rev: January 18, 1999

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17-OH Progesterone ELISA Kit   $425.00/kit  
-sample data-see actual kit insert for batch specific information
Catalogue No : RDI-RE52071 Product group : Gynaecology 12 x 8 Product name : Progesterone, 17-OH **510K** ELISA 96 Method : ELISA Incubation time: 90 min, 30 min Standard curve : 0.1 ng/ml - 4 ng/ml Sample/Prep. : 20µl Serum, EDTA plasma Isotope/Substr.: TMB, 450nm

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 steroide hormone 17-alpha-hydroxyprogesterone (17-OH-progesterone, 17-OHP) is produced in the adrenal cortex and in the gonads. Gestagenic effects exerted by 17-OHP are only small. Nevertheless, this hormone is of clincal significance because it represents the ultimate precursor of 11-desoxycortisol (compounds, CpS). CpS is formed by hydroxylation of the carbon atom C 21. Enyzme activity of 21-hydroxylase in the adrenal cortex may thus be monitored by analyzing the level of 17-OHP in the blood. Deficiencies in 21-hydroxylase, most commonly found in congenital adrenal hyperplasia, result in excessive secretion of 17-OHP consequently in enhanced blood levels. Deficiencies in 11-hydroxlase, however, merely lead to moderately increased values of 17-OHP. The analysis of this steroid hormone, therefore, plays a significant role in the differential diagnosis of congenital adrenal hyperplasia. In adult nonpregnant women, 17-OHP levels in the blood depend on the phase of the menstrual cycle. Like progesterone, 17-OHP is secreted by the mature follicle and the corpus luteum. Concentrations are generally higher after ovulation. In addition, levels of 17-OHP are influenced by daytime rhythms which correlate with the adrenal secretion of cortisol. Maximal levels are found in samples collected between midnight and 8.00 a.m. In adult men, there are few indications of similar fluctuations of 17-OHP levels. During pregnancy, large amounts of 17-OHP are produced by the fetus, the placenta and the adrenal cortex. The hormone is secreted into the fetal and the maternal blood circulation. Maternal values of 17-OHP strongly increase after the 32. week of pregnancy reaching 4-fold higher levels than during the luteal phase of the menstrual cycle. 17-OHP may also be found in the umbilical cord of newborns.

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 rabbit IgG (goat, polyclonal) in foilbag with desiccant. 2. Standards A - G 7 vials 0.2 ml each, ready to use containing the below mentioned concentrations of 17-OHPin human serum and thimerosal (< 0.01%) as preservative. Concentrations: Standard A B C D E F G ng/ml 0 0.25 0.90 1.75 3.50 7.0 14 nmol/l 0 0.76 2.7 5.3 10.6 21.2 42.4 3. 17-OHP Antiserum 1 vial 10 ml, ready to use 17-OHP antiserum (rabbit, polyclonal) in phoshate buffer, blue dye and thimerosal (< 0.01%) as preservative. 4. Enzyme Conjugate, concentrate (9x) 1 vial 2 ml, concentrate, 17-OHP conjugated to the enzyme horseradish peroxidase in phosphate buffer, yellow dye and thimerosal (< 0.01%) as preservative. Dilute 1 to 9 with conjugate buffer prior to use. 5. Conjugate Buffer 1 vial 15 ml, ready to use, containing phosphate buffer and thimerosal (< 0.01%) as preservative. 6. TMB Substrate, concentrate (31x) 1 vial 1 ml, concentrate, containing a solution of tetramethylbenzidine (TMB) in buffer supplemented by stabilizers. Dilute 1 to 31 with TMB substrate buffer prior to use. 7. TMB Substrate Buffer 1 vial 30 ml, ready to use containing a solution of hydrogen peroxide (H2O2) in citrate buffer supplemented by stabilizers. 8. TMB Stop Solution 1 vial 15 ml, ready to use 1 M sulphuric acid (H2SO4) Corrosive! Avoid contact with stop solution, it may cause skin irritations and burns. 9. Wash Buffer, concentrate (20x) 1 vial 50 ml, concentrate containing phosphate buffer with tween and thimerosal (< 0.01%) as preservative.

