rev: January 18, 1999

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Testosterone ELISA Kit   $388.00/kit  
-sample data-see actual kit insert for batch specific information
Catalogue No : RDI-RE52151 Product group : Gynaecology 12 x 8 Product name : Testosterone **510K** ELISA 96 Method : ELISA Incubation time: 1 h, 15 min Standard curve : 0.2-16 ng/ml Sample/Prep. : 50 µl Serum, Plasma Isotope/Substr.: TMB, 450 nm
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 testosterone assay is an Enzyme-Imunoassay (EIA) for the quantitative determination of testosterone in human serum or plasma. Testosterone (17ß-Hydroxy-4-Androstene-3-on) is a C19 steroide hormone with a molecular weight of 288 Dalton. Testosterone is one of the most important male sex hormones. In man it is mainly synthesized by the testes, in women both the ovaries and by the adrenal cortex; it is secreted into circulation. Testosterone is transported in the plasma by a beta-globulin, called testosterone binding globulin. It is estimated that about 98 % of the circulating testosterone is bound. The remainder, present as free testosterone, is assumed to be the metabolic active portion. In the target organ, it is transformed by enzymatic reduction into the physiologically effective androgen dihydrotestosterone. In man the determination of testosterone is used as an indicator for the function of the testes: low hormone levels are found in cases with Klinefelter's syndrome, cryptorchism or anorchia. Male or female patients with an androgen producing tumor (ovaries, adrenal cortex, testes) show increased values. Measurement of testosterone is used to confirm hirsutism in woman. The determination of free or not specifically protein-bound testosterone can be helpful in cases of hyperprolactinemic women or hyperandrogenism.

Contents of the Kit

Back to Contents Do components contain < 250 µl solution, please care that all solution is on the bottom of the vial. 1. Microtiter Strips 12 x 8 wells break apart strips coated with polyclonal anti testosterone antiserum (rabbit, polyclonal) in foilbag with desiccant. 2. Enzyme conjugate 1 vial 22 ml, ready to use, Testosterone conjugated to the enzyme horseradish peroxidase supplemented by enzyme stabilizers and 0.3 % procline. 3. Standard A (Zero Standard) 1 vial 0.5 ml, ready to use proteinmatrix with 0.1 % thimerosal and 0.3 % procline. 4. Standards (B - G) 6 vials 0.25 ml, ready to use, proteinmatrix with 0.1 % thimerosal and 0.3 % procline. Concentration in ng/ml 0.2 0.5 1 2 6 16 5. TMB Substrate Solution 1 vial 22 ml, ready to use, containing a solution of tetramethylbenzidine (TMB) with hydrogen peroxide in buffer supplemented with stabilizers. 6. TMB Stop Solution 1 vial 11 ml, ready to use, 0.5 M sulphuric acid (H2SO4). Avoid contact with stop solution it may cause skin irritations and burns. 7. Wash Buffer, concentrate (20 x) 1 vial 25 ml, concentrate, in phosphat-buffered saline, with nonionic detergent, dilute 1 to 20 with distilled water.

Principle of the Test

Back to Contents This Testosterone 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 enzyme 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 antigen 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. Summary: 1.1. Secure the desired number of coated strips in the holder. 1.2. Pipet 25 µl of standards, controls or samples. 1.3. Incubate 5 minutes at room temperature. 1.4. Add 200 µl of enzyme conjugate. 1.5. Thoroughly mix the plate for 10 seconds. 1.6. Incubate for 60 minutes at room temperature. 1.7. Rinse the wells 3 times with diluted wash buffer. 1.8. Add 200 µl of TMB substrate solution. 1.9. Incubate for 15 minutes at room temperature. 1.10. Add 100 µl of TMB stop solution. 1.11. Determine the absorbance at 450 ñ 10 nm. 2. Detailed Instructions for use 2.1. Secure the desired number of coated strips in the holder. 2.2. Dispense 25 µl of standards into appropriate wells. 2.3. Dispense 25 µl of sample into selected wells. Time between distribution of first standard and last sample can be up to 10 minutes without affecting the results. 2.4. Incubate 5 minutes at room temperature. 2.5. Dispense 200 µl of enzyme conjugate into each well. 2.6. Thoroughly mix the plate for 10 seconds. It is important to have complete mixing in this step. 2.7. Incubate for 60 minutes at room temperature ( 18 - 24 °C) without covering the plate. 2.8. Briskly shake out the contents of the wells. 2.9. Rinse the wells 3x with diluted wash buffer (300 µl per well). Strike the wells sharply on absorbent paper to remove residual droplets. 2.10. Add 200 µl of TMB substrate solution to each well, at timed intervals. 2.11. Incubate for 15 minutes at room temperature (18 - 24 °C). 2.12. Stop the enzymatic reaction by adding 100 µl of TMB stop solution to each well at the same timed intervals as in step 10 and determine the absorbance of each well at 450 ñ 10 nm, within 30 minutes following step 12. Storage and Stability When stored at 2 - 8 °C unbroken reagents will retain reactivity until expiration date. Do not use reagents beyond this date. Enzyme conjugate, standards, substrate solution, wash buffer must be stored at 2 - 8°C. Microtiter strips must be stored at 2 - 8 °C. Once the foilbag has been broken care should be taken to close it tightly again. The immuno-reactivity of the coated microtiter wells is stable for approx. 6 weeks in the broken but tightly closed plastic zip pouch containing the desiccant. Specimen collection and preparation for analysis Serum and plasma Serum or EDTA plasma should be used in the assay. No special pretreatment of sample 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 testosterone concentration higher than 16 ng/ml are to be diluted with zero standard. Attention: Samples containing sodium azide should not be used in this test. PREPARATION OF SAMPLES AND REAGENTS Dilute the wash buffer concentrate with distilled water 1 to 20. Ready to use wash buffer is stable for 2 weeks when stored at room temperature. 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. Pipette standards and samples onto the bottom of the well. For pipetting of enzyme conjugate and stop solution it is recommended to hold the pipette in a vertical position above the well and dispense the correspondent solution into the centre of the well so that a complete mixing of enzyme conjugate with sample or standard and of the stop solution with the substrate solution is achieved. 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. As a general rule the enzymatic reaction is linearly proportional to time and temperature. This makes interpolation possible for fixed physico-chemical conditions. If in a test run the absorbance of zero standard is lower than 1.0 or above the upper performance limit of your microtiterplate spectrophotomter you can extend or reduce the incubation time of the final enzymatic formation of color to 30 or 10 minutes accordingly. Since calibrators are assayed in each run, absorbance fluctuations do not affect the result. The substrate solution should be colourless or slightly blue or green. If the solution is dark blue the reagent is unusable and must be discarded. During incubation with substrate solution avoid direct sunlight on the microtiter plate.

