FOR RESEARCH USE ONLY--NOT FOR USE IN DIAGNOSTIC PROCEDURES

Transcription

1 ENGLSH Androstenedione EA DSL Revision date: March 8, 2005 Diagnostic Systems Laboratories, nc. Corporate Headquarters, 445 Medical Center Blvd. Webster, Texas USA General Business: Tel: Customer Assistance Center: Tel: Fax: Website: FOR RESEARCH USE ONLY--NOT FOR USE N DAGNOSTC PROCEDURES This Package nsert (FU) is intended for Professional Use and must be read completely before product use.. NTENDED USE The DSL ACTVE Androstenedione Enzyme mmunoassay (EA) Kit provides materials for the quantitative measurement of androstenedione in serum or plasma. This assay is intended for in vitro research use in the measurment and treatment of females with excessive levels of androgen production.. SUMMARY AND EXPLANATON OF THE TEST Androstenedione (ASD, 4-Androstene-3,17-dione), a C steroid, is produced in the adrenal gland and gonads. 19 ASD is an immediate precursor to both testosterone and estrone, both of which may be subsequently converted to estradiol. Due to the presence of a 17-oxo (rather than hydroxyl) group, ASD has relatively weak androgenic activity, estimated at <20% of testosterone [1]. Although it is a weak androgen, serum ASD levels may exceed testosterone in both normal and disease states, ASD secretion and production rates exceed those of testosterone in women, and significant extra-adrenal conversion of ASD to testosterone occurs. Furthermore, the affinity of sex hormone-binding globulin for ASD is much less than for testosterone or estradiol [1-3]. The physiologic role of ASD is not well-defined. Serum ASD levels are high in fetal and neonatal serum, decrease during childhood, and increase during puberty. n normal pubertal and adult men, the major portion of ASD is derived from the testis, either directly or from conversion of testosterone, while in normal adult women, essentially equivalent amounts of ASD are produced by the adrenal gland and ovary [2,3]. ncreased ASD levels may play a role in the development of secondary sexual hair during adrenarche. Serum ASD levels show significant diurnal variation dependent on the secretion of ACTH. Ovarian ASD production is stimulated by luteinizing hormone, and serum ASD levels vary with the menstrual cycle [3]. Adrenal ASD production gradually declines with advanced age in both men and women. n addition, ovarian ASD production decreases after menopause [3]. Measurement of serum ASD provides a useful marker of androgen biosynthesis. Serum ASD levels are increased in polycystic ovary syndrome, ovarian stromal hyperthecosis, 3 -hydroxysteroid dehydrogenase deficiency, and other causes of hirsutism in women [3-5]. By definition, ASD levels are normal in idiopathic hirsutism. Elevated serum ASD levels may also occur in adrenal and ovarian virilizing tumors [3]. Assays for ASD include gas-liquid chromatography, mass spectrometry, and immunoassay. The DSL ASD Assay Kit uses a specific and sensitive rabbit anti-human ASD polyclonal antiserum [4-6]. Cross-reactivity to DHEA, DHEA-S, testosterone and androgen metabolites is negligible.. PRNCPLE OF THE TEST The procedure follows the basic principle of enzyme immunoassay where there is competition between an unlabeled antigen and an enzyme-labeled antigen for a fixed number of antibody binding sites. The amount of enzyme-labeled antigen bound to the antibody is inversely proportional to the concentration of the unlabeled analyte present. Unbound materials are removed by decanting and washing the wells. V. REAGENTS The DSL ACTVE Androstenedione EA Kit contains sufficient reagents for 96 wells. Each kit contains the following reagents: A. GARG-Coated Microtitration Strips: One stripholder containing 96 microtitration wells coated with goat anti-rabbit gamma globulin serum. Store at 2-8 C until expiration date in the resealable pouch with a desiccant to protect from moisture. ANDROSTENEDONE EA DSL

