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.

A fixed amount of ¹²⁵I labelled steroid competes with the steroid to be measured present in the sample or in the standard for a fixed amount of antybody sites being immobilized to the wall of a polystyrene tube. Neither extraction nor chromatography are required because of the high specificity of the coated antibodies. After a 3 hours incubation at 37°C, an aspiration step terminates the competition reaction. The tubes are then washed with 3 ml of wash solution and aspirated again. A standard curve is plotted and the E2 concentrations of the samples are determined by dose interpolation from the standard curve.

1. Radioactive material: Radioactive material may be received, acquired, possessed and used only by physicians, clinical laboratories, or hospitals for "In-Vitro" clinical or laboratory tests not involving internal or external administration of the material, or the radiation therefrom, to human beings or animals.

Compliance with these basic rules of radiation safety should provide adequate protection:1. Do not eat, drink, smoke, or apply cosmetics in areas where radioactive material is used.

2. Do not pipet by mouth reagents containing radioactive materials.

3. Wear protective clothing; i.e., lab coats and disposable gloves, in order to avoid direct contact with radioactive reagents.

4. Work with radioactive materials should be performed in a designed area.

5. Radioactive materials should be stored in an acceptable location.

6. A log should be kept for receipt and disposal of radioactive materials.

7. Radioactive spills or accidents should be taken care of immediately according to established procedures.

8. Disposal of radioactive materials must comply with prevailing regulations and guidelines of the agencies holding jurisdiction over the laboratory.

2. Sodium azide: Sodium Azide, used as a bacteriostatic agent, is toxic in acid medium. In addition, it may form potentially explosive lead or copper azides. To avoid dangerous deposits, waste solutions should be flushed away with large volumes of water.

3. Hepatitis and Acquired Immune Deficiency Syndrome (HTLV-III): All Bio-Line reagents included in this kit have been tested and found to be non reactive for hepatitis B surface antigen. They have also been screened and determined to be non-reactive for HTLV-III antibody. However, human serum products should be handled as if potentially capable of transmitting hepatitis, Acquired Immune Deficiency Syndrome, or other infectious agents.

Kit contains sufficient reagents for 100 determinations.

1. Human serum and azide based standards & control: 9 vials ready to use except controls 1.0 ml.

            Standards: 0-15.0-50.0-150.0-500.0-1500.0-3000.0 pg/ml.

2. ¹²⁵I Estradiol tracer: 1 vial (red solution) containing 1 ml. Activity < 4µCi or 142 kBq.

3. Coated tubes: 2 x 50 tubes, coated with Anti-E2

4. Wash solution concentrate: 1 vial of 10 ml of concentrate, to be diluted into 700 ml distilled water and stored at 4° C.

5. Tracer buffer : 1 vial of 105 ml .

The following material is required but not provided in the kit:

1. Distilled water

2. Pipettes for delivery of: 50 µl and 1ml (the use of accurate pipettes with disposable plastic tips is recommended)

3. 5 ml automatic syringe (Cornwall type) for washing

4. Aspiration system (optional)

5. Gamma counter, set for ¹²⁵I counting and Vortex mixer and magnetic stirrer.

Serum and plasma samples must be kept at 2 – 8°C.

If the test is not run within 24 hours, storage at –20°C is recommended and will not result in loss of immuno-reactivity for at least 6 months.

Avoid successive freezing and thawing.

Serum and heparinized plasma provide similar results.

Y (serum) = 0.95 x (hep. plasma) + 3 r = 0.98 n = 16

EDTA plasm provides 25% lower results than serum and heparinized plasma :

Y (serum) = 1.27 x (EDTA plasma) + 12 r = 0.98 n = 16

1. Label coated tubes in duplicate for each standard, sample, control. For determination of total counts, label 2 normal tubes.

2. Vortex mix briefly standards, samples, controls and dispense 100 µl of each into the respective tubes.

            3. Dispense 1 ml of tracer into each tube, including the uncoated tubes for total counts..

4. Shake the tube rack gently to liberate any trapped bubbles.

5. Incubate for 3 hours at 37°C.

6. Aspirate the content of each tube (except total counts). Be sure that the plastic tip of the aspirator reaches the bottom of the coated tube in order to remove all the liquid.

7. Wash the tubes with 3 ml Wash Solution (except total counts) and aspirate. Avoid foaming during the addition of the Wash Solution.

8. In order to increase the reproducibility of the assay, leave the tubes on the table for 2 minutes and aspirate the remaining drop of liquid carefully.

9.. Count the tubes in a gamma counter for 60 seconds.

Using a 3 cycle semi-logarithmic or logit-log graph paper plot the (B/BO x 100) values for each standard point as a function of the TESTO concentration of each standard point. Computer assisted methods can also be used to construct the calibration curve.

By interpolation of the sample (B/BO x 100) values, determine the E2 concentrations of the samples from the reference curve.

5. For each assay, the percentage of total tracer bound in the absence of unlabelled E2 (BO/T) must be checked.

INTRA ASSAY                                                                                             INTER ASSAY

RECOVERY TEST

2. GERRIS J. et al. (1986)A lesion from IVF endocrinology: the importance of the follicular phase to success and failure in non IVF cycle.Acta. Eur. Fertil. 17,4,251-258

3. HENNY A.B.et al (1986)Diagnosis of luteinized unruptured follicle by ultrasound and steroid hormone assays in peritoneal fluid: a comparative study.Fertil. and Steril. 46, 5, 823-827

4. METHA R.R. (1987)Subcellular concentrations of estrone, estradiol, androstenedione and 17-ß-hydroxysteroid dehydrogenose (17BOH-SOH) Activity in malignant and non malignant human breast tissues.Int. J. Cancer 40, 305-308

5. PELLICER A. et al (1987)Outcome of in vitro fertilization in women with low response to ovarian stimulation.Fertil. and Steril. 47, 5, 812-815.

6. SELBY et al (1986)Dose dependent response of symptoms, pituitary, and bone to transferma oestrogen in postmenopausal women.Br. Med. 293, 1337-1339.

7. WRONSLY H. et al (1987)Pregnancy rate in relation to number of cleaved eggs replaced after in vitro fertilization in stimulated cycles monitored by serum levels of estradiol and progesterone as sole index.Hum. Reprod. 4, 325-32