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Serum Estradiol measurements are a valuable index in evaluating a variety of menstrual dysfunctions such as precocious or delayed puberty in girls (11) and AccuDiag™
primary and secondary amenorrhea and menopause (12). Estradiol levels have been reported to be increased in patients with feminizing syndromes (14), Estradiol
gynaecomastia (15) and testicular tumors (16). ELISA
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). Cat# 2046-18
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). Diagnostic Automation, Inc. Estradiol (E2) EIA kits are designed for the measurement of total Estradiol in human serum. TEST PRINCIPLE
Estradiol ELISA
Enzyme Linked
The Diagnostic Automation, Inc. E2 EIA is based on the principle of competitive binding between E2 in the test specimen and E2-HRP conjugate Immunosorbent Assay
for a constant amount of rabbit anti-Estradiol. In the incubation, goat anti- Principle
Competitive Immunoassay
rabbit IgG-coated wells are incubated with 25 µl E2 standards, controls, patient Detection Range
0-1000 pg/mL
samples, 100 µl Estradiol-HRP Conjugate Reagent and 50 µl rabbit anti- 25µL serum
Estradiol reagent at room temperature (18-25°C) for 90 minutes. During the Specificity
incubation, a fixed amount of HRP-labeled E2 competes with the endogenous E2 in the standard, sample, or quality control serum for a fixed number of Sensitivity
binding sites of the specific E2 antibody. Thus, the amount of E2 peroxidase Total Time
conjugate immunologically bound to the well progressively decreases as the 12-14 Months from the
concentration of E2 in the specimen increases. Shelf Life
manufacturing date
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 color development is stopped with the addition of 1N HCl, and the absorbance is INTENDED USE
measured spectrophotometrically at 450 nm. The intensity of the color formed is proportional to the amount of enzyme present and is inversely related to the For the quantitative determination of Estradiol (E2) concentration in human amount of unlabeled E2 in the sample. A standard curve is obtained 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. SUMMARY AND EXPLANATION
Estradiol (E2) is a C18 steroid hormone with a phenolic A ring. This steroid SPECIMEN COLLECTION AND PREPARATION
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 the adrenal cortex, and the male testes (1,2,3). 2. No special pretreatment of sample is necessary. Estradiol (E2) is secreted into the blood stream where 98% of it circulates 3. Serum samples may be stored at 2-8°C for up to 24 hours, and should be bound to sex hormone binding globulin (SHBG). To a lesser extent it is bound frozen at −20°C or lower for longer periods. Avoid grossly hemolytic to other serum proteins such as albumin. Only a tiny fraction circulates as free (bright red), lipemic (milky), or turbid samples. hormone or in the conjugated form (4,5). Estrogenic activity is effected via 4. Please note: Samples containing sodium azide should not be used in the
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 PROCEDURAL NOTES
1. Manual Pipetting: It is recommended that no more than 32 wells be used for each assay run. Pipetting of all standards, samples, and controls should be In non-pregnant women with normal menstrual cycles, estradiol secretion follows a cyclic, biphasic pattern with the highest concentration found 2. Automated Pipetting: A full plate of 96 wells may be used in each assay immediately prior to ovulation (6,7). The rising estradiol concentration is run. However, it is recommended that pipetting of all standards, samples, understood to exert a positive feedback influence at the level of the pituitary and controls be completed within 3 minutes. where it influences the secretion of the gonadotropins, follicle stimulating 3. All standards, samples, and controls should be run in duplicate concurrently hormone (FSH), and luteinizing hormone (LH), which are essential for so that all conditions of testing are the same. follicular maturation and ovulation, respectively (8,9). Following ovulation, 4. It is recommended that the wells be read within 15 minutes following 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 MATERIALS AND COMPONENTS
Materials provided with the test kits
1. Calculate the mean absorbance value (A450) for each set of reference 1. Antibody-Coated Wells (1 plate, 96 wells) Microtiter wells coated with goat anti-rabbit IgG 2. Construct a standard curve by plotting the mean absorbance obtained for 2. Reference Standard Set (0.5 ml/vial) each reference standard against its concentration in pg/ml on a linear-linear