Performance Characteristics

Back to Contents 1. Specificity The specificity of the testosterone assay was assessed according to Abraham's method. Steroid Cross-reactivity (%) Testosterone 100 Androstendione 0.9 Androsterone 0.9 5à- Dihydrotestosterone < 0.05 17-ß-Estradiol < 0.05 Estron < 0.05 Estriol < 0.05 Epitestosterone < 0.05 17-OH-Progesterone < 0.05 Progesterone < 0.05 Cortisol < 0.05 DHEA-SO4 < 0.05 2. Sensitivity The lowest detectable level of testosterone - defined as the testosterone concentration given by the mean absorbance of the zero standrad minus 2 standard deviations was assessed to be approx. 0.1 ng/ml. 3. Precision 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: Mean concentration n CV (%) (ng/ml) 0.83 20 3.9 5.54 20 4.0 9.91 20 5.8 Inter Assay Variation Between run variation was determined by replicate measurements of three different control sera in several differebt assays. The between assay variability is shown below: Mean concentration n CV (%) (ng/ml) 0.82 11 6.4 5.44 21 5.5 13.1 11 8.7 4. Linearity Three patient samples were serially diluted with zero standard in a linearity study. Sample Dilution Measured conc. Expected conc. Recovery (ng/ml) (ng/ml) (%) 1 neat 2.42 -- -- 1:2 1.32 1.21 109 1:4 0.62 0.60 102 1:8 0.31 0.30 102 2 neat 5.13 -- -- 1:2 2.63 2.57 103 1:4 1.29 1.28 101 1:8 0.69 0.64 108 1:16 0.36 0.32 112 3 neat 12.0 -- -- 1:2 6.31 6.00 105 1:4 3.21 3.00 107 1:8 1.73 1.50 115 1:16 0.79 0.75 105 5. Recovery Spiked serum samples were prepared by adding varying levels of testosterone to two different serum samples. Serum Added Testosterone Measured Testosterone Recovery (ng/ml) (ng/ml) (%) 1 0 3.0 -- 1 3.6 90 2 4.9 98 4 7.1 101 2 0 0.6 -- 1 1.3 82 2 2.1 81 4 4.2 92 8 8.0 93

Expected Values

Back to Contents Females 0.1 - 1.2 ng/ml Males 2.4 - 12 ng/ml Conversion factor: 1 ng/ml = 3.47 nmol/l

