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the color formed is proportional to the amount of enzyme present and is inversely
For the quantitative determination of estradiol (E2) concentration in human
related to the amount of unlabeled E2 in the sample. A standard curve is obtained
serum. For in vitro
diagnostic use only.
by plotting the concentration of the standard versus the absorbance. The E2
concentration of the specimens and controls run concurrently with the standards
can be calculated from the standard curve.
Estradiol (E2) 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 ovary, placenta, and in smaller amounts by
Materials provided with the kit:
the adrenal cortex, and the male testes. 1,2,3
1. Goat Anti-Rabbit IgG-coated microtiter wells, 96 wells
Estradiol (E2) is secreted into the blood stream where 98% of it circulates
2. Estradiol Reference Standards: 0, 10, 30, 100, 300, and 1000 pg/ml. Liquid,
bound to sex hormone binding globulin (SHBG). To a lesser extent it is
bound to other serum proteins such as albumin. Only a tiny fraction
3. Rabbit Anti-Estradiol Reagent (pink color), 7 ml
circulates as free hormone or in the conjugated form.4,5 Estrogenic activity is
4. Estradiol-HRP Conjugate Reagent (blue color), 12 ml
affected via estradiol-receptor complexes that trigger the appropriate
5. Estradiol Control 1, Liquid, 0.5 ml, Ready to use.
response at the nuclear level in the target sites. These sites include the
6. Estradiol Control 2, Liquid, 0.5 ml, Ready to use.
follicles, uterus, breast, vagina, urethra, hypothalamus, pituitary and to a
In non-pregnancy women with normal menstrual cycles, estradiol secretion
follows a cyclic, biphasic pattern with the highest concentration found
Materials required but not provided:
immediately prior to ovulation.6,7 The rising estradiol concentration is
1. Precision pipettes: 25 μl, 50 μl, 100μl, 200μl, and 1.0 ml
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
Warnings and Precautions for Users
Test methods are not available which can offer complete assurance that Hepatitis
Serum estradiol measurements are a valuable index in evaluating a variety
B virus, Human Immunodeficiency Virus (HIV/HTLV-III/LAV), or other infectious
of menstrual dysfunctions such as precocious or delayed puberty in girls11
agents are absent from the reagents in this kit. Therefore, all human blood
and primary and secondary amenorrhea and menopause.12 Estradiol levels
products, including patient samples, should be considered potentially infectious.
have been reported to be increased in patients with feminizing syndromes14,
Handling and disposal should be in accordance with the procedures defined by an
gynaecomastia15 and testicular tumors.16
appropriate national biohazard safety guideline or regulation, where it exists (e.g.,
In cases of infertility, serum estradiol measurements are useful for monitoring
USA Center for Disease Control/National Institute of Health Manual, “Biosafety in
induction of ovulation following treatment with, for example, clomiphene
Microbiological and Biomedical Laboratories,” 1984). 22
citrate, LH-releasing hormone (LH-RH), or exogenous gonadotropins.
During ovarian hyperstimulation for in vitro fertilization (IVF), serum estradiol
Specimen Collection and Preparation
concentrations are usually monitored daily for optimal timing of human
chorionic gonadotropin (hCG) administration and oocyte collection.19
2. No special pretreatment of sample is necessary.
3. Serum samples may be stored at 2-8°C for up to 24 hours, and should be
Principle of the Test
frozen at −10°C or lower for longer periods. Do not use grossly hemolyzed
The E2 EIA is based on the principle of competitive binding between E2 in
the test specimen and E2-HRP conjugate for a constant amount of rabbit
4. Please note:
Samples containing sodium azide should not be used in the
anti-estradiol. In the incubation, goat anti-rabbit IgG-coated wells are
incubated with 25μl E2 standards, controls, patient samples, 100μl estradiol-
Storage of Test Kit and Instrumentation
HRP conjugate reagent, and 50μl rabbit anti-estradiol reagent at room
temperature (18-25°C) for 90 minutes. During the incubation, a fixed amount
Unopened test kits should be stored at 2-8°C upon receipt and the microtiter plate
of HRP-labeled E2 competes with the endogenous E2 in the standard,
should be kept in a sealed bag with desiccants to minimize exposure to damp air.
sample, or quality control serum for a fixed number of binding sites of the
Opened test kits will remain stable until the expiration date shown, provided it is
specific E2 antibody. Thus, the amount of E2 peroxidase conjugate
stored as described above. A microtiter plate reader with a bandwidth of 10nm or
immunologically bound to the well progressively decreases as the
less and an optical density range of 0-3 O.D. at 450nm wavelength is acceptable
concentration of E2 in the specimen increases.