Contains 0, 10, 30, 100, 300 and 1,000 pg/ml estradiol in human serum graph paper, with absorbance values on the vertical or Y axis, and
with preservatives, liquid, ready to use concentrations on the horizontal or X axis. 3. Rabbit Anti-Estradiol Reagent (7 ml) 3. Use the mean absorbance values for each specimen to determine the Contains rabbit anti-estradiol in bovine serum albumin (BSA) buffer with corresponding concentration of Estradiol in pg/ml from the standard curve. 4. Any values obtained for diluted samples must be further converted by 4. Estradiol-HRP Conjugate Reagent (12 ml) applying the appropriate dilution factor in the calculations. 5. Estradiol Control 1 and 2 (0.5 ml/vial) EXAMPLE OF STANDARD CURVE
Contains approximately 35 and 350 pg/ml estradiol, respectively, in human serum, liquid, 0.5 ml each, ready to use Results of a typical standard run with optical density readings at 450 nm shown in the Y axis against Estradiol concentrations shown in the X axis. Note: This
Contains 3, 3’, 5, 5’-TMB stabilized in buffer solution 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 Estradiol (pg/ml)
Absorbance (450 nm)
Materials required but not provided
1. Precision pipettes: 25 µl, 50 µl, 100 µl, 200 µl, and 1.0 ml. 2. Disposable pipette tips. 4. Vortex mixer or equivalent. 5. Absorbent paper or paper towel. REAGENT PREPARATION
1. All reagents should be allowed to reach room temperature (18-25°C) before 2. All reagents should be mixed by gentle inversion or swirling prior to use. 3. Samples with expected testosterone concentrations over 1,000 pg/ml may be quantitated by dilution with diluent available from Diagnostic Estradiol Conc. (pg/ml)
ASSAY PROCEDURE
1. Secure the desired number of coated wells in the holder. EXPECTED VALUES
2. Dispense 25 µl of standards, specimens and controls into appropriate wells. 3. Dispense 100 µl of Estradiol-HRP Conjugate Reagent into each well. Each laboratory should establish its own normal range based on the patient 4. Dispense 50 µl of rabbit anti-Estradiol (E2) reagent to each well. population. Diagnostic Automation, Inc. Estradiol EIA was performed on 5. Thoroughly mix for 30 seconds. It is very important to mix them
randomly selected outpatient clinical laboratory samples. The results of these completely.
6. Incubate at room temperature (18-25°C) for 90 minutes. 7. Rinse and flick the microwells 5 times with distilled or deionized water. 8. Dispense 100 µl of TMB Reagent into each well. Gently mix for 10 seconds. 9. Incubate at room temperature (18-25°C) for 20 minutes. 10. Stop the reaction by adding 100 µl of Stop Solution to each well.
11. Gently mix 30 seconds. It is important to make sure that all the blue
color changes to yellow color completely.
12. Read absorbance at 450 nm with a microtiter well reader within 15
minutes.
PERFORMANCE CHARACTERISTICS
IV. Linearity Study
I. Accuracy
Four patient samples were serially diluted to determine linearity. The A statistical study using 80 human serum samples demonstrated good correlation with a commercially available kit as shown below. Expected
Observed
Comparison between the Diagnostic Automation, Inc. Estradiol EIA and Dilution
Expected
the DRG Estradiol kit provided the following data: Undilute
Sensitivity
Mean = 88.4%
The minimum detectable concentration of the Diagnostic Undilute
Automation, Inc. Estradiol ELISA assay as measured by 2 SD from the mean of a zero standard is estimated to be 10 pg/ml. III. Precision
Within-run precision was determined by replicate determinations of four different serum samples in one assay. Within-assay variability Mean = 87.4%
Undilute
Mean = 100.8%
Between-run precision was determined by replicate measurements of four different serum samples over a series of individually calibrated Undilute
assays. Between-assay variability is shown below: Mean = 89.8%
Recovery Studies
3. Serum samples demonstrating gross lipemia, gross hemolysis, or turbidity Various patient serum samples of known Estradiol levels were combined 4. The results obtained from the use of this kit should be used only as an and assayed in duplicate. The mean recovery was 101.0%. adjunct to other diagnostic procedures and information available to the EXPECTED
CLINICAL APPLICATION
[Estradiol]
RECOVERY
[Estradiol]
Information is cited from reference # 19 Assessment of Female Menstrual Dysfunctions:
Hyperestrogenism in girls:
Elevated E
2 can be used in the evaluation of precocious puberty in girls. However, extensive ancillary aids are required for specific E2 measurements are frequently utilized in the assessment of hypoestrogenism in cases of delayed puberty, primary and secondary amenorrhea, and menopause. In hypoestrogenism women, E2
VI. Specificity
concentrations are usually <30 pg/ml.
