BL-12-CT: 100 determinations

Testosterone is a C-19 steroid hormone (molecular weight :288) wich is produced from androstenedione                            in the testes, adrenels and ovaries. Testosterone is a precursor along with androstenedione of the                               estrogen steroids.

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 testosterone 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: 8 vials ready to use except controls 0.5 ml.

                            Standards: 0-0.17-0.57-1.7-5.4-17.0 ng/ml.

2. ¹²⁵I Testosterone tracer: 1 vial (red solution) containing 55 ml. Activity < 5µCi or 190 kBq.

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

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

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

1. Distilled water

2. Pipettes for delivery of: 50 µl, 500 µl (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 heparinozed plasma provide similar results.

                        Y (serum) = 0.96 x (hep. plasma) + 0.12 r = 0.09 n = 23

              EDTA plasm provides 20% lower results than serum and heparinized plasma :

                        Y (serum) = 1.22 x (EDTA plasma) + 0.19 r = 0.99 n = 21

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 50 µl of each into the respective tubes.

            3. Dispense 500 µl 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 TESTO concentrations of the samples from the reference curve.

        INTRA ASSAY                                                                                         INTER ASSAY

Each dilution xas always performed form the undiluted samples which was diluted with the zero calibrator.

                                                        RECOVERY TEST

E. Example of a typical Standard curve

The following data are for illustration only and should never be used in place of the real lime standard curve

1. J.L. ANDREYKO et al.Role of Serum Androgens and Sex Hormone Binding Globulin Capacity in the Evaluation of Hirsutism in Women.Clin. Biochem. Vol. 19.58-61.

2. A. BIZZARO, et al.Influence of Testosterone Therapy on Clinical and Immunological features of Autoimmune Diseases Associated with Klinefelter’s Syndrome..J. Clin. Endocrin. Metab. Vol. 64,N°1 :32-36M. CARRABBA et al. (1985)

3.Abnormalities of Sex hromones in Men with Systemic Lupus ErythematosusClin. Rheumatology, N°4 :422-425.

4. P. HILL. Et al. (1985).Plasma Testosterone and Breast Cancer.Eur. J. Cancer Clin. Oncol, Vol.21, N°10, pp. 1265-1266.

5. CG. MAHLCK et al. (1986)Testosterone, SHBG and Albumin in Patients with ovarian carcinoma.Acta Obstet. Gynecol. Scand. 65 :533-S38.

6. D.M.D. PERERA et 31 (1987)Amniotic Fluid Testosterone end testosterone Glucuronide Levels in the determination of Foetal Sex.J. Steroid Biochem., Vol.26,N°2,pp273-277.

7. J. TRACHTENBERG (1987)Experimental Treatment of Prostatic Cancer by intermittent Hormonal Therapy.J. Urology, Vol. 137, pp. 785-788.