rev: January 18, 1999

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Androstenedione ELISA Kit   $525.00/kit  
-sample data-see actual kit insert for batch specific information

Catalogue No : RDI-RE52161 Product group : Steroids 96 Product name : Androstenedione ELISA 96 Method : ELISA Incubation time: 60 min, 30 min, Standard curve : 0.1 - 10.0 ng/ml Sample/Prep. : 20 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 Androstenedione is produced by the adrenals and gonads. Therefore, the determination of the level of androstenedione in serum is important in the evaluation of the functional state of the glands. Androstenedione is a precursor of testosterone and estrone. Besides the adrenals, in females, the ovaries have been shown to be an important source of androstenedione during the ovulatory cycle.The principle production of testosterone in females is from the conversion of other related androgens, especially androstenedione. An abnormal testosterone level in women should be accompanied by the estimation of serum androstenedione. The use of serum testosterone determination in conjunction with Enzyme Immunoassay of androstenedione can be used to determine if source of excess androgen production is adrenal or ovarian.

Contents of the Kit

Back to Contents For 96 Determinations: 1. Coated microtiterplate 12 x 8 Wells wells coated with anti rabbit antiserum (goat polyclonal) 2. Antiserum 1 vial 11 ml, ready to use, containing anti androstenedione antiserum (rabbit polyclonal) in phosphate buffer, yellow coloured containing 0.01% Kathon OG as preservative 3. Enzyme Conjugate 1 vial 11 ml, ready to use, blue colored, androstenedione conjugated to horseradish peroxidase. 4. Wash Buffer (10x) 1 vial 50 ml, concentrate, dilute 1 to 10 with distilled water 5. Zero Standard (A) 1 vial lyophilized, reconstitute with 0.5 ml distilled water 6. Standards (B-F) 5 vials lyophilized, reconstitute with 0.5 ml distilled water mix well after 15 min at room temperature in following concentrations in ng/ml: B C D E F 0.1 0.3 1.0 3.0 10 7. Substrat Solution A 1 vial 11 ml ready to use, H2O2 solution containing stabilisers, 8. TMB Substrat Solution B 1 vial 11 ml, TMB, ready to use. 9. TMB Stop Solution 1 vial 15 ml, ready to use 4 N H2SO4 Avoid contact with stop solution it may cause skin irritations and burns. Storage and Stability Store all reagents at 2 - 8 °C and use before expiry date. Unused microtiter strips must always be stored at 2 - 8 °C in the resealable bag provided. After reconstitution the standards are stable for max. 3 month at -20 °C. Allow reagents and required number of strips to reach room temperature prior to use. Specimen Collection and Storage Collect blood by venipuncture, allow to clot, and separate serum by centrifugation at room temperature. Avoid hemolysis. Specimens should be capped and may be stored for up to 24 hours at 2-8 °C prior to assaying. Specimen held for a longer time should be frozen only once at -20 °C prior to assay. Thawed samples should be inverted several times prior to testing. Do not use grossly hemolyzed or lipemic specimens. Preparation of Samples and Reagents The lyophilized standards have to be reconstituted with 0.5 ml distilled water each. After 15 minutes at room temperature mix thoroughly. Dilute the wash buffer concentrate 1 to 10 with distilled water. All standards, samples, and controls should be run in duplicate concurrently so that all conditions of testing are the same. Once the test has been started, all steps should be completed without interruption. All reagents and specimens must be allowed to come to room temperature before use.

