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DRG® Estradiol ELISA (EIA-2693)
REVISED 23 MAY 2005

1 INTRODUCTION
The DRG® Estradiol Enzyme Immunoassay Kit provides materials for the quantitative determination of Estradiol in
serum and plasma.
This assay is intended for in vitro diagnostic use only.

Estradiol (1,3,5(10)-estratriene-3,17β-diol; 17β-estradiol; E21) is a C18 steroid hormone with a phenolic A ring. This
steroid hormone has a molecular weight of 272.4. It is the most potent natural Estrogen, produced mainly by the Graffian
follicle of the female ovary and the placenta, and in smaller amounts by the adrenals, and the male testes (1,2,3).
Estradiol (E2) is secreted into the blood stream where 98% of it circulates bound to sex hormone binding globulin
(SHBG) and to a lesser extent to other serum proteins such as albumin. Only a small fraction circulates as free hormone or
in the conjugated form (4,5). Estrogenic activity is affected via estradiol-receptor complexes which trigger the appropriate
response at the nuclear level in the target sites. These sites include the follicles, uterus, breast, vagina, urethra,
hypothalamus, pituitary and to a lesser extent the liver and skin.
In non-pregnant women with normal menstrual cycles, estradiol secretion follows a cyclic, biphasic pattern with the
highest concentration found immediately prior to ovulation (6,7). The rising estradiol concentration is understood to exert
a positive feedback influence at the level of the pituitary where it influences the secretion of the gonadotropins, follicle
stimulating hormone (FSH), and luteinizing hormone (LH), which are essential for follicular maturation and ovulation,
respectively (8,9). Following ovulation, estradiol levels fall rapidly until the luteal cells become active resulting in a
secondary gentle rise and plateau of estradiol in the luteal phase. During pregnancy, maternal serum Estradiol levels
increase considerably, to well above the pre-ovulatory peak levels and high levels are sustained throughout pregnancy
(10).
Serum Estradiol measurements are a valuable index in evaluating a variety of menstrual dysfunctions such as precocious
or delayed puberty in girls (11) and primary and secondary amenorrhea and menopause (12). Estradiol levels have been
reported to be increased in patients with feminizing syndromes (14), gynaecomastia (15) and testicular tumors (16).
In cases of infertility, serum Estradiol measurements are useful for monitoring induction of ovulation following treatment
with, for example, clomiphene citrate, LH-releasing hormone (LH-RH), or exogenous gonadotropins (17,18). During
ovarian hyperstimulation for in vitro fertilization (IVF), serum estradiol concentrations are usually monitored daily for
optimal timing of human chorionic gonadotropin (hCG) administration and oocyte collection (19).
2 PRINCIPLE OF THE TEST
The DRG® Estradiol ELISA Kit is a solid phase enzyme-linked immunosorbent assay (ELISA), based on the principle of
competitive binding. The microtiter wells are coated with an antibody directed towards a unique antigenic site on the
Estradiol molecule.
Endogenous Estradiol of a patient sample competes with an Estradiol horseradish peroxidase conjugate for binding to the
coated antibody. After incubation the unbound conjugate is washed off.
The amount of bound peroxidase conjugate is reverse proportional to the concentration of Estradiol in the sample. After
addition of the substrate solution, the intensity of colour developed is reverse proportional to the concentration of
Estradiol in the patient sample.
DRG International, Inc., USA Fax: (908) 233 0758 e-mail: [email protected] 1
DRG® Estradiol ELISA (EIA-2693)
REVISED 23 MAY 2005
3 PRECAUTIONS
• This kit is for in vitro diagnostic use only. • For information on hazardous substances included in the kit please refer to Material Safety Data Sheets. • 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 be treated as potential biohazards in use and for disposal. • Avoid contact with Stop Solution containing 0.5 M H2SO4. It may cause skin irritation and burns. • Never pipet by mouth and avoid contact of reagents and specimens with skin and mucous membranes. • Do not smoke, eat, drink or apply cosmetics in areas where specimens or kit reagents are handled. • Wear disposable latex gloves when handling specimens and reagents. Microbial contamination of reagents or • Handling should be in accordance with the procedures defined by an appropriate national biohazard safety guideline or • Do not use reagents beyond expiry date as shown on the kit labels. • All indicated volumes have to be performed according to the protocol. Optimal test results are only obtained when • Do not mix or use components from kits with different lot numbers. It is advised not to exchange wells of different 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. • Chemicals and prepared or used reagents have to be treated as hazardous waste according the national biohazard safety • Safety Data Sheets for this product are available upon request directly from DRG Instruments GmbH. • The Safety Data Sheets fit the demands of: EU-Guideline 91/155 EC. DRG International, Inc., USA Fax: (908) 233 0758 e-mail: [email protected] 2
DRG® Estradiol ELISA (EIA-2693)
REVISED 23 MAY 2005
4 KIT COMPONENTS
4.1 Contents of the Kit
1. Microtiterwells, 12x8 (break apart) strips, 96 wells
Wells coated with anti-Estradiol polyclonal rabbit antibody 2. Standard (Standard 0-6), 7 vials, 1 ml, ready to use
Concentration: 0, 25; 100; 250; 500; 1000; 2000 pg/ml Conversion: 1 pg/ml = 3.67 pmol/l 3. Enzyme Conjugate, 1 vial, 25 ml, ready to use
Estradiol conjugated to horseradish peroxidase 4. Substrate Solution, 1 vial, 14 ml, ready to use
5. Stop Solution, 1 vial, 14 ml, ready to use
Avoid contact with the stop solution. It may cause skin irritations and burns. 6. Wash Solution, 1 vial, 30 ml (40X concentrated)

Note: Additional Standard 0 for sample dilution is available on request.