2 B. Androstenedione Antiserum: (BLUE) One vial, 11 ml, containing rabbit anti-androstenedione serum in a protein-based (BSA) buffer with a non-mercury preservative. Store at 2-8 C until expiration date. C. Androstenedione Standards A-F: Six vials, 0.5 ml each, labeled A-F, containing concentrations of approximately 0.0, 0.1, 0.3, 1.0, 3.0 and 10.0 ng/ml androstenedione in a buffer containing a protein stabilizer with a non-mercury preservative. Refer to vial labels for exact concentrations. Store unopened at 2-8ºC until kit expiration date. Store opened vials at 2-8 C for up to 3 weeks or at 20ºC or lower for longer periods. STANDARDZATON NOTE: Due to the lack of universally accepted material, the reference preparation of the Androdstenedione Standard s & Controls was obtained from Steraloids, USA, purified by HPLC, purity verifi ed (single spot) by Thin Layer Chromotography and performance verified by immunoassay. D. Androstenedione Controls: Two vials, 0.5 ml each, Levels and, containing low and high concentrations of androstenedione in a buffer containing a protein stabilizer with a non-mercury preservative. Refer to vial labels for Control ranges. Store unopened at 2-8ºC until kit expiration date. Store opened vials at 2-8 C for up to 3 weeks or at 20ºC or lower for longer periods. E. Androstenedione Enzyme Conjugate-Ready-to-Use (RTU): One bottle, 11 ml, containing androstenedione conjugated to the enzyme horseradish peroxidase in a protein-based (BSA) buffer with a non-mercury preservative. Store at 2-8 C until expiration date. F. TMB Chromogen Solution: One bottle, 11 ml, containing a solution of tetramethylbenzidine (TMB) in citrate buffer with hydrogen peroxide. Store at 2-8 C until labeled expiration date. H. Wash Concentrate: One bottle, 100 ml, containing buffered saline with a nonionic detergent. Store at 2-8 C until expiration date. Dilute 10-fold with deionized water prior to use.. Stopping Solution: One bottle, 11 ml, containing 0.2 M sulfuric acid. Store at 2-8 C until expiration date. NOTE: All reagents and samples must be allowed to reach room temperature (~25 C) and mixed thoroughly by gentle inversion before use. V. PRECAUTONS For in vitro use only. Not for nternal or External Use in Humans or Animals The following Good Laboratory Practices should be observed: Do not eat, drink, smoke, or apply cosmetics where immunomeasurment materials are being handled. Do not pipet by mouth. Wear lab coats and disposable gloves when handling immunomeasurment materials. Wash hands thoroughly afterwards. Cover working area with disposable absorbent paper. Wipe up spills immediately and decontaminate affected surfaces. Avoid generation of aerosols. Provide adequate ventilation. Handle and dispose of all reagents and material in compliance with applicable regulations. WARNNG: POTENTAL BOHAZARDOUS MATERAL This kit may contain some reagents made with human source material (e.g. serum or plasma) or used in conjunction with human source materials. The material in this kit has been tested by FDA recommended methods and found to be non-reactive for HV-1/2 Antibodies, HCV and HBsAg. o available test method can offer complete assurance of eliminating potential biohazardous risk. Handle all reagents and samples at a Biosafety Level 2, as recommended for any potentially infectious human material in the Centers for Disease Control/National nstitutes of Health manual "Biosafety in Microbiological and Biomedical Laboratories," 4 th Edition, April WARNNG: POTENTAL CHEMCAL HAZARD Some reagents in this kit contain ProClin as a preservative. ProClin, TMB, hydrogen peroxide, and sulfuric acid, in concentrated amounts are irritants to skin and mucous membranes. These substances are in diluted form and therefore may minimize exposure risks significantly but not completely. Provide adequate ventilation. Avoid contact with skin, eyes, and clothing. n case of contact with any of these reagents, wash area thoroughly with water and seek medical advice. Dispose of all nonradioactive reagents by flushing with large volumes of water to prevent buildup of chemical hazards in the plumbing system. For further information regarding hazardous substances in the kit, please refer to the component specific MSDS, either at DSLabs.com or by request. V. SPECMEN COLLECTON AND PREPARATON ANDROSTENEDONE EA DSL