Principle of the Test

Back 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 enzym-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 a) Short Instructions 1. Secure the desired number of coated microtiter wells in the holder. 2. Dispense 20 µl of standards, sample or controls into appropriate wells. 3. Dispense 100 µl of enzyme conjugate into each well. 4. Dispense 100 µl of antiserum into each well. 5. Thoroughly mix the plate for 20 seconds. It is important to have complete mixing in this step. 6. Incubate for 60 minutes at room temperature (18-24 °C). 7. Briskly shake out the contents of the wells. 8. Rinse the wells 4 times with diluted wash buffer. (300 µl per well). Strike the wells sharply on absorbent paper to remove residual droplets. 9. Add 100 µl of substrate solution A and substrate solution B each, at timed intervals. 10. Incubate for 30 minutes at room temperature (18-24 °C). 11. Stop the enzymatic reaction by adding 50 µl of stop solution to each well at the same timed intervals as in step 1.9 and determine the absorbance of each well at 450ñ10 nm. Final Reaction Stability: It is recommended that the wells be read within 15 minutes following step 1.11. Calculation of Results Any ELISA reader capable of determining the absorbance at 450ñ10 nm may be used. The androstenedione conceentration for each serum 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 ng/ml. Use the average absorbance of each serum sample to determine the corresponding Androstendione value by simple interpolation from this standard curve, multiplying by the initial sample dilution, if necessary. Alternativly the use of electronic device is possible. The result 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. Typical Calibration Curve Standard [ng/ml] OD 450 nm 0 1.92 0.1 1.53 0.3 1.17 1.0 0.88 3.0 0.59 10 0.17

Performance Characteristics

Back to Contents 1. Specificity The cross-reactivity of the androstenedione antiserum has been measured against various related steroids. The percent cross- reactivity is expressed as the ratio of the androstenedione concentration to the concentration of the reacting compound at 50% binding of the 0 ng/ml standard. Compound %Cross-Reactivity Androstenedione 100 Testosterone 0.55 Androsterone 0.48 DHEA-S 0.27 17alpha-Hydroxyprogesterone 0.18 Progesterone 0.11 Estrone 0.08 Cortisol 0.05 Cortisone 0.04 Estradiol < 0.01 Etiocholanolone < 0.01 2. Sensitivity The minimal detectable concentration of andostenedione by this assay is estimated to be 0.04 ng/ml (defined as 2 x Standard Deviation to the zero satandard) 3. Precision The intra-assay variation was determined from the mean of 10 replicates each. 3.1. Intra Assay: Sample n Mean [ng/ml] SD [ng/ml] CV [%] I 10 0.49 0.062 12.6 II 10 2.38 0.201 8.5 III 10 5.66 0.806 14.2 The inter assay variation was determined from the mean of average duplicates for 14 separate runs. 3.2. Inter Assay: Sample n Mean [ng/ml] SD [ng/ml] CV [%] I 14 0.20 0.044 21.6 II 14 2.31 0.279 12.1 III 14 6.22 0.541 8.7 4. Linearity Two serum samples were diluted with the 0 ng/ml standard and assayed. Sample Dilution Factor Expected [ng/ml] Observed [ng/ml] Recovery [%] I - - 9.90 - 1 : 2 4.95 4.10 82.8 1 : 3 3.30 2.92 88.5 1 : 6 1.65 1.82 110 1 : 12 0.84 1.01 120 1 : 24 0.42 0.51 121 1 : 48 0.27 0.34 124 II - - 2.54 - 1 : 2 1.87 1.83 98 1 : 4 1.25 1.25 100 1 : 8 0.62 0.76 121 1 : 16 0.32 0.42 131 5. Recovery Three serum samples containing different levels of endogenous androstenedione were spiked with known quantities of androstenedione and assayed. Sample Endogeneous Added Expected Observed Recovery (ng/ml) [ng/ml] [ng/ml] [ng/ml] [%] I 0.68 0.12 0.8 0.76 95 0.48 1.16 1.22 105 2.40 3.08 3.17 103 II 1.74 0.48 2.28 2.36 104 2.40 4.14 4.38 106 3.60 5.34 5.22 98 III 3.22 1.20 4.42 4.66 106 2.40 5.62 5.77 103

