rev: January 18, 1999

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DHEA-Sulfate ELISA Kit   $388.00/kit  
-sample data-see actual kit insert for batch specific information

Catalogue No : RDI-RE52181 Product group : Gynaecology 12 x 8 Product name : DHEA-S **510K** ELISA 96 Method : ELISA Incubation time: 60 min, 15 min Standard curve : 0.1 - 10 µg/ml Sample/Prep. : 25 µl Serum, Plasma Isotope/Substr.: TMB, 450ñ10nm


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


Back to Contents The IBL DHEA-S Enzyme Immunoassay Kit provides materials for the quantitative determination of DHEA-S in serum and plasma. This assay is intended for in vitro diagnostic use. Dehydroepiandrosterone sulfate (DHEA-S) is the major C-19 adrenal steroid primarily secreted by the zona reticularis of the adrenal cortex. DHEA-S is thought to be a biologically weak androgen, but because of its high concentration in serum, it contributes significantly to the androgenization process. Its secretion is regulated by ACTH and possibly other pituitary factors. DHEA-S is the sulfate form of DHEA. Its concentration in serum exceeds that of any other steroid hormone and is about five hundred times that of DHEA. DHEA-S is not subject to diurnal variations or day to day differences. No cyclic change in DHEA-S level has been found during the menstrual cycle. DHEA-S in serum is bound to albumin and under normal circumstances, is stable partly due to slow metabolic clearance. DHEA-S is cleared by the kidneys and its metabolites can be measured in urine as 17-Ketosteroids (17-KS). DHEA-S levels are high at birth but fall rapidly to low levels during infancy and childhood. During the prepubertal years, there is a gradual, but progressive increase and adult levels are reached shortly after puberty. After the third decade, there is a gradual decline in both sexes. During pregnancy, serum DHEA-S levels gradually decline due to placental clearance, reaching approximately 50 % of its initial value at term. The physiological role of DHEA-S is not well known, but it seems to be intricately involved in adrenarche (axillary and pubic hair growth). Low serum DHEA-S levels have been found in premenopausal women with primary operable breast cancer and therefore it has been suggested that its measurement might serve as a tumor marker. DHEA-S appears to be an excellent indicator of adrenal androgen production. Therefore its measurement in conjunction with cortisol can give a more comprehensive evaluation of adrenocortical function since cortisol levels assess function of zona fasciculata while DHEA-S measures function of zona reticularis. Although there is a reasonable correlation between serum DHEA-S and urine 17-KS, serum DHEA-S is a better and more sensitive indicator of adrenal androgen secretion. Elevated levels of DHEA-S have been reported in states of excess androgen production such as cystic acne, hirsutism, infertility, enzymatic adrenal defects. Cushing's syndrome due to bilateral adrenal hyperplasia and virilizing adrenal tumors. DHEA-S measurements have been very helpful in proper identification of source of hyperandrogenism and also assesses adequacy of treatment when dexamethasone suppression therapy is utilized.

Contents of the Kit

Back to Contents Do components contain < 250 µl solution, please care that all the solution is on the bottom of the vial. 1.Microtiter Strips, 12 x 8 wells break apart strips coated with anti DHEA-S antiserum (rabbit polyclonal) in foilbag with desiccant 2.Standard A (Zero Standard) 1 vial 0.5 ml, ready to use human serum (steroid free) 3.Standards (B - G) 6 vials 0.25 ml each, ready to use containing the below mentioned concentrations of DHEA-S in human serum, steroid free Standard B C D E F G Concentration in µg/ml 0.1 0.5 1.0 2.5 5 10 4.Enzyme Conjugate 1 vial 22 ml, ready to use DHEA-S conjugated to the enzyme horseradish peroxidase 5.TMB Substrate Solution 1 vial 11 ml, ready to use containing a solution of tetramethylbenzidine (TMB) in citrate buffer with hydrogen peroxide 6.TMB Stop Solution 1 vial 6 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 (20x) 1 vial 25 ml, concentrate containing buffered saline with a nonionic detergent Dilute 1 to 20 with distilled water prior to use Storage and Stability When stored at 2 - 8 °C unopened reagents will retain reactivity until expiration date. Do not use beyond this date. 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 strips are stable for approx. 6 weeks in the broken, but tightly closed bag. 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 24 hours, and should be frozen at - 10 °C or lower for longer periods Samples suspected to contain DHEA-S concentration higher than 40 µg/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. Please note: Samples containing sodium azide should not be used in the assay. Preparation of Samples and Reagents Wash Buffer Dilute the wash buffer concentrate with distilled water 1 to 20. Ready to use wash buffer has to be stored at 2 - 8 °C.

