rev: January 18, 1999

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ESTRADIOL ELISA Kit   $400.00/kit  
-sample data-see actual kit insert for batch specific information

Catalogue No : RDI-RE52041 Product group : Steroids 12 x 8 Product name : Estradiol, 17ß **510K** ELISA 96 Method : ELISA Incubation time: 120min, 15min Standard curve : 25 pg/ml - 2000 pg/ml Sample/Prep. : 25 µl Serum, Plasma Isotope/Substr.: TMB, 450nm
CONTENTS Introduction Contents of the Kit Principle of the Test Test Procedure Performance Characteristics Expected Values Alternative Applications (More) Clinical Background Sales Arguments Product Literature Miscellaneous

Introduction

Back to Contents The IBL Estradiol Enzyme Immonoassay Kit provides material for the quantitative determination of estradiol in serum and plasma. This assay is intended for in vitro diagnostic use. Estradiol [1, 3, 5 (10) -estratriene-3, 17ß-diol; 17ß-estradiol; E2] is a C18 steroid hormone with a phenolic A ring. Only 1-3 % of estradiol circulating in plasma is present in its free form being principally bound to sex hormone binding globuline (SHBG) and to Serum Albumin. Estradiol is the most important natural estrogen and is present in females and males. In the former, the estrogens stimulate the growth of sex organs and the development of secondary sexual characteristics, and they also affect the gonadotrophin secretion. In the males, the role of estradiol is less well defined although it seems to be involved in the regulation of gonadotrophin secretion. In the males, the role of estradiol is less well defined although it seems to be involved in the regulation of gonadotrophin together with testosterone. In non-pregnant women estradiol is almost exclusively produced by ovary. Consequently, its serial (i.e. daily) measurement during the ovulatory cycle is helpful to evaluate the ovarian function for accurate assessment of follicular growth and also for monitoring the exponential increase in estradiol serum occuring either in spontaneous cycles or cycles stimulated for induction of ovulation or "superovulation" before "in vitro" fertilization. In other clinical conditions including delayed puberty, primary and secondary amenorrhoea, and menopausa other hormone assays are required for proper interpretation and differential diagnosis.

Contents of the Kit

Back to Contents Contents of the Kit 1. Coated Microtiterstrips 12 x 8 wells break apart wells coated with anti estradiol antiserum (rabbit polyclonal) in foilbag with desiccant. 2. Standard A (zero standard) 1 vial ready to use 2 ml of steroid free human serum 3. Reference Standard Set (B - G) 6 vials ready to use, Six vials 0.5 ml each, containing the below mentioned concentrations of estradiol in human serum (steroid free) and 0.01 % sodium azide. Standard B C D E F G Concentration in pg/ml 25 100 250 500 1000 2000 4. Enzyme Conjugate 1 vial 22 ml, ready to use, Estradiol conjugated to the enzyme horseradish peroxidase in buffer, supplemented by enzyme stabilizers. 5. TMB Substrate Solution 1 vial 13 ml, ready to use Containing a solution of tetramethylbenzidine (TMB) with hydrogen peroxide in buffer supplemented by stabilizers Do not exchange "LS" from different kits with "HS"! 6. TMB Stop Solution 1 vial 13 ml, ready to use, 0.3 M sulfuric acid (H2SO4) Avoid contact with stop-solution it may cause skin irritations and burns 7. Wash Buffer, Concentrate (10x) 1 vial 50 ml, concentrate, containing borate-citrate buffer with a nonionic detergent, dilute 10-fold with distilled water prior use. Storage: 2 - 8°C Storage and Stability When stored at 2 - 8 °C unopened reagents will retain reactivity until expiration date. Do not use reagents beyond this date. Enzyme conjugate, standard solution, substrate solution, wash solution and zero standard must be stored at 2 - 8°C. Microtiter wells must be stored at 2 - 8 °C. Once the foilbag has been broken care should be taken to close it tightly again. The immunoreactivity of the coated microtiter wells is stable for approx. 6 weeks in the broken, but tightly closed bag. Preparation of Specimens and Storage Serum or EDTA plasma should be used, and the usual precautions for venipuncture should be observed. No special sample pretreatment is necessary. The specimen may be stored at 2 - 8 °C for up to 24 hours, and should be frozen at -10 °C or lower for longer periods. Do not use grossly hemolyzed or grossly lipemic specimens. Samples suspected to contain estradiol concentration higher than 2000pg/ml are to be diluted with zero standard. Thawed samples should be inverted several times prior to testing. Do not use grossly hemolyzed or grossly lipemic specimens.