Principle of the Test

Back to Contents The 17-alpha-hydroxyprogesterone ELISA (17-OH-progesterone ELISA) is a competitive enzyme immunoassay for the direct, quantitative determination of 17-OHP in serum. The wells of the microtiter strips are coated with goat anti rabbit antibodies. The samples and standards are mixed with 17-OHP which is coupled to peroxidase, and a specific rabbit anti 17-OHP antiserum. The assay is based on the competition between the non labelled and the labelled antigen for a fixed number of antibody binding sites. The amount of labelled antigen bound to the antibody is inversely proportional to the analyte concentration of the sample. The immunocomplexes are immobilized by binding to the goat anti rabbit-IgG coated wells. When the system is in equilibrium, free antigens are removed by washing. The bound enzymatic activity is subsequently determined using TMB as substrate by measuring the optical density at 450 nm. Quantification of unknowns is achieved by comparing the enzymatic activity of unknowns with a reference curve of known standards.

Test Procedure

Back to Contents Storage and Stability Store all reagents at 2 - 8 °C and use before expiry date. When stored at 2 - 8 °C unopened reagents will retain reactivity until expiration date. Do not use reagents beyond this date. Once the foilbag of the coated microtiter strips 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. Test Procedure: GENERAL REMARKS: 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 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. 1. Summary 1.1 Wash microtiter strips three times with wash buffer 1.2 Pipet 20 µl of standards and patient samples. 1.3 Add 100 µl of conjugate solution. 1.4 Add 100 µl of antiserum. 1.5 Incubate for 90 minutes at room temperature (18 - 24 °C) on a shaker (250 rpm). 1.6 Wash three times with wash buffer. 1.7 Pipet 200 µl TMB substrate solution. 1.8 Incubate for 30 minutes at room temperature (18 - 24 °C) in the dark. 1.9 Add 100 µl of TMB stop solution. 1.10 Read the absorbance at 450 nm. 2. Detailed Instructions for use Allow reagents to reach room temperature before use. Store reagents at 2 - 8 °C when not required. Assays in duplicate are recommended. 2.1 Perform assay on microtiter strips which are placed on the microtiter plate provided. Unused strips should be resealed carefully and stored at 2 - 8 °C. 2.2 Wash microtiter strips three times with ready to use wash buffer before starting the assay. 2.3 Pipet 20 µl of standards and patient samples into two wells each (see pipetting scheme). 2.4 Add 100 µl of conjugate solution to each well. 2.5 Add 100 µl of antiserum to each well. 2.6 Seal plate with adhesive foil and incubate for 90 minutes at room temperature (18 - 24 °C) on a microtiter plate shaker. 2.7 Wash each well three times with wash buffer. Remove residual fluids from the well by draining plate on tissue pad. Important: Sensitivity and precision of this assay strongly depend on correct washing procedures. 2.8 Pipet 200 µl of freshly prepared TMB substrate solution to each well. 2.9 Seal plate with adhesive foil and incubate for 30 minutes at room temperature (18 - 24 °C) in the dark. 2.10 Add 100 µl of TMB stop solution to each well. Colour changes from blue to yellow. 2.11 Read the absorbance at 450 nm. Preparation of Samples and Reagents The amounts of reagents refer to one assay of 32 determinations. 1. Wash Buffer Mix 15 ml of wash buffer concentrate (2) with distilled water ad 300 ml. Redissolve any phosphate cristals precipitated in the buffer (due to storage at 2 - 8 °C) at room temperature before diluting with water. The ready to use wash buffer may be stored at 2 - 8 °C for 4 weeks. 2. Conjugate Solution Dilute 500 µl of peroxidase conjugate (5) with 4 ml of conjugate buffer (4) and mix well. Prepare solution just before use and use only once. 3. TMB Substrate Solution Dilute the required amount of TMB substrate with TMB substrate buffer 1 to 31 (e.g. 100 µl of TMB substrate + 3 ml TMB substrate buffer). Prepare just before use and use only once. Specimen Collection 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 7 days, and should be frozen at - 20 °C or lower for longer periods. - Samples suspected to contain 17-OHP concentration higher than standard G 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. 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 ng/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.