Principle of the Test

Back to Contents This assay is based on the competition principle and the microtiter plate 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 wells are washed to stop the competition reaction. Having added the TMB 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 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 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. Manual pipetting: It is recommended that not more than 32 wells be used for each assay run. Pipetting of all standards, samples, and controls should be completed within 3 minutes. Automated pipetting: A full plate of 96 wells may be used in each assay run. However, it is recommended that pipetting of all standards, samples, and controls be completed within 3 minutes. All standards, samples, and controls should be run in duplicate concurrently so that all conditions of testing are the same. It is recommended to run tests in duplicate. Leave sufficient microtiter strips in the strip holder to enable the running of standards, controls, and samples in dublicate, plus one well for the chromogen blank. Secure the desired number of microtiter strips in the holder. a) Short Instructions 1. Pipet 25 µl standard or sample 2. Pipet 200 µl enzyme conjugate in each well, mix shortly 3. Incubate for 1 hour at room temperature without covering the plate 4. * Decant supernatant, wash 3x with 400 µl diluted wash buffer 5. ** Pipet 100 µl TMB substrate solution in each well 6. Incubate for 15 minutes at room temperature 7. ** Add 50 µl TMB stop solution and shake plate for homogenizing 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. Pipet 25 µl of standards, controls and samples into the appropriate wells of the strips. Time between distribution of first standard and last sample can be up to 10 minutes without affecting the results. 2. Add 200 µl of enzyme conjugate to each well in sequence. 3. Thoroughly mix the plate for 10 seconds. It is important to have complete mixing in this step. 4. Incubate for 60 minutes at room temperature (18 - 24 °C) without covering the plate. 5. Briskly shake out the contents of the wells. 6. Rinse the wells 3 times with diluted wash buffer (400 µl per well) Strike the wells sharply on absorbent paper to remove residual droplets. 7. Promptly pipet 100 µl of the TMB substrate solution into the rinsed wells. 9. Incubate for 15 minutes at room temperature (18 - 24 °C) 10.Stop the reaction by adding 50 µl of TMB stop solution to each well. 11.Shake gently the microtiter strips being careful not to let the content come from the wells and read at 450 ñ 10 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 µg/ml. For construction of the standard curve we recommend a four parameter logistic function. 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. Do not use this calibration curve. In the laboratory the standard curve should be established in each assay run. Standards OD 450 nm (µg/ml) 0 1.92 0.1 1.53 0.5 1.17 1.0 0.88 2.5 0.59 5.0 0.35 10 0.17

Performance Characteristics

Back to Contents 1. Specificity The following hormones were tested for crossreactivity of the assay: The percentage indicate crossreactivity at 50 % displacement compared to DHEA-S. Steroid % Crossreactivity Steroid % Crossreactivity DHEA-S 100 Estrone 0.9 Androstenedione 20.9 Estriol <0.1 Androsterone 8.5 Estradiol <0.1 Androsterone Sulfate <0.1 Estradiol-Sulfate <0.1 Progesterone 4.7 Cortisol <0.1 Testosterone 0.3 2. Sensitivity The lowest detectable level of human DHEA-S concentration given by the mean absorbance of the zero calibrator minus 3 standard deviations was assessed to be approx. 0.05 µg/ml. 3. Precision 3.1. 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 n Mean Standard CV (%) (µg/ml) Deviation (µg/ml) 1 10 0.52 0.04 7.4 2 10 3.21 0.14 4.3 3 10 5.61 0.15 2.7 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 Standard CV (%) (µg/ml) Deviation (µg/ml) 1 10 0.54 0.047 8.8 2 10 3.59 0.19 5.2 3 10 5.48 0.17 3.1 4. Linearity Three patient samples were serially diluted with zero standard in a linearity study. Sample Dilution Measured Expected Recovery conc. conc. (%) (µg/ml) (µg/ml) neat 2.74 1:2 1.42 1.37 104 1 1:4 0.63 0.69 92 1:8 0.35 0.34 104 neat 4.23 1:2 2.23 2.12 105 2 1:4 1.01 1.06 96 1:8 0.51 0.53 96 1:16 0.21 0.26 79 neat 7.19 1:2 3.32 3.60 90 3 1:4 1.82 1.80 101 1:8 0.92 0.90 102 1:16 0.41 0.45 91 5. Recovery Spiked serum samples were prepared by adding varying levels of DHEA-S to two different serum samples. Sample Endogenous Measured DHEA-S Recovery DHEA-S (µg/ml) (µg/ml) (%) 4.08 112 1 2.0 1.04 108 0.52 106 4.16 106 2.08 113 2 0.28 1.04 97 0.52 95 6. Method Comparison The IBL DHEA-S ELISA was compared to another commercially available DHEA-S assay. A correlation coefficient of r = 0,94 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 = 0,94 IBL ELISA + 0,357

Expected Values

Back to Contents The following values can be used as preliminary guidelines until each laboratory establishes its own normal ranges. µg/ml µmol/l Females Premenopausal 0.8 - 3.9 2.1 - 10.1 Pregnancy (3. Trimenon) 0.2 - 1.2 0.5 - 3.1 Postmenopausal 0.1 - 0.6 0.3 - 1.6 Newborns (both sexes) 1.7 - 3.6 4.4 - 9.4 Males 1.0 - 4.2 2.6 - 10.9 Conversion factor: 1 µg/ml = 2,6 µmol/l Limitations of use During incubation with substrate solution avoid direct sunlight on the microtiter plate. Samples containing sodium azide should not be used in the assay. Do not use grossly hemolyzed or lipemic specimens.