Unbound E2 peroxidase conjugate is then removed and the wells washed.
Next, a solution of TMB Reagent is added and incubated at room
temperature for 20 minutes, resulting in the development of blue color. The
1. All reagents should be brought to room temperature (18-25°C) before use.
color development is stopped with the addition of 1N HCl, and the
2. Samples with expected estradiol concentrations over 1000 pg/ml may be
absorbance is measured spectrophotometrical y at 450nm. The intensity of
quantitated by dilution with diluent available from your vendor.
Phone: 734-487-8300 • Toll Free: 800-445-9853 • Fax: 734-483-1592 • www.pointescientific.com
1. Secure the desired number of coated wells in the holder.
Dispense 25 μl of standards, specimens and controls into appropriate
3. Dispense 100 μl of estradiol-HRP conjugate reagent into each well.
4. Dispense 50μl of rabbit anti-estradiol (E2) reagent to each well.
5. Thoroughly mix for 30 seconds. It is very important to mix
Incubate at room temperature (18-25°C) for 90 minutes.
7. Rinse and flick the microwells 5 times with distilled or deionized
water (Do not use tap water).
8. Dispense 100 μl of TMB Reagent into each well. Gently mix for 10
Estradiol Conc. (pg/ml)
9. Incubate at room temperature (18-25°C) for 20 minutes.
Expected Values and Sensitivity
10. Stop the reaction by adding 100μl of Stop Solution to each well.
Each laboratory should establish its own normal range based on the patient
10. Gently mix 30 seconds. It is important to make sure that all the blue
population. The estradiol EIA was performed on randomly selected outpatient
color changes to yellow color completely.
clinical laboratory samples. The results of these determinations are as follows:
11. Read absorbance at 450 nm with a microtiter well reader within 15
Calculation of Results
1. Calculate the mean absorbance value (A450) for each set of reference
2. Construct a standard curve by plotting the mean absorbance obtained
for each reference standard against its concentration in pg/ml on a
linear-linear graph paper
, with absorbance values on the vertical or Y
axis, and concentrations on the horizontal or X axis.
Use the mean absorbance values for each specimen to determine the corresponding concentration of estradiol in pg/ml from the standard
The minimum detectable concentration of the estradiol ELISA assay as measured
by 2SD from the mean of a zero standard is estimated to be 10 pg/ml.
Any values obtained for diluted samples must be further converted by applying the appropriate dilution factor in the calculations.
Example of Standard Curve
Within-run precision was determined by replicate determinations of four
Results of a typical standard run with optical density readings at 450nm
different serum samples in one assay. Within-assay variability is shown
shown in the Y axis against estradiol concentrations shown in the X axis.
This standard curve is for the purpose of illustration only, and should
not be used to calculate unknowns. Each laboratory must provide its own
data and standard curve in each experiment.
Between-run precision was determined by replicate measurements of four
different serum samples over a series of individually calibrated assays.
Between-assay variability is shown below:
Assessment of Excessive Estrogen Production in Women:
Various patient serum samples of known estradiol levels were
In pregnant women, E2 concentration will be >1,000 pg/ml. In non-
combined and assayed in duplicate. The mean recovery was 101.3%.
pregnant women, excessive estrogen may indicate ovarian neoplasms.
E2 is often measured to monitor ovulation induction and for patient follow-
up during fertility therapy, e.g. in vitro fertilization (IVF).
E2 measurement is used in the differential diagnosis of gynecomastia,
feminizing syndromes, hypogonadism and testicular tumors.
112.6 Limitations of the Procedure
Reliable and reproducible results will be obtained when the assay
procedure is carried out with a complete understanding of the package
insert instructions and with adherence to good laboratory practice.
The wash procedure is critical. Insufficient washing will result in poor
The following materials have been checked for cross reactivity. The
precision and falsely elevated absorbance readings.
percentage indicates cross reactivity at 50% displacement compared to
Serum samples demonstrating gross lipemia, gross hemolysis, or turbidity
The results obtained from the use of this kit should be used only as an
Data on the cross-reactivity for several endogenous and
adjunct to other diagnostic procedures and information available to the
pharmaceutical steroids are summarized in the following table:
Cross-reactivity (%) = Observed Estradiol Concentration × 100
Good laboratory practice requires that controls are run with each calibration curve.
A statistically significant number of controls should be assayed to establish mean
values and acceptable ranges to assure proper performance.
We recommend using Bio-Rad Lyphocheck Immunoassay Control Sera as
controls. The estradiol EIA kit includes internal controls, Level 1 and 2.
1. Tsang, B.K., Armstrong, D.T. and Whitfield, J.F., Steroid biosynthesis by
isolated human ovarian follicular cells in vitro
, J. Clin. Endocrinol. Metab.
