rev: January 18, 1999
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Dihydrotestosterone ELISA Kit $612.00/kit -sample data-see actual kit insert for batch specific information
Catalogue No : RDI-DB52021 Product group : Steroids 12 x 8 Product name : Dihydrotestosterone ELISA 96 Method : ELISA Incubation time: 1 h, 15 min Standard curve : 25-500 pg/ml Sample/Prep. : 50 µl Serum, Plasma Isotope/Substr.: TMB 450nm
CONTENTSIntroduction Contents of the Kit Principle of the Test Test Procedure Performance Characteristics Expected Values Alternative Applications (More) Clinical Background Sales Arguments Product Literature Miscellaneous
IntroductionBack to Contents The 5-alpha dihydrotestosterone Enzyme Immunoassay Kit provides materials for the quantitative determination of dihydrotestosterone in serum and plasma. This assay is intended for in vitro diagnostic use. 5-alpha dihydrotestosterone (DHT) is a steroid similar to testosterone and androstenedione, which belongs to a class called androgens. 5-alpha dihydrotestosterone is a C19 steroid and possesses androgenic activity. The bulk of androgen production takes place mainly in the leydig cells of the testes. Androgens circulate in the blood bound to proteins, especially sex hormone binding globulin (SHBG) and albumin. A trace amount of these steroids circulate in the unbound (free) form in blood. Dihydrotestosterone has at least 3 times the binding affinity to SHBG than does testosterone. In males about 70 % of DHT is derived from peripheral conversion of testosterone. However, in females most of the DHT is derived from androstenedione. In the body testosterone is converted to DHT, estradiol and 5-alpha-androstane-3-alpha,17-beta-diol. The major organ to metabolize androgens is the liver. Therefore in the liver the steroid hormones undergo structural modifications which is generally regarded as prerequisites for their biological inactivation. Some metabolites are formed and some are returned to the circulation before renal excretion. Therefore, elimination of steroids from the body is done through the urine. In Klinefelter's syndrome the DHT level is much lower than that found in normal men. In idiopathic hirsutism about 40 % of the patients have an increased level of DHT. In polycystic ovaries (PCO) about 35 % of the patients have an increased DHT level. The DHT levels of young people are much higher than those found in normal older people, hence androgens production increases at puberty which gives rise to masculinizing characteristics. It has been demonstrated that the human testes produces DHT, which appears to originate in the seminiferous tubules. Therefore, in tubular damage the production of DHT is impaired, which causes a decrease in the levels of plasma DHT (patients with germinal cell aplasia and azoospermia). There is a very low level of plasma DHT in patients with anorchia. It has been reported that in some prostate cancer, especially in stage D, the determination of DHT could be useful in predicting the response to anti-androgen therapy.
Contents of the KitBack to Contents 1. Coated Microtiter Strips 12 x 8 wells wells coated with anti-Dihydrotestosterone antiserum (rabbit, polyclonal) 2. Standard A (Zero Standard) 1 vial 1 ml, ready to use containing steroid free serum and buffer. 3. Standards (B-F) 5 vials 0.6 ml each, ready to use containing the below mentioned concentrations of dihydrotestosterone in serum based buffer. Standards B C D E F Concentration in pg/ml 25 100 500 1000 2500 4. Control 1 vial lyophilized, reconstiute the control with 0.5 ml distilled water. (Concentration is mentioned on the separate QC sheet). 5. Enzyme Conjugate, concentrated (100x) 1 vial 0.2 ml, concentrate, Dihydrotestosterone conjugated to the enzyme horseradish peroxidase 6. TMB Substrate Solution 1 vial 16 ml, ready to use Containing a solution of tetramethylbenzidine (TMB) with hydrogen peroxide in buffer supplemented by stabilizers 7. TMB Stop Solution 1 vial 6 ml, ready to use, 0.3 M sulfuric acid (H2SO4). Avoid contact with stop solution it may cause skin irritations and burns. 8. Wash Buffer, concentrate (10x) 1 vial 50 ml, concentrate, containing borate-citrate buffer with a nonionic detergent, dilute 1 to 10 with distilled water prior to use. 9. Assay Buffer 1 vial 11 ml, ready to use, vortex immediately before use Storage and Stability Store all reagents at 2 - 8 °C protected from intense light and use before expiry date. Control should be stored after reconstitution up to 6 month at - 20 °C. Unused microtiter strips must always be stored at 2 - 8 °C in the resealable bag provided. Allow all reagents and required number of strips to reach room temperature prior to use. Collection 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 5 hours, and should be frozen at - 20 °C or lower for longer periods. Samples suspected to contain DHT concentration higher than 2500 pg/ml are to be diluted with zero standard. Repeated freeze - thawing should be avoided. Thawed samples should be inverted several times prior to testing. Do not use grossly hemolyzed or grossly lipemic specimens. Preparation of Samples and Reagents Store all reagents at 2 - 8 °C. Allow all components to reach room temperature. 1. Enzyme Conjugate : Dilute 1 to 100 in assay buffer before use (e.g. 10 µl of conjugate in 1 ml of assay buffer). If the whole plate is used, dilute 110 µl of conjugate in 11 ml of assay buffer. Store unused diluted conjugate up to 4 days at 2 - 8 °C. 2. Wash Buffer, concentrate: Dilute 1 to 10 with distilled water before use. Occasionally crystals will form. Warm solution to ensure completely dissolved before use. 3. Control Reconstitue the lyophilized control with 0.5 ml distilled water.