4.1.1 Equipment and material required but not provided
− A microtiterplate calibrated reader (450±10 nm)(e.g. the DRG Instruments Microtiterplate Reader).
− Calibrated variable precision micropipettes.
4.2 Storage and stability of the Kit
When stored at 2-8°C unopened reagents will retain reactivity until expiration date. Do not use reagents beyond this date.
All opened reagents must be stored at 2-8°C. Microtiter wells must be stored at 2-8°C. Once the foilbag has been opened,
care should be taken to close it tightly again.
4.3 Preparation of Reagents
Allow all reagents and required number of strips to reach room temperature prior to use.
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.
4.4 Disposal of the Kit
The disposal of the kit must be made according to the national official regulations. Special information for this product is
given in the Material Safety Data Sheets (see chapter 13).
DRG International, Inc., USA Fax: (908) 233 0758 e-mail: [email protected] 3
DRG® Estradiol ELISA (EIA-2693)
REVISED 23 MAY 2005
4.5 Damaged Test Kits
In case of any severe damage of the test kit or components, DRG® have to be informed written, latest one week after
receiving the kit. Severely damaged single components should not be used for a 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
Serum or plasma (EDTA-, Heparin- or citrat plasma) can be used in this assay.
Do not use haemolytic, icteric or lipaemic specimens.
5.1 Specimen Collection
Serum:
Collect blood by venipuncture (e.g Sarstedt Monovette # 02.1388.001), allow to clot, and separate serum by centrifugation
at room temperature.
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 Citrat plasma Sarstedt Monovette – green cap - # 02.167.001.)
5.2 Specimen Storage
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.
5.3 Specimen Dilution
If in an initial assay, a serum specimen is found to contain more than the highest standard, the specimens can be diluted
10-fold or 100 fold with Standard 0 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 0 (mix thoroughly) 10 µl dilution a) 1:10 + 90 µl Standard 0 (mix thoroughly). 6 TEST PROCEDURE
6.1 General Remarks
− All reagents and specimens must be allowed to come to room temperature before use. All reagents must be mixed
− 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 cross contamination DRG International, Inc., USA Fax: (908) 233 0758 e-mail: [email protected] 4
DRG® Estradiol ELISA (EIA-2693)
REVISED 23 MAY 2005
− Absorbance is a function of the incubation time and temperature. Before starting the assay, it is recommended 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. 6.2 Assay Procedure
All standards, samples, and controls should be run in duplicate concurrently so that all conditions of testing are the same.
1. Secure the desired number of Microtiterwells in the holder.
2. Dispense 25 µl of each Standard, controls and samples with new disposable tips into appropriate wells.
3. Dispense 200 µl Enzyme Conjugate into each well.
4. Thoroughly mix for 10 seconds. It is important to have a complete mixing in this step.
5. Incubate for 120 minutes at room temperature without covering the plate.
6. 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 the washing
procedure!
7. Add 100 µl of Substrate Solution to each well.
8. Incubate for 15 minutes at room temperature.
9. Stop the enzymatic reaction by adding 50 µl of Stop Solution to each well.
10. Read the OD at 450±10 nm with a microtiterplate reader within 10 minutes after adding the Stop Solution.
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 the standard 4. Automated method: Computer programs using cubic spline, 4 PL (4 Parameter Logistics) or Logit-Log can generally 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. For the calculation of the concentrations this dilution factor has to be taken into account. DRG International, Inc., USA Fax: (908) 233 0758 e-mail: [email protected] 5
DRG® Estradiol ELISA (EIA-2693)
REVISED 23 MAY 2005
Below is listed a typical example of a standard curve with the Estradiol ELISA.
