? Ann Clin
Biochem. 1986; 23(Pt 5):590–5.
13. Kricka LJ. Human Anti-Animal Antibody Interferences in Immunological Assays. Clin Chem. 1999;
45(7):942–56.
14. 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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