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E2-easia - 080522-1.docRead entire protocol before use.
I. INTENDED USE
The BioSource E2-EASIA is a competitive binding immunoassay for the quantitative determination of estradiol in serum and plasma. II. GENERAL INFORMATION
Proprietary name :
Catalogue number :
Manufactured by :
BioSource Europe S.A. Rue de l'Industrie, 8, B-1400 Nivelles, Belgium. For technical assistance or ordering information contact :
Tel : +32 (0)67 88.99.99
Fax : +32 (0)67 88.99.96
17-beta-estradiol (E2) is a C-18 steroid hormone (molecular weight 272.4) produced mainly by the ovary and placenta, and in small amounts by adrenals and testes. Estradiol is in equilibrium with estrone, which can be converted to estriol by the liver and placenta. B.
Clinical applications of the measurement of E2 levels
Like LH, FSH and progesterone, measurement of estradiol concentration in serum, peritoneal fluid and follicular fluid is an essential biochemical tool for the investigation of fertility, tumor and sexual diseases and disorders of hypothalamic/pituitary/gonadal axis. to check the effectiveness of the induction of ovulation (with ultrasound) and the level of E2 in follicular fluid. It allows normal detection or dysfunctional ovulation induction (the empty follicle synfrome may reflect a dysfonctional ovulation induction); to diagnose the luteinized unruptured follicle (LUF) syndrome (by the estimation of 17 beta-estradiol and progesterone levels in peritoneal fluid); to aid in the diagnosis of breast tumors (total estrogens – E1-E2 – and 17 beta-hydroxysteroïd dehydrogenase activity are significantly higher in malignant than in non malignant breast tissues); other areas of investigation are: premature adrenarche, gynecomastie and menopausal period. IV. PRINCIPLE OF THE TEST
frozen. Three freezing-thawing cycles are allowed. • Store the unused strips at 2 - 8°C in the closed bag containing the E2-EASIA is an enzyme immunoassay performed in a microtiter plate. A fixed amount of estradiol labeled with horseradish peroxidase (HRP) • The concentrated wash solution is stable at room temperature until competes with unlabeled estradiol present in calibrators or samples for expiration date. Prepare fresh diluted wash solution each day. a limited number of binding sites of a specific antibody. The • The freshly prepared substrate solution is stable for a maximum of E2-HRP-antibody complex is simultaneously fixed on the wells of the 15 minutes at room temperature and must be discarded after use. microtiter plate coated with an excess of anti-rabbit-gammaglobulins. Neither extraction nor chromatography are required due to the high specificity of the antibody. VII. SUPPLIES REQUIRED BUT NOT PROVIDED
After 2 hours incubation at room temperature the microtiter plate is 1. Microtiter plate reader capable of measurement at or near 450 nm. washed to stop the competition reaction. 2. Calibrated adjustable precision pipettes, preferably with disposable plastic tips. (A manifold multi-channel pipette is desirable for large The substrate solution (tetramethylbenzidine (TMB) – H202) is added and incubated for 30 minutes. The reaction is stopped with H2S04 and the absorbance is measured at the appropriate wavelength. The amount of substrate turnover is inversely proportional to the estradiol 4. Plate washer: automated or manual (squirt bottle, manifold concentration in the sample. A calibration curve is plotted and estradiol 5. Data analysis and graphing software. Graph paper: linear concentrations in samples are determined by interpolation from the calibration curve. (Cartesian), log-log, or semi-log, as desired. 6. Calibrated beakers and graduated cylinders in various sizes. V. REAGENTS PROVIDED
• The human blood components included in this kit have been tested Reagent
and found non reactive for HBsAg and anti-HIV. Nevertheless, no Microtiter Plate with 96 wells 96 wells known method can offer complete assurance that human blood derivatives will not transmit hepatitis, AIDS or other infections. Therefore, handling of reagents, serum or plasma specimens should be in accordance with local safety procedures, e.g., CDC/HIH Health Manual: “Biosafety in Microbiological and Biomedical • Do not eat, drink, smoke or apply cosmetics where kit reagents are IX. PROCEDURAL NOTES/LAB QUALITY CONTROL
• Do not use kit components beyond the expiration date. • Do not mix materials from different kit lots. • Do not mix strips from different plates. Bring all the reagents and specimens to room temperature • Thoroughly mix the reagents and samples before use by gentle • Use a clean disposable plastic pipette tip for each reagent, calibrator, control or specimen addition in order to avoid solution, avoid pipettes with metal parts. • Use a clean plastic container to prepare the wash solution. The TMB solution in substrate buffer should be colorless. A dark blue color indicates that the reagent is unusable and must be Note: Calibrator 0 pg/mL is recommended for sample dilutions.
