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De2693 estradiol 101207 m

Product information
Information about other products is available at: www.demeditec.com Demeditec Diagnostics GmbH • Lise-Meitner-Straße 2 • D-24145 Kiel (Germany) Phone: +49 (0)431/71922-0 • Fax. +49 (0)431/71922-55 Email: info@demeditec.de • http://www.demeditec.com
Contents


1 INTRODUCTION . 3 2 PRINCIPLE OF THE TEST. 3 3 WARNINGS AND PRECAUTIONS. 4 4 REAGENTS . 4 5 SPECIMEN COLLECTION AND PREPARATION . 5 6 ASSAY PROCEDURE . 6 7 EXPECTED VALUES. 7 8 QUALITY CONTROL . 7 9 PERFORMANCE CHARACTERISTICS . 8 10 LIMITATIONS OF USE . 10 11 LEGAL ASPECTS. 10 12 REFERENCES / LITERATURE . 11 SYMBOLS USED WITH DEMEDITEC ASSAY´S . 12 Demeditec Diagnostics GmbH • Lise-Meitner-Straße 2 • D-24145 Kiel (Germany) Phone: +49 (0)431/71922-0 • Fax. +49 (0)431/71922-55 Email: info@demeditec.de • http://www.demeditec.com 1 INTRODUCTION
Intended Use
The DEMEDITEC Estradiol ELISA is an enzyme immunoassay for the quantitative in vitro diagnostic
measurement of Estradiol in serum and plasma
Summary and Explanation
Estradiol (1,3,5(10)-estratriene-3,17β-diol; 17β-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 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, vagine, 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 luteinising hormone (LH), which are essential for folicular 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 feminising 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 fertislisation (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 DEMEDITEC 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 a polyclonal [rabbit] antibody directed towards an 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 inversely proportional to the
concentration of Estradiol in the sample. After addition of the substrate solution, the intensity of colour
developed is inversely proportional to the concentration of Estradiol in the patient sample.
Demeditec Diagnostics GmbH • Lise-Meitner-Straße 2 • D-24145 Kiel (Germany) Phone: +49 (0)431/71922-0 • Fax. +49 (0)431/71922-55 Email: info@demeditec.de • http://www.demeditec.com 3 WARNINGS AND 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 • 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 1 N acidic solution. It may cause skin irritation and • Never pipet by mouth and avoid contact of reagents and specimens with skin and mucous • 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 specimens may give false results. • Handling should be in accordance with the procedures defined by an appropriate national biohazard safety guideline or regulation. • 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 using calibrated pipettes and microtiterplate readers. • 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 guideline or regulation. • For information on hazardous substances included in the kit please refer to Material Safety Data Sheets. Material Safety Data Sheets for this product are available upon request directly from DEMEDITEC. 4 REAGENTS
Reagents provided
1. Microtiterwells, 12x8 (break apart) strips, 96 wells;
Wells coated with a anti-Estradiol antibody (polyclonal). 2. Standard (Standard 0-6), 7 vials, 1 ml, ready to use;
Concentrations: 0, 25; 100; 250; 500; 1000; 2000 pg/ml Conversion: 1 pg/ml = 3.67 pmol/L contain 0.03% Proclin 300 + 0.005% gentamicin sulfate as preservatives. 3. Enzyme Conjugate, 1 vial, 25 ml, ready to use;
Estradiol conjugated to horseradish peroxidase; * contains 0.03% Proclin 300, 0.015% BND and 0.010% MIT as preservatives. 4. Substrate Solution, 1 vial, 14 ml, ready to use;
5. Stop Solution, 1 vial, 14 ml, ready to use;
contains 1 N acidic solution. 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 upon request.
Demeditec Diagnostics GmbH • Lise-Meitner-Straße 2 • D-24145 Kiel (Germany) Phone: +49 (0)431/71922-0 • Fax. +49 (0)431/71922-55 Email: info@demeditec.de • http://www.demeditec.com Material required but not provided
− A microtiter plate calibrated reader (450 ± 10 nm), − Calibrated variable precision micropipettes. − Absorbent paper. − Distilled or Deionized water − Timer − Semi-logarithmic graph paper or software for data reduction Storage Conditions
When stored at 2°C - 8°C unopened reagents will retain reactivity until expiration date. Do not use reagents beyond this date. Opened reagents must be stored at 2°C - 8°C. Microtiter wells must be stored at 2°C - 8°C. Once the foil bag has been opened, care should be taken to close it tightly again. Preparation of Reagents
Allow all reagents and required number of strips to reach room temperature prior to use.
Wash Solution
Add deionized water to the 40X concentrated 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.
Disposal of the Kit
The disposal of the kit must be made according to the national regulations. Special information for this product is given in the Material Safety Data Sheets (see chapter 13). Damaged Test Kits
In case of any severe damage to the test kit or components, DEMEDITEC has to be informed in writing, at the 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 COLLECTION AND PREPARATION
Serum or plasma (EDTA-, Heparin- or citrate plasma) can be used in this assay.
Do not use haemolytic, icteric or lipaemic specimens.
Please note: Samples containing sodium azide should not be used in the assay.
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. Do not centrifuge before complete clotting has occurred.
Patients receiving anticoagulant therapy may require increased clotting time.
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 Citrate plasma Sarstedt Monovette – green cap - # 02.167.001.) Specimen Storage d Preparation
Specimens should be capped and may be stored for up to 5 days at 2°C - 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. Demeditec Diagnostics GmbH • Lise-Meitner-Straße 2 • D-24145 Kiel (Germany) Phone: +49 (0)431/71922-0 • Fax. +49 (0)431/71922-55 Email: info@demeditec.de • http://www.demeditec.com Specimen Dilution