Assessment of Excessive Estrogen Production in Women:
The following materials have been checked for cross reactivity. The In pregnant women, E2 concentrations will >1,000 pg/ml. In non-
percentage indicates cross reactivity at 50% displacement compared to pregnant women, excessive estrogen may indicate ovarian neoplasms. Monitoring Ovulation:
E2 is often measured to monitor ovulation induction and for patient Data on the cross-reactivity for several endogenous and pharmaceutical follow-up during infertility therapy, e.g. in vitro fertilization (IVF). steroids are summarized in the following table: Estradiol Measurement in Male:
E2 measurement is used in the differential diagnosis gynecomastia, Cross-reactivity (%) = Observed Estradiol Concentration × 100 feminizing syndromes, hypogonadism and testicular tumors. Cross-Reactivity
PRECAUTIONS
1. CAUTION: This kit contains human material. The source material used
for manufacture of this kit tested negative for HBsAg, HIV 1/2 and HCV by FDA-approved methods. However, no method can completely assure absence of these agents. Therefore, all human blood products, including serum samples, should be considered potentially infectious. Handling and disposal should be as defined by an appropriate national biohazard safety guideline or regulation, where it exists.21 2. Do not use reagents after expiration date and do not mix or use components 3. Do not use the reagent when it becomes cloudy or contamination is 4. Do not use the reagent if the vial is damaged. 5. Replace caps on reagents immediately. Do not switch caps. 6. Each well can be used only once. 7. Do not pipette reagents by mouth. QUALITY CONTROL
8. Solutions containing additives or preservatives, such as sodium azide, should not be used in the enzyme reaction. Good laboratory practice requires that controls are run with each calibration 9. Avoid contact with 1N HCl. It may cause skin irritation and burns. If curve. A statistically significant number of controls should be assayed to contact occurs, wash with copious amounts of water and seek medical establish mean values and acceptable ranges to assure proper performance. attention if irritation persists. 10. For in vitro diagnostic use. We recommend using Bio-Rad Lyphochek Immunoassay Control Sera as controls. Diagnostic Automation, Inc. Estradiol EIA kit also provides with internal controls, Level 1 and 2. 1. Store the unopened kit at 2-8°C upon receipt and when it is not in use, until LIMITATIONS OF THE PROCEDURE
the expiration shown on the kit label. Refer to the package label for the expiration date. 1. Reliable and reproducible results will be obtained when the assay procedure 2. The opened and used reagents are stable until the expiration date if stored is carried out with a complete understanding of the package insert instructions and with adherence to good laboratory practice. 3. Keep microtiter plate in a sealed bag with desiccant to minimize exposure 2. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings. 18. USA Center for Disease Control/National Institute of Health Manual, INSTRUMENTATION
“Biosafety in Microbiological and Biomedical Laboratories”, 1984. 19. ICN Guide to Endocrine Testing. Diagnostic Division, ICN Biomedicals, A microtiter well reader with a bandwidth of 10 nm or less and an optical density range (OD) of 0 to 3 OD or greater at 450 nm wavelength is acceptable for absorbance measurement. REFERENCES
1. Tsang, B.K., Armstrong, D.T. and Whitfield, J.F., Steroid biosynthesis by ISO 13485
isolated human ovarian follicular cells in vitro, J. Clin. Endocrinol. 2. Gore-Langton, R.E. and Armstrong, D.T., Follicular steroidogenesis and its control. In: Knobil, E., and Neill, J. et al., ed. The Physiology of Reproduction. Raven Press, New York; 1988: 331-385. 3. Hall, P.F., Testicular steroid synthesis: Organization and regulation. In: Knobil, E., and Neill, J. et al., ed. The Physiology of Reproduction. Raven Diagnostic Automation/