3 Serum or plasma should be used and the usual precautions for venipuncture should be observed. The serum or plasma samples may be stored at 2-8 C for up to 1 week or frozen at -20 C or lower for up to 2 months. Avoid repeated freezing and thawing of samples. Do not use hemolyzed, icteric or lipemic samples. Frozen samples should be thawed and mixed thoroughly by gentle swirling or inversion prior to use. V. PROCEDURAL NOTES A thorough understanding of this package insert is necessary for successful use of the product. Reliable results will only be obtained by using precise laboratory techniques and accurately following the package insert. A standard curve must be included with each assay. Bring all kit reagents and specimens to room temperature (~25 C) before use. Thoroughly mix the reagents and samples before use by gentle inversion. Do not mix various lots of any kit component within an individual assay. Do not use any component beyond the expiration date shown on its label. ncomplete washing will adversely affect the outcome and assay precision. To minimize potential assay drift due to variation in the substrate incubation time, care should be taken to add the stopping solution into the wells in the same order and speed used to add the TMB Chromogen Solution. Avoid microbial contamination of reagents, especially of the conjugate concentrate and the conjugate diluent (Assay Buffer). Avoid contamination of the TMB Chromogen Solution and with the Antigen-Enzyme Conjugate. Use a clean disposable pipette tip for each reagent, Standard, Control or specimen. For dispensing sulfuric acid and TMB Chromogen Solution, avoid pipettes with metal parts. Containers and semi-automatic pipette tips used for the Antigen-Enzyme Conjugate Solution and the TMB Chromogen Solution can be reused provided they are thoroughly rinsed with distilled water and dried prior to and after each usage. The enzyme used as the label is inactivated by oxygen, and is highly sensitive to microbial contamination, sodium azide, hypochlorous acid and aromatic chlorohydrocarbons often found in laboratory water supplies. Use high quality water. Avoid exposure of the reagents to excessive heat or direct sunlight during storage and incubation. V. TEST PROCEDURE A. Materials Supplied: Materials supplied in the DSL ACTVE Androstenedione EA Kit, Catalog No. DSL MATERAL QUANTTY CATALOG NO. Androstenedione Standard A Androstenedione Standard B Androstenedione Standard C Androstenedione Standard D Androstenedione Standard E Androstenedione Standard F GARG-Coated Microtitration Strips Androstenedione Antiserum Androstenedione Enzyme Conjugate--RTU Wash Concentrate TMB Chromogen Solution Stopping Solution A Androstenedione Control Level Androstenedione Control Level One Plate B. Materials Required But Not Supplied: Microtitration plate EA reader capable of absorbance measurement at 450 nm and preferentially capable of dual wavelength correction at 600 or 620 nm Deionized water Precision pipette to deliver 50 µl Repeating pipette to deliver 100 µl Automatic microtitration plate washer Vortex mixer Microtitration plate shaker capable of orbital revolutions per minute (rpm) Absorbent materials for blotting the strips Graph paper for manual data reduction C. Preparation of Reagents: 1. Wash Solution Pour 100 ml of the Wash Concentrate into a clean container and dilute by adding up to 1000 ml of deionized water. The Wash Solution is stable for one month at room temperature (~25 C) provided the bottle is kept tightly sealed. 2. Microtitration Wells Select the number of coated wells required for the assay. The remaining unused wells should be placed in ANDROSTENEDONE EA DSL

4 the resealable pouch with a desiccant. The pouch must be resealed to protect from moisture. D. Assay Procedure: Allow all specimens and reagents to reach room temperature (~25 C) and mix thoroughly by gentle inversion before use. Standards, Controls and unknowns should be assayed in duplicate. 1. Mark the Microtitration Strips to be used. 2. Pipet 25 µl of the Standards, Controls and unknowns to the appropriate wells. 3. Add 100 µl of the Conjugate RTU to each well using a semi-automatic dispenser. 4. Add 100 µl of the Androstenedione Antiserum to each well using a semi-automatic dispenser. 5. ncubate the wells, for 1 hour at room temperature (~25 C) on a shaker set at rpm. 6. Aspirate and wash each well five times with the diluted Wash Solution using an automatic microplate washer. Blot dry by inverting the plate on absorbent material. NOTE: Use of an automatic microplate washer is strongly recommended. ncomplete washing will adversely affect assay precision. f a microplate washer is not available, (a) completely aspirate the liquid from each well, (b) dispense 0.35 ml of the Wash Solution into each well, and (c) repeat steps (a) and (b) five times. 7. Add 100 µl of the TMB Chromogen Solution to each well using a semi-automatic dispenser. 8. ncubate the wells, for minutes at room temperature (~25 C) on a shaker set at rpm. Avoid exposure to direct sunlight. 9. Add 100 µl of the Stopping Solution to each well using a semi-automatic dispenser. Shake gently by hand. 10. Read the absorbance of the solution in the wells within 30 minutes, using a microplate reader set to 450 nm. NOTE: f wavelength correction is available, set the instrument to dual wavelength measurement at 450 nm with background wavelength correction set at 600 or 620 nm. X. CALCULATON OF RESULTS A. Calculate the mean absorbance for each Standard, Control or unknown. B. Using a linear-log graph paper, plot the mean absorbance readings for each of the Standards along the y-axis versus the androstenedione concentrations in ng/ml along the x-axis. Alternatively, any data reduction software designed for immunoassays could be used. Four-parameter curve-fit is recommended. C. Draw the best fitting curve through the mean of the duplicate points. D. Determine the androstenedione concentrations of the unknowns and controls from the standard curve by matching their mean absorbance readings with the corresponding androstenedione concentrations. E. Any sample reading higher than the highest Standard should be appropriately diluted with the 0 ng/ml Standard and reassayed. F. Any sample reading lower than the lowest Standard should be reported as such. G. Multiply the value by the dilution factor if required. f the microplate reader is not capable of reading absorbance greater than the absorbance of the zero Standard, a second reading at 490 or 492 nm is needed (reference fi lter 600 or 620, if available). n this case, proceed to cons truct a second standard curve as above with the abs orbance readings of all of the standards at 490 nm. The concentration o f the off-scale samples at 450 nm are then read from the new standard curve. The readings at 490 nm should not replace the on-scale readings at 450 nm. X. LMTATONS The reagents supplied in this kit are optimized to measure androstenedione levels in serum or plasma. Avoid repeated freezing and thawing of reagents and specimens. Hemolyzed, icteric or lipemic specimens may give false values and should be avoided. The results of this assay should be used in conjunction with other pertinent information. X. QUALTY CONTROL The DSL Controls or other commercial controls results should fall within the established confidence limits. The confidence limits for the DSL Controls are printed on the Control vial labels. Low and high level controls should be run with each assay. The TMB Solution should be colorless. Development of a blue color may indicate reagent contamination or instability. ANDROSTENEDONE EA DSL