Principle of the TestBack to Contents This assay 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 ProcedureBack to Contents It is recommended to use control samples according to state and federal regulations. The use of control sera or plasma is advised to assure the day to day validity of results. Use controls at both normal and pathological levels. 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 pipete tips for each reagent, standard or specimen in order to avoid cross contamination. For the dispensing of the TMB substrate solution and the TMB 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. a) Short Instructions 1. Pipet 50 µl standards, controls or unknowns 2. Pipet 100 µl enzyme conjugate in each well 3. Incubate for 60 minutes at room temperature (18 - 24 °C) on a shaker 4. * Decant supernatant, wash 3x with 250 µl diluted wash buffer 5. ** Pipet 150 µl TMB substrate solution in each well 6. Incubate for 5 - 15 minutes at room temperature (18 - 24 °C) on a shaker 7. ** Add 50 µl TMB stop solution and shake plate for homogenising 8. Measure absorbance at 450 nm * wash procedure is essential for the assay results ** stop solution should be pipetted in the same time schedule as substrate solution b) Detailed Instructions 1. Leave sufficient microtiter strips in the strip holder to enable the running of standards, controls, and samples, plus one well for the chromogen blank. 2. Pipet 50 µl of standards, controls and samples into the appropriate wells of the strips. 3. Add 100 µl of enzyme conjugate to each well in sequence. 4. Incubate for 1 hour at room temperature (18 - 24 °C) on a shaker. 5. Washing: discard the incubation solution, rinse the wells 3x with the wash buffer (dilute concentrate 1 to 10 with distilled water) and remove any residual. 6. Promptly pipet 150 µl of the TMB substrate solution into the rinsed wells. 7. Incubate for 10 - 15 minutes at room temperature (18 - 24 °C) on a shaker. 8. Stop the reaction by adding 50 µl of TMB stop solution to each well. 9. Shake gently the microtiter strips being careful not to let the content come from the wells and read at 450 nm within 30 minutes from the stopping. Calculation of Results Any ELISA reader capable of determining the absorbance at 450 ñ 10 nm may be used. The antigen concentration of each sample is obtained as follows: 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. Use the average absorbance of each sample to determine the corresponding antigen value by simple interpolation from this standard curve, multiplying by the initial sample dilution, if necessary. Alternatively the use of electronic device is possible. The results can also be calculated with normal programs for automatic data processing, i.e. 4 Parameter, Spline, Logit-Log. Any sample reading greater than the highest standard should be diluted appropriately with the zero standard and reassayed. Do not use this calibration curve. In the laboratory the standard curve should be established in each assay run. Standards (pg/ml) OD 450 nm 0 2.096 25 1.528 100 1.166 500 0.512 1000 0.347 2500 0.250
Performance CharacteristicsBack to Contents 1. Specificity The specificity of the DHT assay was assessed according to Abraham's method. The percentage indicate cross-reactivity at 50 % displacement compared to DHT. Steroid % cross-reactivity Steroid % cross-reactivity Dihydrotestosterone 100.0 17beta-Estradiol < 0.01 Testosterone (32*) Estriol < 0.01 5beta-Dihydrotestosterone 2 Estrone < 0.01 Androstenedione 0.2 Progesterone < 0.01 Dehydroepiandrosterone < 0.01 17-OH-Progesterone < 0.01 sulfat Pregnenolone < 0.01 Cortisol < 0.01 * This crossreactivity does not influence the testresults due to a specific complexing buffer system, which blocks the binding of testosterone to the antibody. 2. Sensitivity The minimal detectable concentration of DHT by this assay is estimated to be 6 pg/ml (defined as 2x standard deviation to the zero standard). 3. Precision 3.1. Intra Assay Variation Within run variation was determined by replicate determination of two different control sera in one assay. The within assay variability is shown below: Sample n Mean S.D. C.V. (%) 1 10 348.62 38.96 11.2 2 10 913.39 91.93 10.1 3.2. 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 n Mean S.D. C.V. (%) 1 10 343.09 41.32 11.1 2 10 32.60 3.88 12.0 4. Linearity Three patient samples were assayed, both undiluted and diluted with calibrator A. The results (in pg/ml) are tabulated below: Sample Measured conc. Expected conc. Recovery (%) (pg/ml) (pg/ml) A neat 495.8 A 1:2 248.8 247.9 100 A 1:4 132.0 124.0 106 A 1:8 60.4 62.0 97 B neat 569.6 B 1:2 264.2 284.8 93 B 1:4 133.5 142.2 94 B 1:8 62.6 71.2 88 C neat 641.0 C 1:2 295.2 320.5 92 C 1:4 154.8 160.3 97 C 1:8 100.4 80.1 125 5. Recovery Dihydrotestosterone Standards (D, E, F) were used to spike 3 different samples (1 : 3). The results (in pg/ml) are tabulated below: Sample Measured Expected conc. Recovery % conc. (pg/ml) (pg/ml) 1 neat 766.4 1+D 728.3 729.1 100 1+E 823.2 799.1 103 1+F 987.2 1009.1 98 2 neat 79.9 2+D 134.0 138.8 97 2+E 174.4 208.8 84 2+F 401.2 418.8 96 3 neat 439.8 3+D 447.0 448.2 100 3+E 512.6 518.2 99 3+F 671.1 728.2 92
