DHEA SULFATE ENZYME IMMUNOASSAY TEST KIT; Page 1
Atlas Link
12720 Dogwood Hills Lane
Fairfax, VA 22033 USA
Phone: (703) 266-5667, FAX: (703) 266-5664
http://www.atlaslink-inc.com, info@atlaslink-inc.com
DHEA-SULFATE ENZYME IMMUNOASSAY
TEST KIT
Catalog Number: 2055
ENZYME IMMUNOASSAY FOR THE QUANTITATIVE
DETERMINATION OF
DEHYDROEPIANDROSTERONE-3-S ULFATE IN
SERUM
FOR IN VITRO USE ONLY.
Store at 2 to 8 °C
EXPLANATION OF THE TEST
Dehydroepiandrosterone-3-sulfate (DHEA-Sulfate) is
produced mainly (>90%) in the adrenals and is therefore
a marker for adrenal androgen biosynthesis.
DHEA-S is a valuable parameter for detecting
adrenal hormone producing tumors, mainly in the
diagnosis of female virilization.
DHEA-S serum
concentrations > 6.6 µg/mL plasma or serum indicate the
presence of an adrenal androgen producing tumor.
Normal range values do not necessarily exclude an
adrenal tumor. Therefore a parallel determination of
testosterone is indicated.
NORMAL RANGE
Males:
Prepubescent
Adult
0.10 - 0.60 µg/mL
2.00 - 3.35 µg/mL
Females:
Prepubescent 0.10 - 0.60 µg/mL
Premenopausal 0.70 - 1.17 µg/mL
3rd Trimester 0.23 - 0.60 µg/mL
Newborns:
Both sexes
1.67 - 3.64 µg/mL
PRINCIPLE OF THE TEST
The Diagnostic Automation DHEA-Sulfate Enzyme
Immunoassay is a direct, competitive enzyme
immunoassay using peroxidase as enzyme and TMB
(3,5,3',5'-tetramethyl benzidine) as substrate.
It is
designed for determination of DHEA-S in serum or
plasma without sample predilution. Due to microwell
technique, small sample numbers can be assayed
repeatedly. The microwells are coated with a second
antibody thereby eliminating any edge effects. The whole
incubation time is 45 minutes.
REAGENTS
1. 12 framed microstrips (8 microwells each):
Coated with affinity purified goat anti-rabbit IgG.
2. 1 vial antiserum:
11 mL (in 0.15 mol/L phosphate buffer, pH 7.4,
containing 0.01% Kathon CG as preservative) yellow
colored, ready to use.
3. 1 vial of enzyme conjugate diluent:
11 mL (0.15 mol/L phosphate buffer, containing
Kathon CG as preservative, pH 7.4) blue colored,
ready to use.
4. 1 vial of concentrated DHEA -S HRP-conjugate:
110 µl.
Dilute before use 1:101 with enzyme
conjugate diluent.
5. 5 vials of DHEA-S standards (lyophilized):
Standard 1
0.10 µg/mL
Standard 2
0.30 µg/mL
Standard 3
1.00 µg/mL
Standard 4
3.00 µg/mL
Standard 5
10.00 µg/mL
6. 1 vial of sample diluent (lyophilized).
7. 1 vial plasma control (lyophilized).
8. 2 vials of substrate:
Solution A (containing H202 - urea) 11 mL, ready for
use. Solution B (containing 3,5,3',5'-tetramethyl
benzidine) 11 mL ready for use.
9. 1 vial of stop solution:
(4 mol/L H2SO4) 6 mL, ready for use.
10. 1 vial of wash solution:
50 mL (containing NaCl and Tween 20) 10-fold
concentrate, dilute before use 1:10 with distilled
water.
WARNINGS AND PRECAUTIONS FOR USERS
1. CAUTION: Test methods are not available that can
offer complete assurance that Hepatitis B virus,
Human Immunodeficiency Virus (HIV-1), or other
infectious agents are absent from the reagents in
this kit. Therefore, all human blood products should
be considered potentially infectious. Handling should
be in accordance with the procedures defined by an
appropriate national biohazard safety guideline or
regulation, where it exists e.g., USA Center for
Disease Control/National Institute of Health Manual,
“Biosafety in Microbiological and Biomedical
Laboratories”, 1988.