5 WELL NO. A1,A2 B1,B2 C1, C2 D1, D2 E1, E2 F2,F2 TYPCAL ACTVE ANDROSTENEDONE EA STANDARD CURVE DATA WELL CONTENTS STANDARDS A B C D E F MEAN ABSORBANCE CONCENTRATON CAUTON: The above data must not be employed in lieu of data obtained by the use r in the laboratory. X. EXPECTED VALUES Each laboratory should establish its own normal ranges. The results of an expected range study conducted with the DSL ACTVE Androstenedione EA kit with apparently normal healthy adults are as follows: POPULATON N MEAN Male Female S.D MEDAN Central 95% X. PERFORMANCE CHARACTERSTCS All performance characteristics are stated in ng/ml. To convert to nmol/l: ng/ml x 3.45 = nmol/l A. Sensitivity: The theoretical sensitivity, or minimum detection limit, calculated by the interpolation of the mean minus two standard deviations of 20 replicates of the 0 ng/ml Androstenedione Standard,is 0.03 ng/ml. B. Precision: The intra-assay precision was determined from the mean of 11 replicates each with three human serum samples. SAMPLE N MEAN STANDARD DEVATON COEFFCENT OF VARATON(%) The inter-assay precision was determined from 10 separate runs over a twelve-day period with three human serum samples. SAMPLE N MEAN STANDARD DEVATON COEFFCENT OF VARATON (%) C. Recovery: Three human serum samples containing different levels of endogenous androstenedione were spiked with known amounts of androstenedione and assayed. SAMPLE ENDOGENOUS ADDED EXPECTED OBSERVED RECOVERY (%) ANDROSTENEDONE EA DSL

6 D. Linearity: Three human serum samples were diluted with the 0 ng/ml Androstenedione Standard and assayed. SAMPLE DLUTON FACTOR EXPECTED OBSERVED RECOVERY (%) 1:2 1:4 1:8 1:16 1:2 1:4 1:8 1:16 1:2 1:4 1:8 1:16 1: F. Method Comparison: The DSL ACTVE Androstenedione EA has been compared to a commercially available RA kit (Method X). Ninety-nine serum samples ranging in value from 0.08 to ng/ml were assayed and linear regression analysis of the results yielded the following: Regression: [DSL ] = 1.1 [Method X] r = 0.97 XV. REFERENCES 1. Dorfman R, Shipley RA: Androgens. John Wiley and Sons, New York, pp , Horton R, Tait J: Androstenedione production and interconversion rates measured in peripheral blood and studies on the possible site of its conversion to testosterone. J Endocrinol nvest 45: , Pang S, Riddick L: Hirsutism. N Lifshitz F (ed): Pediatric Endocrinology, A Clinical Guide, second edition. Marcel Dekker, nc., New York, 1990, pp Rittmaster RS, Thompson DL: Effects of leuprolide and dexamethasone on hair growth and hormone levels in hirsute women: The relative importance of the ovary and adrenal in the pathogenesis of hirsutism. J Clin Endocrinol Metab 70: , Zwicker H, Rittmaster RS: Androsterone sulfate: Physiology and significance in hirsute women. J Clin Endocrinol Metab 76: , Rosen M. Nouri N, Alexander L: Evaluation of a direct assay for the measurement of androstenedione. Clin Chem 33:891, 1987 (abstract 054) ANDROSTENEDONE EA DSL