2. Gore-Langton, R.E. and Armstrong, D.T., Follicular steroidogenesis and its
control. In: The Physiology of Reproduction, Ed.: Knobil, E., and Neill, J. et
al., pp. 331-385. Raven Press, New York (1988)
Hall, P.F., Testicular Steroid Synthesis: Organization and Regulation. In: The Physiology of Reproduction, Ed.: Knobil, E., and Neill, J. et al., pp 975-998.
4. Siiteri, P.K. Murai, J.T., Hammond, G.L., Nisker, J.A., Raymoure, W.J. and
Information is cited from reference #19.
Kuhn, R.W., The serum transport of steroid hormones, Rec. Prog. Horm.
Assessment of Female Menstrual Dysfunctions
5. 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
Elevated E2 can be used in the evaluation of precocious puberty in
Giusti, G. pp 125-133, Academic Press, New York (1976)
girls. However, extensive ancillary aids are required for specific
6. McNatty, 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.
E2 measurements are frequently utilized in the assessment of
7. Abraham, G.E., Odell, W.D., Swerdloff, R.S., and Hopper, K., Simultaneous
hypoestrogenism in cases of delayed puberty, primary and secondary
radioimmunoassay of plasma FSH, LH, progesterone, 17-
amenorrhea, and menopause. In hypoestrogenism women, E2
hydroxyprogesterone and estradiol-17β during the menstrual cycle, J.Clin.
concentrations are usually <30 pg/ml.
8. March, C.M., Goebelsmann, U., Nakumara, R.M., and Mishell, D.R., Roles of
Oestradiol and progesterone in eliciting midcycle luteinizing hormone and
follicle stimulating hormone surges. J. Clin. Endocrinol. Metab. 49:507-513
Phone: 734-487-8300 • Toll Free: 800-445-9853 • Fax: 734-483-1592 • www.pointescientific.com
9. Simpson, E.R., and McDonald, P.C., Endocrinology of Pregnancy. In:
Textbook of Endocrinology, Ed.: Williams, R.H., pp. 412-422, Saunders Company, Philadelphia (1981).
10. Jenner, M.R., Kelch, R.P., et al., Hormonal changes in prepubertal
children, pubertal females and in precocious puberty, premature
thelarche, hypergonadoism and in a child with feminizing tumor, J. Clin.
11. Goldstein, D. et al., Correlation between estradiol and progesterone in
cycles with luteal phase deficiency, Fertil. Steril. 37:348-354 (1982)
12. Kirschner, M.A., The role of hormones in the etiology of human breast
13. Odell, W.D. and Swerdloff, R.D., Abnormalities of gonadal function in
14. McDonald, P.C., Madden, J.C., Brenner, P.F., Wilson, J.D. and Siiteri,
P.K. Origin of estrogen in normal men and women with testicular feminization, J.Clin. Endocrinol. Metab., 49:905-916 (1979).
15. Fishel, S.B., Edwards, R.G., Purdy, J.M., Steptoe, P.C., Webster, J.
Walters, E., Cohen, J. Fehiliy, C. Hewitt, J., and Rowland, G., Implantation, abortion and birth after in vitro
fertilization using the natural menstrual cycle or follicular stimulation with clomiphene citrate and human menopausal gonadotropin, J. In Vitro Fertil. Embryo Transfer, 2: 123-131 (1985).
16. 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).
17. Tietz, N.W. ed., Clinical Guide to Laboratory Tests, 3rd Edition, W.B.
Saunders, Co., Philadelphia, 1995: 216-217.
18. USA Center for Disease Control/National Institute of Health Manual,
“Biosafety in Microbiological and Biomedical Laboratories”, 1984.
19. ICN Guide to Endocrine Testing. Diagnostic Division, ICN Biomedicals,
Manufactured for Pointe Scientific, Inc.
5449 Research Drive, Canton, MI 48188 European Authorized Representative: Obelis s.a. Boulevard Général Wahis 53 1030 Brussels, BELGIUM Tel: (32)2.732.59.54 Fax: (32)2.732.60.03 email: [email protected]
Rev. 5/10 P803-S1111-01
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Thresholds for therapies: highlights of the St GallenInternational Expert Consensus on the Primary Therapyof Early Breast Cancer 2009A. Goldhirsch1,2*, J. N. Ingle3, R. D. Gelber4, A. S. Coates5, B. Thu¨rlimann6, H.-J. Senn7& Panel members 1International Breast Cancer Study Group, Oncology Institute of Southern Switzerland, Bellinzona, Switzerland; 2European Institute of Oncology, Milan, It