• During incubation with substrate solution, avoid direct sunlight on VI. STORAGE AND STABILITY
• Follow the incubation times described in the assay procedure. Store the kit at 2 - 8°C until expiration date printed on the kit label. Dispense the substrate solution within 15 minutes following the Once opened, store the concentrated estradiol-HRP conjugate vial at 2 - 8°C. Diluted estradiol-HRP conjugate is stable for 4 hours at room temperature or 24 hours at 2 - 8°C when protected from direct • After reconstitution, store anti-estradiol, calibrators and controls at 2 - 8°C for 1 week maximum. For prolonged storage they must be X. SAMPLE COLLECTION AND PREPARATION
XIII. CALCULATIONS AND RESULTS INTERPRETATION
• No special pretreatment of the sample is necessary. Prior to use, all • Calculate the mean absorbance of duplicate determinations, the samples should be at room temperature. It’s recommended to • For each calibrator and sample calculate the percent bound: B/Bo x 100 = OD (calibrator or sample) x 100 • Samples may be stored for up to 72 hours at 2 - 8°C prior to testing. Samples held for longer time should be frozen at -20°C prior to • Using either linear-linear or semi-log graph paper, plot the (B/BO x 100) values for each calibrator point as a function of the estradiol • Serum, heparinized plasma or EDTA plasma provide similar • By interpolation of the samples (B/Bo x 100) values, determine the estradiol concentrations of the samples from the reference curve. If using curve fitting software, the four-parameter algorithm provides XI. REAGENT PREPARATION
• Calibrators and Controls:
XIV. EXAMPLE OF TYPICAL REFERENCE CURVE
Reconstitute the lyophilized calibrators and controls to the volume The following data are for demonstration purposes only and can not be specified on the vial label with distilled water (4 mL for the zero used in place of data generated at the time of assay. calibrator and 0.5 mL for the other calibrators and controls). Allow them to remain undisturbed until completely dissolved, then mix • HRP-Estradiol Conjugate:
Pipette 0.1 mL of the concentrated HRP-estradiol solution into one of the vials of conjugate buffer. Prepare immediately prior to use. Maximum stability is 4 hours at room temperature or 24 hours at 2 - 8°C when protected from direct exposure to sunlight. • Wash Buffer:
Dilute 2 mL in 400 mL distilled water or the content of the entire vial in 2000 mL distilled water (use a magnetic stirrer). • Substrate Solution:
Pipette 0.2 mL of the chromogen (TMB) into one of the vials of XV. EXPECTED VALUES
substrate buffer (H2O2 in acetate/citrate buffer). Prepare immediately prior to use. Maximum stability is 15 minutes at room temperature when protected from direct exposure to sunlight. XII. ASSAY PROCEDURE
A calibration curve must be run with each assay. It is recommended that the assays be performed in duplicate and that instructions for the assay procedure be followed exactly to obtain 1. Select sufficient strips to accommodate calibrators, controls and all 2. Fit the strips into the holding frame. 3. Dispense 50 µL of each calibrator, control or sample into the appropriate wells. Vertical alignment is recommended. 4. Dispense 50 µL of estradiol-HRP conjugate into all wells. 5. Dispense 50 µL of anti-estradiol into each well. 6. Incubate for 2 hours at room termperature on a horizontal shaker set a) aspirating the liquid from each well; XVI. PERFORMANCE CHARACTERISTICS
b) dispensing 0.4 mL of wash solution into each well; c) aspirating the contents of each well. Repeat steps b) and c) 4 Minimum detectable concentration (MDC) of estradiol in 10 different 8. Dispense 200 µL of the freshly prepared substrate solution into assays was 5 ± 2 pg/mL (mean ± SD). MDC is defined as the each well immediately after the washing step. concentration of estradiol corresponding to 95% of maximum binding. 9. Incubate the plate for 30 minutes at room temperature, protecting from direct sunlight, on a horizontal shaker set at 700 ± 100 RPM. 10. Dispense 50 µL of stop reagent into each well. The percentage of cross-reaction was estimated under physiological 11. Read the absorbances of each well at 450 nm (reference wavelength conditions in serum by comparison of the concentration yielding a 50% at 650 nm) within 1 hour after addition of stop reagent. Incubate for 2 hours at RT with continuous shaking Wash 5 times with 0.4 mL of wash solution and aspirate Incubate 30 minutes at RT with continuous shaking Read the microtiter plate 450 nm (versus 650 nm) Sample Added Recovery Recovery Serum Theoreti Measured Recovery XVII. BIBLIOGRAPHY
1. Abraham, G.E. (1974) Ovarian and adrenal contribution to peripheral androgens during the menstrual cycle. J. Clin. Endocrinol. Metab. 39:340-346. 2. Baird, D.T., Fraser, I.S. (1974) Blood production and ovarian secretion rates of estradiol-17 beta and estrone in wormen throughout the menstrual cycle. J. Clin. Endocrinol. Metab. 38:1009-1017. 3. Haning, R.V. et al. (1985) Maternal serum progesterone, 17 Beta-estradiol and estriol are increased in pregnancies which follow treatment with human menopausal gonadotropins; effects of multiple gestation and maternal endocrine status. J. Steroid Biochem. 22:833-829. 4. Mehta, R.R. (1987) Subcellular concentrations of estrone, estradiol, (17BOH-SOH). Activity in malignant and non-malignant human breast tissues. Int. J. Cancer 40:305-308. 5. Selby, et al. (1986) Dose dependent response of symptoms, pituitary, and bone to transferma oestrogen in postmenopausal women. Br. Med. J. 293:1337-1339.
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