Alternative Applications

Back to Contents 1. Extraction of Steroids from Serum Freshly prepared serum should be used for extraction. In case of frozen sera, they should be mixed and centrifuged after thawing. The supernatant is used for extraction. Extraction procedure: 1. Mix 150 µl serum with 1 ml ether 2. Vortex thoroughly for 2 x 20 sec 3. Centrifuge to reach complete separation of the phases 4. Pipete the upper organic phase (ether) into a new tube and evaporate (e.g. with nitrogen) in a water bath at 35 °C. 5. Reconstitute the residue with 75 µl zero standard. 6. This extract may be used as described in the assay procedure. Note: The result obtained by this procedure has to be multiplied by 2 to consider the different volumes (serum and reconstituted residue). 2. 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 3500 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 Clinical Applications 1. Virilizing Disorders: DHEA-S concentrations exceeding 6.6 µg/ml strongly support the diagnosis of adrenal neoplasm and indicate the need for computerized tomography of the adrenal cortex. Normal range DHEA-S concentrations, however, do not entirely rule out the diagnosis of adrenal virilizing tumor since there are cases reports of such tumors secreting excesses of other androgens such as testosterone. For this reaction, the diagnostic survey should include both DHEA-S and testosterone measurements along with suitable x-ray studies, vascular catheterization and surgical exploration. DHEA-S concentrations in upper normal or modestly elevated ranges are frequently seen in benign androgenizing disorders such as polycystic ovarian disease. DHEA-S concentrations are usually in the normal range in patients afflicted with late onset 21-hydroxylase deficiency adrenal hyperplasia. 2. Ovulation Induction Resistant To Clomiphene Citrate: Some infertility patients with polycystic ovarian disease are minimally androgenized but are very resistant to induction of ovulation with traditional drugs such as clomiphene citrate or human menopausal gonadotropin (HMG). Such patients may have elevated DHEA-S concentrations and can be induced to ovulate following suppression of DHEA-S with mini doses of corticosteroids. Ovulation may resume following mini dose corticosteroids alone or may still require clomiphene citrate or HMG although the doses are usually reduced, sometimes at a considerable savings of expensive medications. Thus serial DHEA-S measurements are frequently useful in setting doses of corticosteroids in patients with polycystic ovarian disease who are otherwise resistant to ovulation stimulation. Factors Affecting Normal Values: Drugs: Corticosteroids: DHEA-S concentrations are suppressed in patients with a gradual falloff with advancing age. Age: Peak DHEA-S concentrations are seen during the third decade with a gradual falloff with advancing age. Malnutrition: DHEA-S concentrations are low. Obesity: DHEA-S concentrations are frequently elevated in obese women. Hyperprolactinemia: DHEA-S concentrations are frequently elevated in women with hyperprolactinemia

Sales Arguments

Back to Contents DHEA-S ELISA Cat.-No. RDI-RE52181 Assay advantages for the IBL assay - 510K exempt by FDA - Break apart wells for individual determinations - Reagents ready for use - Incubation at room temperature - Short incubation time - Suitable for automation - Excellent correlation to RIA Potential customers University-labs, Private-labs, Gynaecologists, Endocrinologists Clinical Applications Diagnosis and identification of the origin of hyperandrogenemia and therapy follow up at: - Hirsutism and virilism - Adrenogenital syndrome - Cushing's-tumor-Syndrom caused by adrenal hyperplasia - Tumors of the adrenal cortex

Product Literature

Back to Contents 1. Abraham GE. J. Clin. Endocrinol. Metabol. 39: 340, ( 1974). 2. Abraham GE. Progress in Gynecology VI, 111-113, (1975). 3. Chakmakjian ZH et al. Obstet. Gynecol. 46: 544, (1975). 4. Nelson DH. The Adrenal Cortex, XVIII, 102, (1980). 5. Abraham GE et al. J. Clin. Endocrinol. Metabol. 37: 581 (1973). 6. Abraham GE et al. Obstet. Gynecol. 44: 171 (1974). 7. Lobo RA et al. Obstet. Gynecol. 57: 69 (1981). 8. Maroulis GB et al. Obstet. Gynecol. 49: 454 (1976). 9. Forst MG et al. The Endocrine Function of the Human Cortex, Academic Press, 561-562 (1978). 10.Vermeulen A. Adrenal Androgens, Raven Press, 199-200 (1980). 11.Parrini D et al. Ibid. 199-200. 12.Zumoff B et al. Cancer Res. 41: 3360 (1981). 13.Abraham GE et al. The Infertile Female, 213-215 (1979). 14.Marynik SP et al. N. Engl. J. Med. 308: 981 (1983). 15.Schindler AE. Androgenization in Women, Excerpta Medica, 93-95 (1980). 16.Yamaji T et al.. I. Clin. Endocrin. 29: 273 (1969). 17.Abraham GE et al. The Role of Adrenal Cortex in Hirsutism, Academic Press, 33-35 (1978). 18.Abraham GE et al. Obstet. Gynecol. 57: 158, (1981).


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