If in an initial assay, a specimen is found to contain more than the highest standard, the specimens can be diluted 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 ASSAY PROCEDURE
General Remarks
− All reagents and specimens must be allowed to come to room temperature before use. All reagents − 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 − 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. Test Procedure
Each run must include a standard curve. 1. Secure the desired number of Microtiter wells in the frame holder. 2. Dispense 25 µL of each Standard, Control and samples with new disposable tips into
3. Dispense 200 µL Enzyme Conjugate into each well.
Thoroughly mix for 10 seconds. It is important to have a complete mixing in this step. 4. Incubate for 120 minutes at room temperature (without covering the plate).
5. 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!
6. Add 100 µL of Substrate Solution to each well.
7. Incubate for 15 minutes at room temperature.
8. Stop the enzymatic reaction by adding 50 µL of Stop Solution to each well.
9. Determine the absorbance (OD) of each well at 450±10 nm with a microtiter plate reader.
It is recommended that the wells be read within 10 minutes after adding the Stop Solution.
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 4. Automated method: The results in the IFU have been calculated automatically using a 4 PL (4 Parameter Logistics) curve fit. 4 Parameter Logistics is the preferred method. Other data reduction functions may give slightly different results. 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. Demeditec Diagnostics GmbH • Lise-Meitner-Straße 2 • D-24145 Kiel (Germany) Phone: +49 (0)431/71922-0 • Fax. +49 (0)431/71922-55 Email: info@demeditec.de • http://www.demeditec.com Example of Typical Standard Curve
The following data is for demonstration only and cannot be used in place of data generations at the
time of assay.
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 DEMEDITEC Estradiol ELISA
the following values are observed:
Population
5 – 95% Percentile
The results alone should not be the only reason for any therapeutic consequences. The results should be correlated to other clinical observations and diagnostic tests. 8 QUALITY CONTROL
Good laboratory practice requires that controls be 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.
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
DEMEDITEC directly.
Demeditec Diagnostics GmbH • Lise-Meitner-Straße 2 • D-24145 Kiel (Germany) Phone: +49 (0)431/71922-0 • Fax. +49 (0)431/71922-55 Email: info@demeditec.de • http://www.demeditec.com 9 PERFORMANCE CHARACTERISTICS
Assay Dynamic Range
The range of the assay is between 9.7 pg/ml – 2000 pg/ml. Specificity of Antibodies (Cross Reactivity)
The following substances were tested for cross reactivity of the assay: Compound
Compound
reactivity
reactivity
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. Reproducibility
Intra Assay
The within assay variability is shown below: Inter Assay
The between assay variability is shown below: Demeditec Diagnostics GmbH • Lise-Meitner-Straße 2 • D-24145 Kiel (Germany) Phone: +49 (0)431/71922-0 • Fax. +49 (0)431/71922-55 Email: info@demeditec.de • http://www.demeditec.com 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)
Linearity
Dilution
Mean Conc. (pg/ml)
Recovery (%)
Demeditec Diagnostics GmbH • Lise-Meitner-Straße 2 • D-24145 Kiel (Germany) Phone: +49 (0)431/71922-0 • Fax. +49 (0)431/71922-55 Email: info@demeditec.de • http://www.demeditec.com 10 LIMITATIONS OF USE
Reliable and reproducible results will be obtained when the assay procedure is performed with a
complete understanding of the package insert instruction and with adherence to good laboratory
practice.
Any improper handling of samples or modification of this test might influence the results.
10.1 Interfering Substances
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. 10.2 Drug Interferences
Until today no substances (drugs) are known to us, which have an influence to the measurement of Estradiol in a sample. 10.3 High-Dose-Hook Effect
No hook effect was observed in this test. 11 LEGAL ASPECTS
11.1 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 DEMEDITEC. 11.2 Therapeutic Consequences
Therapeutic consequences should never be based on laboratory results alone even if all test results are in agreement with the items as stated under point 11.1. 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 therapeutic consequences be derived. The test result itself should never be the sole determinant for deriving any therapeutic consequences. 11.3 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 11.2. 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. Demeditec Diagnostics GmbH • Lise-Meitner-Straße 2 • D-24145 Kiel (Germany) Phone: +49 (0)431/71922-0 • Fax. +49 (0)431/71922-55 Email: info@demeditec.de • http://www.demeditec.com 12 REFERENCES / LITERATURE
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 oestradiol and progesterone in cycles with luteal phase deficiency, Fertil. Steril. 37: 348-54 (1982). 13. Kirschner, M.A., therole of hormones in the etiology of human breast cancer, Cancer 39:2716 26 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 17. Taubert, H.d. and Dericks-Tan, J.s.E., Induction ofr ovulation by clomiphene citrate in combination with high doses of oestrogens 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). 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. Demeditec Diagnostics GmbH • Lise-Meitner-Straße 2 • D-24145 Kiel (Germany) Phone: +49 (0)431/71922-0 • Fax. +49 (0)431/71922-55 Email: info@demeditec.de • http://www.demeditec.com SYMBOLS USED WITH DEMEDITEC ASSAY´S

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Consult instructions for Gebrauchsanweisung instructions d’utilisation Instrucciones Date limite d’utilisation Fecha de caducidad Conjugué enzymatique Conjugado enzimático Demeditec Diagnostics GmbH • Lise-Meitner-Straße 2 • D-24145 Kiel (Germany) Phone: +49 (0)431/71922-0 • Fax. +49 (0)431/71922-55 Email: info@demeditec.de • http://www.demeditec.com

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