Expected Values

Back to Contents It is recommended that each laboratory should establish its own normal range. N Mean [ng/ml] Absolute Range [ng/ml] Males 38 1.75 0.35 - 3.15 Females (18-49 years) 47 2.15 0.70 - 3.50 Females (50-80 years) 33 1.80 0.20 - 3.40 Conversion factor: To convert to nmol/L: ng/ml x 3.45 = nmol/l Limitations of use Once the assay has been started, all steps should be completed without any interruption to obtain reliable results. Wash procedure is essential for the assay results. Strike the wells sharply on absorbent paper to remove residual droplets. Substrate solution should be colourless or slightly green/blue. If solution B is dark blue the reagent is unusable and must be discarded. During incubation with substrate avoid direct sunlight exposure of the microtiter plate.

Alternative Applications

Back to Contents

(More) Clinical Background

Back to Contents Clinical Applications: 1. Androgenizing Disorders: Increased serum concentrations of androstenedione are seen in association with androgenizing disorders in women. 2. Congenital Adrenal Hyperplasia: Elevated serum concentrations of androstenedione are seen both in virilized newborns and children as well as late onset cases in adult women who are not virilzed. Androstenedione measurements in adult androgenized women with late onset 21-hydroxylase deficiency are seen in association with elevated 17à-hydroxyprogesterone elevations and may be useful in reconfirmation of the diagnosis. 3. Polycystic Ovarian Disease: Serum concentrations of androstenedione are modestly elevated or normal in uncomplicated case of polycystic ovarian disease. 4. Virilizing Adrenal And Ovarian Tumors: Serum concentrations of androstenediones can range from normal to markedly elevated in cases of virilizing adrenal or ovarian neoplasm. Factors Affecting Normal Values: 1. Combination Oral Contraceptives: Androstenedione concentrations are suppressed by administration of oral contraceptives. 2. Corticosteroids: Androstenedione concentrations are suppressed by administration of corticosteroids.

Sales Arguments

Back to Contents Androstenedione ELISA Cat.-No. RE 521 61 Assay advantages for the IBL assay: - Direct determination without extraction - Low sample volume necessary (20µl) - Enzyme conjugate ready for use - Break apart wells for individual determinations - Incubation at room temperature - Short incubation time - High sensitivity - Reagents are sufficient to perform several runs - Suitable for automation - Excellent correlation with RIA Potential customers University-labs, Private-labs, Gynaecologists, Endocrinologists, Clinical Applications - Androgenizing disorders in females - Congenital adrenal hyperplasia - Polycystic ovarian disease - Virilizing adrenal and ovarial tumors

Product Literature

Back to Contents 1. Berel, E., et al., J. Steroid Biochem. 13: 89, 1980. 2. Bermudez, J. A., et al., J. Steroid Biochem. 6: 283, 1975. 3. Hampl, R., et al., J. Steroid Biochem. 9: 771, 1978. 4. Hummer, L., et al., Scand. J. Clin. Lab. Invest. 43: 301, 1983. 5. Hennam, J. F., et al., Acta Endocrinol. 76: 597, 1974. 6. Judd, H. L., et al., J. Endo. Metabo. 36: 475, 1973. 7. Lejeune-Lenair, C., et al., Clin. Chem. Acta. 94: 327, 1979. 8. Parker, L. N., et al., Steroids 29: 715, 1977. 9. Pizarro, M. A., et al., J. Steroid Biochem. 12: 509, 1980. 10.Rao, P. N., et al., Steroids 24: 793, 1974. 11.Putz, Z., et al., J. Clin. Chem. Clin. Biochem. 20: 761, 1982. 12.Schandader, B. D., et al., Endocrinology 97: 787, 1975. 13.Slaats, E. H., et al., Clin. Chem. 33: 300, 1987. 14.Swinkles, L. M., et al., Am. Clin. Biochem. 23: 354, 1988. 15.Thorneycroft, I. H., et al., Steroids 21: 111, 1973.

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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