4. Siiteri, P.K., Murai, J.T., Hammond, G.L., Nisker, J.A., Raymoure, W.J. Cortez Diagnostics, Inc.
and Kuhn, R.W., The serum transport of steroid hormones, Rec. Prog. 23961 Craftsman Road, Suite E/F,
Calabasas, California 91302 USA
5. Baird, D.T., Ovarian steroid secretion and metabolism in women. In: Date Adopted
Cat # 2046-18
James, V.H.T., Serio, M. and Giusti, G., eds. The Endocrine Function of AccuDiag™- Estradiol
the Human Ovary. Academic Press, New York; 1976: 125-133. 2007-07-05
ELISA-2013
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 CEpartner4U, Esdoornlaan 13,
venous plasma and follicular fluid throughout the menstrual cycle, J. 3951DB Maarn. The Netherlands.
7. Abraham, G.E., Odell, W.D., Swerdloff, R.S., and Hopper, K., Simultaneous radioimmunoassay of plasma FSH, LH, progesterone, 17- hydroxyprogesterone and estradiol-17β during the menstrual cycle, J. Clin. Endocrinol. Metab., 1972; 34: 312-318. 8. March, C.M., Goebelsmann, U., Nakumara, R.M., and Mishell, D.R. Jr., Roles of estradiol and progesterone in eliciting the midcycle luteinizing hormone and follicle-stimulating hormone surges. J. Clin. Endocrinol. Metab., 1979; 49: 507-513. 9. Simpson, E.R., and MacDonald, P.C., Endocrinology of pregnancy. In: Williams, R.H., ed., Textbook of Endocrinology. Saunders Company, Philadelphia; 1981: 412-422. 10. Jenner, M.R., Kelch, R.P., Kaplan, S.L. and Grumbach, M.M., Hormonal changes in puberty: IV. Plasma estradiol, LH, and FSH in prepubertal children, pubertal females and in precocious puberty, premature thelarche, hypogonadism and in a child with feminizing ovarian tumor. J. Clin. Endocrinol. Metab., 197 2; 34: 521-530. 11. Goldstein, D., Zuckerman, H., Harpaz, S., et al., Correlation between estradiol and progesterone in cycles with luteal phase deficiency. Fertil. Steril., 1982; 37: 348-354. 12. Kirschner, M.A., The role of hormones in the etiology of human breast cancer. Cancer, 1977; 39: 2716-2726. 13. Odell, W.D. and Swerdloff, R.S., Abnormalities of gonadal function in men. Clin. Endocr., 1978; 8: 149-180. 14. MacDonald, P.C., Madden, J.D., Brenner, P.F., Wilson, J.D. and Siiteri, P.K., Origin of estrogen in normal men and in women with testicular feminization, J.Clin. Endocrinol. Metabl., 1979; 49: 905-916. 15. 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 fertilization using the natural menstrual cycle or follicular stimulation with clomiphene citrate and human menopausal gonadotropin, J. In Vitro Fertil. Embryo Transfer, 1985; 2: 123-131. 16. Ratcliffe, W.A., Carter, G.D., Dowsett, M., et al., Oestradiol assays: applications and guidelines for the provision of a clinical biochemistry service, Ann. Clin. Biochem., 1988; 25:466-483. 17. Tietz, N.W. ed., Clinical Guide to Laboratory Tests, 3rd Edition, W.B. Saunders, Co., Philadelphia, 1995: 216-217.

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