Principle of the Test

Back to Contents The Estradiol ELISA KIT is based on the competition principle and the microtiterplate separation. An unknown amount of antigen present in the sample and a fixed amount of enzym labelled antigen compete for the binding sites of the antibodies coated onto the wells. After an incubation the microtiterplate is washed to stop the competition reaction. Having added the substrate solution the concentration of estradiol is inversely proportional to the optical density measured. The measured ODs of the standards are used to construct a calibration curve against which the unknown samples are calculated.

Test Procedure

Back to Contents 1. Leave sufficient strips in the strip holder to enable the running of calibrators, controls, and samples in duplicate, plus one well for the chromogen blank. Secure the desired number of coated microtiter strips in the holder. 2. Pipette 25 µl of standards, controls and samples into the appropriate wells of the strips. 3. Add 200 µl of enzyme conjugate to each well in sequence. 4. Incubate for 120 min at 37 °C without covering the plate. 5. Washing: discard the incubation solution, rinse the wells with wash buffer (300 µl) three times, and remove any residual. 6. Promptly pipette 100 µl of the TMB substrate solution into the rinsed wells. 7. Incubate for 15 minutes at room temperature. 8. Stop the reaction by adding 100 µl of TMB stop solution to each well. 9. Shake gently the microplate being careful not to let the content come from the wells and read at 450 nm within one hour from the stopping. General Remarks: All reagents and specimens must be allowed to come to room temperature before use. All reagents must be mixed without foaming. Once the test has been started, all steps should be completed without interruption. Use new disposable plastic pipette tips for each reagent, standard or specimen in order to avoid cross contamination. For the dispensing of the substrate - solution and the stop - solution avoid pipettes with metal parts. Absorbance is a function of the incubation time and temperature. Before starting the assay, it is recommended that all reagents be ready, caps removed, all needed wells secured in holder, etc. This will ensure equal elapsed time for each pipetting step without interruption. If in a test run the absorbance of the zero standard is lower than 1.3 or higher than 2.0, because the lab temperature is lower than 21 °C o higher than 28 °C, the next runs should be carried out setting the incubation time for the colour development to 20 and 10 minutes respectively. However, in case of absorbances higher than 2.0 it is not necessary to shorten the incubation time for the enzymatic reaction when the microplate reader is linear up to 2.5 - 3.0 absorbance units. Calculation of Results Any ELISA reader capable of determining the absorbance at 450 +/- 10 nm may be used. The estradiol concentration of each sample is obtained as follows : 1. Using linear-linear or semi log graph paper, construct an standard curve by plotting the average absorbance (Y) of each reference standard against its corresponding concentration (X) in pg/ml. 2. Use the average absorbance of each sample to determine the corresponding estradiol value by simple interpolation from this standard curve, multiplying by the initial sample dilution, if necessary. The results can also be calculated with normal programs for automatic data processing, i.e. 4 Parameters, Spline, Logit- Log. Should estradiol value exceed the highest standard value, dilute the sample with the zero calibrator and re-run the assay. Multiply the result obtained by the sample dilution factor. 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. Standards OD 450 nm [pg/ml] 0 2.08 25 1.77 100 1.30 250 0.85 500 0.60 1000 0.43 2000 0.28