Standard
Optical Units (450 nm)
7 EXPECTED 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 DRG® Estradiol ELISA the following values are
observed:
Population
5 – 95% Percentile
8 ASSAY CHARACTERISTICS
8.1 Assay Dynamic Range
The range of the assay is between 0 – 2000 pg/ml.
DRG International, Inc., USA Fax: (908) 233 0758 e-mail: [email protected] 6
DRG® Estradiol ELISA (EIA-2693)
REVISED 23 MAY 2005
8.2 Specificity of Antibodies (Cross Reactivity)
The following substances were tested for cross reactivity of the assay:
Compound
% Cross reactivity
Compound
% Cross reactivity
8.3 Analytical Sensitivity
The analytical sensitivity was calculated from the mean minus two standard deviations of twenty (20) replicate analyses of
Standard 0 and was found to be 9.714 pg/ml.
8.4 Precision
8.4.1 Intra Assay Variation
The within assay variability is shown below:
Sample
Mean (pg/ml)
8.4.2 Inter Assay Variation
The between assay variability is shown below:
Sample
Mean (pg/ml)
DRG International, Inc., USA Fax: (908) 233 0758 e-mail: [email protected] 7
DRG® Estradiol ELISA (EIA-2693)
REVISED 23 MAY 2005

8.5 Accuracy
8.5.1 Quality Control
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 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 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 to ensure 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 patient 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 DRG® directly.
8.5.2 Recovery
Samples have been spiked by adding Estradiol 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 and half 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.
Added Concentration
Measured Conc.
Expected Conc.
Recovery (%)
1:1 (v/v) (pg/ml)
DRG International, Inc., USA Fax: (908) 233 0758 e-mail: [email protected] 8
DRG® Estradiol ELISA (EIA-2693)
REVISED 23 MAY 2005

8.5.3 Linearity
Dilution
Mean Conc. (pg/ml)
Recovery (%)
9 LIMITATIONS OF USE
9.1 Interfering Substances
Any improper handling of samples or modification of this test might influence the results.
Haemoglobin (up to 4 mg/ml), Bilirubin (up to 0.5 mg/ml) and Triglyceride (up to 30 mg/ml) have no influence on the
assay results.
9.2 Drug Interferences
Until today no substances (drugs) are known to us, which have an influence to the measurement of Estradiol in a sample.
10 LEGAL ASPECTS
10.1 Reliability of Results
The test must be performed exactly as per the manufacturer’s instructions for use. Moreover the user must strictly adhere
to the rules of GLP (Good Laboratory Practice) or other applicable national standards and/or laws. This is especially
relevant for the use of control reagents. It is important to always include, within the test procedure, a sufficient number of
controls for validating the accuracy and precision of the test.
The test results are valid only if all controls are within the specified ranges and if all other test parameters are also within
the given assay specifications. In case of any doubt or concern please contact DRG®.
DRG International, Inc., USA Fax: (908) 233 0758 e-mail: [email protected] 9
DRG® Estradiol ELISA (EIA-2693)
REVISED 23 MAY 2005
10.2 Therapeutical Consequences
Therapeutical consequences should never be based on laboratory results alone even if all test results are in agreement with
the items as stated under point 10.1. Any laboratory result is only a part of the total clinical picture of a patient.
Only in cases where the laboratory results are in acceptable agreement with the overall clinical picture of the patient
should therapeutical consequences be derived.
The test result itself should never be the sole determinant for deriving any therapeutical consequences.
10.3 Liability
Any modification of the test kit and/or exchange or mixture of any components of different lots from one test kit to
another could negatively affect the intended results and validity of the overall test. Such modification and/or exchanges
invalidate any claim for replacement.
Claims submitted due to customer misinterpretation of laboratory results subject to point 10.2. are also invalid.
Regardless, in the event of any claim, the manufacturer’s liability is not to exceed the value of the test kit. Any damage
caused to the test kit during transportation is not subject to the liability of the manufacturer.