2. Do not use reagents after expiration date.
3. Do not mix or use components from kits with
different lot numbers.
4. Replace caps on reagents immediately. Do not
switch caps.
5. Do not pipette reagents by mouth.
6. Some reagents contain sodium azide which may
react with lead or copper plumbing to form potentially
explosive metal azides. When disposing these
materials always flush with large volumes of water to
prevent azide buildup.
7. For in vitro use only.
Atlas Link, 12720 Dogwood Hills Lane, Fairfax, VA 22033 USA
Phone: (703) 266-5667, FAX: (703) 266-5664
http://www.atlaslink-inc.com, info@atlaslink-inc.com
DHEA SULFATE ENZYME IMMUNOASSAY TEST KIT; Page 2
MATERIALS AND EQUIPMENT REQUIRED
BUT NOT SUPPLIED
1. Pipettes capable of delivering volumes of 20, 50 100,
200, and 300 µl.
2. Microwell reader equipped with a filter for 450 nm.
SPECIMEN COLLECTION AND PREPARATION
Serum should be used in this EIA procedure.
It is important to preserve the chemical integrity of a
blood specimen from the moment it is collected until it is
assayed.
Perform phlebotomy using a non-traumatic
venipuncture technique with either a sterile syringe or a
vacuum tube. If a syringe is used, remove the needle
and carefully transfer the blood to a tube. Allow blood to
clot at room temperature for 20-30 minutes, until the clot
just
begins
to
retract.
Immediately following
centrifugation, transfer the serum to a storage tube, cap
tightly and refrigerate. If it cannot be assayed within 24
hours, it should be frozen.
1. Precautions
As with any sample that may contain pathogens,
care must be taken to prevent contact with open
wounds.
2. Additives and Preservatives
No additives or preservatives are necessary to
maintain the integrity of the specimen.
3. Storage, Handing and Shipping
Specimens should be capped, stored at 2-8 ° C and
assayed within 24 hours after collection. If the assay
cannot be performed within 24 hours or if the
specimen is to be shipped, cap and keep it frozen.
As in the case of most proteinaceous material,
repeated freezing and thawing should be avoided.
The use of hemolyzed or lipemic specimens is not
recommended.
Specimens should be allowed to come to room
temperature and mixed thoroughly by gentle
inversion before assaying.
ASSAY PROCEDURE
NOTE:
FOR PLASMA CONTROL VALUES, PLEASE SEE
ENCLOSED CERTIFICATE OF ANALYSIS. PLEASE NOTE
THESE VALUES CHANGE FOR EACH LOT NUMBER.
Reagent Preparation
1. Reconstitute standards and plasma control with 500
µl of distilled water and let them stand for 15 minutes,
then mix carefully. Store redissolved standards at -20
°C.
2. Reconstitute the sample diluent with 1 mL of distilled
water and treat it like the standards.
3. Dilute the conjugate concentrate 1:101 with
conjugate diluent (e.g. take 20 µl of concentrate and
add 2.00 mL of enzyme diluent). Any unused diluted
conjugate must be discarded after each assay.
4. Dilute wash solution 1:10 with distilled water (50 mL
of concentrated wash solution plus 450 mL of distilled
water). Store at 2-8 °C not longer than 3 months.
5. Immediately before adding the substrate solution mix
substrate solutions A and B 1:1. Unused mixed
substrate solution must be discarded after each
assay. Note: Use clean glassware, rinse with
distilled water before mixing the substrate solutions.
Assay
1. Pipette 20 µl of standards, control and serum
samples in duplicate into appropriate wells.
2. Add 100 µl of diluted conjugate solution.
3. Add 100 µl of antibody solution.
4. Assay blank: Pipet 20 µl of standard 1, 100 µl diluted
conjugate solution and 100 µl of water, in duplicates.
5. Incubate 30 minutes at room temperature. Do not
shake the microwell plate.