Performance Characteristics

Back to Contents 1. Specificity The specifity of this kit was assessed according to Abraham's method. The percentage indicate cross reactivity at 50 % displacement compared to estradiol. Steroids % Cross- Steroids % Cross- Reactivity Reactivity Estradiol 100.0 Cortisone < 0.1 Estrone 2.1 Progesterone < 0.1 Estriol 1.5 Testosterone < 0.1 17-Estradiol 0.3 DHEA-S < 0.1 Cortisol < 0.1 5-Dihydrotestosterone < 0.1 2. Sensitivity The minimal detectable concentration of estradiol by this assay is estimated to be 16 pg/ml (defined as 3x standard deviation to the zero standard). 3. Precision a. Intra Assay Variation Within run variation was determined by replicate determination of three different control sera in one assay. The within assay variability is shown below: Sample 1 2 3 n 20 20 20 Mean (pg/ml) 62.86 177.77 514.72 Standard Deviation (pg/ml) 4.11 6.93 12.43 CV (%) 6.50 3.90 2.40 b. Inter Assay Variation Between run variation was determined by replicate measurements of three different control sera in several different assay. The between-assay variability is shown below: Sample 1 2 3 n 20 20 20 Mean (pg/ml) 91.8 236.3 589.8 Standard Deviation (pg/ml) 9.2 16.4 26.0 CV (%) 10.0 6.9 4.4 4. Recovery Spiked serum samples were prepared by adding varying levels of estradiol to three different serum samples. Serum Added Estradiol Measured Recovery (pg/ml) Estradiol (%) (pg/ml) 1 0 34 -- 89 96 69.7 246 267 94.7 315 369 106.3 2 0 32 -- 89 97 73.0 246 245 86.6 315 363 105.1 3 0 183 -- 89 245 134.8 246 476 119.1 315 557 118.7 5. Linearity Three patient samples were serially diluted with zero-standard in a linearity study. Patient Dilution Measured conc. Expected Recovery factor (pg/ml) conc. (%) (pg/ml) neat 991 -- -- 1:2 577 495.5 116.4 1 1:4 314 247.8 126.7 1:8 148 123.9 119.4 1:16 81 61.9 130.8 neat 443 -- -- 1:2 220 221.5 99.3 2 1:4 111 110.7 100.3 1:8 61 55.4 110.1 neat 232 -- -- 3 1:2 115 116 99.1 1:4 65 58 89.2 6. Method Comparison The IBL-Estradiol ELISA was compared to another commercially available estradiol assay. Serum samples of 47 obviously healthy non pregnant females and 25 pregnant females were analysed according in both test systems. A correlation coefficient of r = 0.92 (r2 = 0.85) was found between the two test systems. The linear regression curve was calculated from y = mx + b where m is the slope and b the y - intercept. The resulting equation is: y = 0.95 IBL-ELISA + 78 Serum samples of 19 obviously healthy non pregnant females and 16 pregnant females were compared to their plasma samples using the IBL-Estradiol ELISA. A correlation coefficient of r = 0.989 (r2 = 0.99) was found between the two. The linear regression curve was calculated from y = mx + b where m is the slope and b the y - intercept. The resulting equation is: Plasma = 0.99 Serum + 1.98

Expected Values

Back to Contents The following values can be used as preliminary guidelines until each laboratory establishes ist own normal ranges: Women (> 18 n pg/ml years) Follicular Phase 34 30 - 120 Ovulatory Peak 42 130 - 370 Luteal Phase 29 70 - 250 Menopause 25 15 - 60 Men 95 15 - 60

Alternative Applications

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(More) Clinical Background

Back to Contents Clinical Applications 1. Precocious puberty in girls: E2 measurements may be utilized in the assessment of precocious puberty in girls. Although elevated E2 measurements themselves are meaningful, extensive ancillary aids are required in the rendering of specific diagnoses. 2. Gynecomastia in males: E2 measurements may be helpful in the assessment of unexplained gynecomastia. 3. Assessment of hypoestrogenism in women: E2 measurements are frequently utilized to document hypoestrogenism in cases of delayed puberty, primary and secondary amenorrhea, and menopause. Gonadotropin measurements must be utilized to localize the cause of the hypoestrogenism to the ovaries or to a hypoestrogenic woman. 4. Assessment of excessive Estrogen production in women: E2 concentrations exceeding 1000 pg/ml are usually observed only during pregnancy or in rare cases of estrogen secreting neoplasms. Factors affecting normal values: 1. Combination oral contraceptives: Estradiol concentrations are suppressed to chronically anovulatory levels. 2. Superovulatory drugs: Estradiol concentrations reflect on development of multiple follicles and rise superphysiologically as a product of the number of follicles recruited. Clomiphene citrate produces values in the 500 to 1000 pg/ml range whereas human menopause gonadotropin (HMG) may produce values in the 500 - 3000 pg/ml range. 3. Estrogen replacement therapy: Estradiol concentrations rise from menopausal values into low follicular phase range depending on doses. Conjugated estrogens contain estrone sulfate which is converted in vivo to estradiol. 4. GnRH analogous: Estradiol concentrations are suppressed into the postmenopausal range during pituitary down regulation with analogous. 5. Surgical oophorectomy: Estradiol concentrations fall into the postmenopausal range immediately after oophorectomy.

Sales Arguments

Back to Contents 17ß-Estradiol ELISA Cat.-No. RE 520 41 Assay advantages for the IBL assay 510K exempt by FDA Break apart wells for individual determinations Reagents ready for use Short incubation time Suitable for automation Excellent correlation to RIA After extraction suitable for the determination in rat samples Potential customers University-labs, Private-labs, Gynaecologists, Endocrinologists, Clinical Applications - Differential diagnosis in gynaecological and andrological disorders - Im vitro fertilisation - Follow up in therapy of infertility

Product Literature

Back to Contents 1. Tietz, N.W..: Textbook of Clinical Chemistry. Saunders 1986

Miscellaneous

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