DRG International, Inc., USA Fax: (908) 233 0758 e-mail: [email protected] 10
DRG® Estradiol ELISA (EIA-2693)
REVISED 23 MAY 2005
11 REFERENCES
1. Tsang, B.K., Armstrong, D.T. and Whitfield, J.F., Steroid biosyntheses by isolated human ovarian follicular cells in
vitro, J. Clin. Endocrinol. Metab. 51:1407 - 11 (1980). 2. Gore-Langton, R.E. and Armstrong, D.T., Follicular stoidogenesis and its control. In: The physiology of Reproduction, Ed.: Knobil, E., and Neill, J. et al., pp. 331-85. Raven Press, New York (1988). 3. Hall, P.F., Testicular Steroid Synthesis: Organization and Regulation. In: The Physiology of Reproduction, Ed.: Knobil, E., and Neill, J. et al., pp 975-98. Raven Press, New York (1988). 4. Siiteri, P.K. Murai, J.T., Hammond, G.L., Nisker, J.A., Raymoure, W.J. and Kuhn, R.W., The serum transport of steroid hormones, Rec. Prog. Horm. Res. 38:457 - 510 (1982). 5. Martin, B., Rotten, D., Jolivet, A. and Gautray, J-P-. Binding of steroids by proteins in follicular fluid of the human ovary, J.Clin. Endicrinol. Metab. 35: 443-47 (1981). 6. Baird, D.T., Ovarian steroid secretion and metabolism in women. In: The Endocrine Function of the Human Ovary. Eds.: James, V.H:T., Serio, M. and Giusti, G. pp. 125-33, Academic Press, New York (1976). 7. McNastty, K.P., Baird, D.T., Bolton, a., Chambers, P., Corker, C.S. and McLean, H., concentration of oestrogens and androgens in human ovarian venous plasma and follicular fluid throughout the menstrual cycle, J. Endocrinol. 71:77-85 (1976). 8. Abraham, G.E., Odell, W.D., Swerdloff, R.S., and Hopper, K., Simultaneous radioimmunoassay of plasma FSH, LH, progesterone, 17-hydroxyprogesterone and estradiol-17ß during the menstrual cycle, J.Clin. Endocrinol. Metab. 34:312-18 (1972). 9. March, C.M., Goebelsmann, U., Nakumara, R.M., and Mishell, D.R., Roles of oestradiol and progesterone in eliciting midcycle luteinising hormone and follicle stimulating hormone surges. J. clin. Endicrinol. Metab. 49:507-12 (1979). 10. Simpson, E.R., and McDonald, P.C., Endocrinology of Pregnancy. In: Textbook of Endocrinology, Ed.: Williams, R.H. pp412-22, Saunders Company, Philadelphia (1981). 11. Jenner, M.R., Kelch, R.P., et al., Hormonal Changes in prepubertal children, pubertal females and in precocious puberty, premature thelarche, hypogonadism and in a child with feminising tumour, J. clin. Endocrinol. 34: 521 (1982). 12. Goldstein, D. et al., Correlation between estradiol and progesterone in cycles with luteal phase deficiency, Fertil. 13. Kirschner, M.A., The role of hormones in the etiology of human breast cancer, Cancer 39:2716 26 (1977). 14. Odell, W.D. and Swerdloff, R.D., Abnormalities of gonadal function in men, 15. McDonald, P.C., Madden, J.C., Brenner, P.F., Wilson, J.D. and Siiteri, P.K. Origin of oestrogen in normal men and women with testicular feminisation, J.Clin. Endcrinol. Metabol. 49:905 (1979). 16. Peckham, M.J: and McElwain, T.J:, Testicular tumours, J.Clin. Endocrinol. Metab. 4:665-692 (1975). 17. Taubert, H.d. and Dericks-Tan, J.S. E., Induction for ovulation by clomiphene citrate in combination with high doses of estrogens or nasal application of LH-RH. In: Ovulation in the Human. Eds.: Crosignandi, P.G. and Mishell, D.R., pp.265-73, Academic Press, New York (1976). DRG International, Inc., USA Fax: (908) 233 0758 e-mail: [email protected] 11
DRG® Estradiol ELISA (EIA-2693)
REVISED 23 MAY 2005
18. Fishel, S.B., Edwards, R.G., Purdy, J.M., Steptoe, P.C., Webster, J. Walters, E., cohen, J. Fehilly, C. Hewitt, J., and
Rowland, G., Implantation, abortion and birth after in-vitro fertilisation using the natural menstrual cycle or follicular stimulation with clomiphene citrate and human menopausal gonadotropin, J. In Vitro Fertil. Embryo Transfer, 1:24-28 (1985). 19. Wramsby, H., Sundstorm, P- and Leidholm, P., Pregnancy rate in relation to number of cleaved eggs replaced after in vitro-fertilisation of stimulating cycles monitored by serum levels of oestradiol and progesterone as sole index. Human Reproduction 2: 325-28 (1987). 20. Ratcliff, W.A., Carter, G.D., et al., Estradiol assays: applications and guidelines for the provision of clinical biochemistry service, Ann. Clin. Biochem. 25:466-483 (1988). 21. Tietz, N.W. Textbook of Clinical Chemistry. Saunders, 1986. DRG International, Inc., USA Fax: (908) 233 0758 e-mail: [email protected] 12

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