6. Wash the plate: either 2 cycles with a microplate
washer or decant the liquids and add 300 µl of
prediluted wash solution, incubate for 30 seconds and
decant liquids. Repeat manual washing three times in
total. Gently tap the inverted microstrips a few times
on a clean paper towel.
7. Add 200 µl of the prepared substrate solution to each
well.
8. Incubate 15 minutes at room temperature. Do not
shake the microwell plate.
9. Add 50 µl of stop solution to each well.
10. Read the absorbance at 450 nm.
CALCULATION OF RESULTS
For establishing the standard curve plot the mean
absorbance values of each standard concentration in a
linear manner versus the given standard concentrations
in a logarithmic manner. The mean absorbance values
of the samples can be converted to µg/mL by means of
the standard curve.
Assay Blank: The assay blank is to control the
effectiveness of the washing procedure and also of
possible contamination to the substrate solution due to
insufficiently clean glassware. The assay blank values
should not exceed >0.200 extinction units.
Note: Samples with DHEA-S values > 10 µg/mL
should be diluted 1:2 or 1:4 with sample diluent and
assayed again.
PERFORMANCE CHARACTERISTICS
Sensitivity:
0.10 µg/mL
Competitor
Cross Reaction (%)
Dehydroepiandrosterone-3-sulfate
100.0
Dehydroepiandrosterone
180.0
Androstenedione
12.0
Androsterone
6.0
Testosterone
0.3
Atlas Link, 12720 Dogwood Hills Lane, Fairfax, VA 22033 USA
Phone: (703) 266-5667, FAX: (703) 266-5664
http://www.atlaslink-inc.com, info@atlaslink-inc.com
DHEA SULFATE ENZYME IMMUNOASSAY TEST KIT; Page 3
Estrone
Estradiol
Estriol
Progesterone
Hydrocortisone
<0.1
<0.1
<0.1
<0.1
<0.1
Atlas Link, 12720 Dogwood Hills Lane, Fairfax, VA 22033 USA
Phone: (703) 266-5667, FAX: (703) 266-5664
http://www.atlaslink-inc.com, info@atlaslink-inc.com
DHEA SULFATE ENZYME IMMUNOASSAY TEST KIT; Page 4
Reproducibility:
Intra-Assay Variation:
Sample
in Mean (µg/mL)
1
20
0.18
2
20
1.70
3
20
7.61
SD (µg/mL) CV (%)
0.019
10.7
0.098
5.8
0.439
5.9
Inter-Assay Variation:
Sample
in Mean (µg/mL)
1
10
0.16
2
10
1.59
3
10
7.62
SD (µg/mL)
0.024
0.116
0.518
CV (%)
15.0
7.25
6.81
REFERENCES
1. Abraham, G.E. (1974): "Ovarian and adrenal
contribution to peripheral androgens during
menstrual cycle". J. Chem., 39, p. 349.
2. Lobo, R.A., Paul, W.L., Goebelsmann, U. (1981):
"Dehyroepiandrosterone sulfate as indicator of
adrenal function". Obstet, Gynecol. 57, p. 69.
3. Simon,
J.A.,
Buster,
J.E.
(1983):
"Dehydroepiandrosterone sulfate concentrations in
normal ovulatory women". Clin. Chem. 29, 1, p. 318.
4. Yuen, B.H., Moon, Y.S., Mincey, E.K., Li, D. (1983):
"Adrenal and sex hormone production by virilizing
adrenal adenoma and its diagnosis with
computerized tomography". Am J. Obstet. Gynecol.
145, p. 169.
Technical Consultation
Call or Write:
Atlas Link
12720 Dogwood Hills Lane, Fairfax, VA 22033 USA
Phone: (703) 266-5667, FAX: (703) 266-5664
http://www.atlaslink-inc.com, info@atlaslink-inc.com
Atlas Link, 12720 Dogwood Hills Lane, Fairfax, VA 22033 USA
Phone: (703) 266-5667, FAX: (703) 266-5664
http://www.atlaslink-inc.com, info@atlaslink-inc.com