Expected ValuesBack to Contents The following values can be used as preliminary guidelines until each laboratory establishes its own normal ranges. pg/ml Females: Premenopausal 24-368 Postmenopausal 10-181 Males: 250-990
Alternative ApplicationsBack to Contents
(More) Clinical BackgroundBack to Contents Dihydrotestosterone (DHT) DHT is responsible for the development of the genitals and prostate in men as well as the physical changes during puberty. Furthermore it is essential for the development and maintenance of libido and sexual potency. With the possibility to determine DHT directly, the measurement of 3- Androstanediol as well as the corresponding glucuronide is unnecessary, especially in serum these only represent the DHT metabolism of skin. Clinical Applications: Diagnosis and follow up at: Male pseudohermaphroditism Enzyme deficiency of testosterone production Congenital adrenal hyperplasia Late-onset adrenal hyperplasia Adiposity Virilizing adrenal tumours Follow up of finasterid (therapy in patients with BPH) Clinical consequences of hyperandrogenemia: Androgenizing disorders (Acne, Hirsutism) Amenorrhoe Infertility Increased risk for breast tumours Increased risk for carcinoma of the prostate and BPH
Sales ArgumentsBack to Contents Dihydrotestosterone (DHT) ELISA Cat.-No. DB 520 21 Assay advantages for the IBL assay Low sample volume necessary (50 µl) No extraction necessarry No oxidation required No organic solvents Incubation at room temperature Short incubation time High sensitivity Suitable for automation Potential customers University-labs, Private-labs, Gynaecologists, Endocrinologists, Pediatricians, Dermatologists Clinical Applications Diagnosis and follow up at: - Androgen-related disorders with disturbed development of the genitals - Functional hyperandrogenemia - Organic hyperandrogenemia
Product LiteratureBack to Contents 1. Bassett RM. A simple chromatographic method for the radioimmunoassay of four androgenic steroids. Medical Laboratory Sciences, 37: 31 (1980). 2. Baxendale PM et al. Plasma and salivary androstenedione and dihydrotestosterone in women with hyperandrogenism. Clinical Endocrinology, 18: 447 (1983). 3. Brooks RV. Androgens. Physiology and Pathology. In: Makin, HLJ (ed.) Biochemistry of Steroid Hormones, 2nd ed., Oxford Blackwell Scientific Publications, 565: (1984). 4. Cameron EHD. In proceedings of the fifth tenovous workshop. Steroid Immunoassay (ed.: Cameron EHD et al, Alpha Omega Publishing, Cardiff, (1975). 5. Condom R et al. The preparation of three 5 dihydrotestosterone - BSA conjugates, a comparison of the antigenic properties. Journal of Steroid Biochemistry 8: 1085 (1977). 6. Dunn JF et al. Transport of Steroid Hormones: binding of 21 endogenous steroids to both SHBG and CBG in human plasma. J. Clin. Endocr. Metab. 53: 58 (1981). 7. Hammond GL et al. The simultaneous radioimmunoassay of seven steroids in human spermatic and peripheral venous blood. J. Clin. Endocr. Metab. 45: 16 (1977). 8. Ito T et al. Dihydrotestosterone in Human Peripheral Plasma. J. Clin. Endocr. 31: 362 (1970). 9. Llewelyn DEH et al. The use of multivariable standard curves in the radioimmunoassay of testosterone and 5 dihydrotestosterone. Steroid, 28: 339 (1976). 10.Mean F et al. Study of the Binding of Dihydrotestosterone, Testosterone and Oestradiol with Sex Hormone Binding Globulin. Clinica Chimica Acta 80: 171 (1977). 11.Mooradian AD et al. The Biological actions of Androgens. Endocr. Rev. 8: 1 (1987). 12.Pazzagli M et al. Radioimmunoassay of plasma dihydrotestosterone in normal and hypogonadal men. Clin. Endocr. 82: 380 (1976). 13.Wakelin K et al. Relationship of 5-alpha-dihydrotestosterone and 5-dihydro-testosterone to testosterone in health and disease. J. Endocrinol. 87: 450 (1980). 14.Wang C et al. Solitary androgens in hirsutism: are they of use in routine evaluation. Ann. Clin. Biochem. 23: 590 (1986).
MiscellaneousBack 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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