Alternative Applications

Back to Contents Determination of Steroids in Saliva Preparation of Saliva Samples The saliva samples have to be extracted prior to use. Patients should rinse their mouth with tap water 15 min before taking the sample. It is recommended to freeze the sample. After thawing, the sample should be centrifuged to separate mucines. The following extraction procedures are suitable for the determination of DHEA-S, progesterone, 17-OH-progesterone, estradiol, testosterone and cortisol in saliva. The supernatant should be used for extraction. In general concentrations of steroids in saliva are about 10 times lower compared to serum. Therefore we recommend to reconstitute the extract in a lower volume (e.g. 20 µl zero standard if the original volume was 100 µl saliva) of zero standard. To compensate possible loss due to extraction, standards should be treated in the same way. The following extraction methods are recommended: Method 1: 1. Mix 100 µl saliva with 2 ml ether (*). 2. Shake for 20 minutes at room temperature. 3. Freeze-out organic phase at -20 °C (*). 4. Pipette the supernatant into a test tube and evaporate (e.g. with nitrogen) in a water bath at 35 °C. 5. Reconstitute the residue with 100 µl zero standard. 6. This extract may be used as described in the assay procedure. (*) If H2O saturated ether is used, the separation of the phases may be performed without the freezing step (3). Method 2: 1. Mix 150 µl saliva with 1.5 ml Methylenchloride. 2. Shake for 20 minutes at room temperature. Note: If the sample is cloudy, centrifuge 10 minutes at 500 x g. 3. Aspirate 1 ml of the lower organic phase and evaporate (e.g. with nitrogen) in a water bath at 40 °C. 4. Reconstitute the residue with 100 µl zero standard. 5. This extract may be used as described in the assay procedure.

(More) Clinical Background

Back to Contents Monitoring of androgen suppressing drugs: Testosterone measurements may also be utilized in women for the adjustment of androgen suppressing drugs and their dosages. Common oral contraceptives drugs and drugs containing cyproterone acetate (CPA) are very effective in the suppression of testosterone concentrations. Factors affecting normal values: 1. Combination oral contraceptives: Testosterone concentrations are suppressed by administration of oral contraceptives. 2. Corticosteroids: Testosterone concentrations are suppressed in patients taking adrenal corticosteroids even with very small doses. 3. GnRH analogous: Testosterone concentrations are suppressed during pituitary down regulation with analogous. 4. Danazol: Testosterone concentrations will be artifactually high in women taking danazol. Although danazol (17à-pregna 2,4-dien-20-yno-(2,3-d)-isoxazol -17-0l ) shows negligible cross reactivity, its metabolic by-products may display a high cross-reactivity. There is rarely, if ever, an indication to determine testosterone concentrations in serum of women taking danazol. Pregnancy: Testosterone concentrations are relatively unchanged over the three trimesters of pregnancy.

Sales Arguments

Back to Contents Assay advantages for the IBL assay: - 510k exempt by FDA - Low sample volume necessary (25 µl) - Reagents ready to use - Break apart wells for short assay runs - Enlarged standard range - Incubation at room temperature - Short incubation time - High sensitivity - Suitable for automation - Suitable for the determination in rat and dog samples Potential customers: University-labs, Private-labs, Gynaecologists, Endocrinologists Clinical Applications: - Androgenizing disorders in females - Monitoring of androgen suppressing drugs - Virilizing adrenal and ovarial tumors - Evaluation of infertility in males

Product Literature

Back to Contents 1. Griffin JE, Wilson JD: The androgen resistance syndromes: 5alpha-reductase deficiency, testicular feminization, and related syndroms. In Scriver CR, Beaudet AI, Sly WS, Valle D [eds.]: The Metabolic Basis of Inherited Disease, 6th edition. McGraw-Hill, New York, 1989, 1919-1944. 2. Chen A, Bookstein JJ, Meldrum DR: Diagnosis of testosterone-secreting adrenal adenoma by selective venous catherization. Fertil. Steril., 55: 1202-1203 (1991). 3. Dorfman RI, Shipley RA: Androgens. Jahn Wiley and Sons, Inc. New York, 116-128 (1956). 4. Granoff AB, Abraham GE: Peripheral and adrenal venous levels of steroids in a patient with virilizing adenoma.Obstet. Gynecol. 53: 111 (1979). 5. Yen SSC: Chronic anovulation caused by peripheral endocrine disorders. In Yen SSC and Jaffe RB (eds.):Reproductive Endocrinology Chapter 17. W. B. Saunders, Philadelphia (1991). 6. Bricaire C, Raynaud A, Benotmane A, Clair F, Paniel B, Mowszowicz I, Wright F, Moreau JF, Kutten F, Mauvais-Jarvis P: Selective venous catherization in the evaluation of hyperandrogenism. J. Endocrinol. invest. 14: 949-956 (1991). 7. Heinonen PK: Androgen production by epithelial ovarian tumors in postmenopausal woman. Maturitas 13: 117-122 (1992).

Miscellaneous

Back to Contents Limitations of use Reliable and reproducible results will be obtained when the assay procedure is carried out with a complete understanding of the package insert instructions and with adherence to good laboratory practice. Azide and thimerosal at concentrations higher than 0.1 % interfere in this assay. Therefore control sera or samples containing higher concentrations of the above mentioned components may give false results. Reagents from different kits or lots should not be mixed, due to possible different shipping or storage conditions. Any improper handling of samples or modification of this test might influence the results. Interferences caused by improper sample handling are explained in chapter `Specimen Collection and Storage'. For diagnostic purpose results obtained by this assay should be used in conjunction with other test results, the overall clinic presentation to the physician, and all other appropriate information.
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