Performance Characteristics

Back to Contents 1. Sensitivity The minimal detectable concentration of 17-OHP by this assay is estimated to be 0.1 ng/ml (defined as 2x standard deviation to the zero standard). 2. Linearity Three patient samplkes were serially diluted with zero standard in a linearity study. Sample Dilution Measured Expected Recovery conc. (ng/ml) conc. (ng/ml) (%) -------------------------------------------------------------- 1 neat 8.9 -- -- 1:2 4.1 4.45 92.1 1:4 2.05 2.22 92.3 1:8 1.19 1.11 107.2 2 neat 6.4 -- -- 1:2 3.4 3.2 106.2 1:4 1.47 1.6 91.8 1:8 0.78 0.8 97.5 3 neat 4.2 -- -- 1:2 2.1 2.1 100.0 1:4 1.12 1.05 106.7 1:8 0.57 0.52 109.6 3. Precision 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. (All data in ng/ml) Sample Mean Standard deviation CV (%) n 1 1.84 0.12 6.4 10 2 8.26 0.25 3.1 10 Inter Assay Variation Between run variation was determined by replicate measurements of two different control sera in several different assay. The between assay variability is shown below: (All data in ng/ml) Sample Mean Standard deviation CV (%) n 1 1.82 0.15 8.2 10 2 8.22 0.31 3.8 10 4. Cross-Reactivity The specificity of the 17-OHP ELISA was assessed according to Abraham's method. The percentage indicate cross reactivity at 50% displacement compared to 17-OHP. Component Cross-Reactivity (%) 17 OH-Pregnenolone (5-Pregnene-3,17-diol-20-one) 1.7 Progesterone (4-Pregnene-3,20-dione) 1.3 11-Desoxycortisol (4-Pregnene-17,21-diol-3,20-dione) 1.3 Desoxycorticosterone (4-Pregnene-21-ol-3,20-dione) 0.12 Cortisol (4-Pregnene- Pregnenolone (5-Pregnene-3-ol-20-one) 0.011 DHEA (5-Androstene-3-ol-17-one) 0.0012 DHEA-sulfate (5-Androstene-3-ol-17-on sulfate) < 0.001 Danazol < 0.001 Estrone (1,3,5(10)-Estratriene-3-ol-17-one) < 0.001 Estriol (1,3,5(10)-Estratriene-3,16,17-triole) < 0.001 Cholesterol (5-Cholesten-3-ole) < 0.001 5. Recovery Spiked serum samples were prepared by adding varying levels of 17-OHP to three different serum samples. Sample Added Measured Recovery (ng/ml) (ng/ml) (ng/ml) (%) --------------------------------------------------- 1 (0.89) 1.25 2.47 111.2 2.5 3.39 100.0 5.0 6.57 111.5 10.0 10.35 95.0 2 (1.43) 1.25 2.68 92.2 2.5 3.93 101.3 5.0 6.43 114.0 10.0 11.43 105.2 6. Method Comparison The IBL 17-OHP ELISA was compared with another commercially available 17-OHP RIA. Serum samples of 27 obviously healthy (non pregnant) females, 26 pregnant females and 16 obviously healthy males were analysed according in both test systems. A correlation coefficient of r = 0.98 (r2 = 0.95) 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 = 1.14 IBL-ELISA + 0.05 35 serum samples were compared with their plasma samples using the IBL 17-OHP ELISA. A correlation coefficient of r = 0.97 (r2 = 0.94) was found between the two kinds of samples. The linear regression curve was calculated from y = mx + b where m is the slope and b the y - intercept. The resulting equation is: Plasma = 0.996 serum + 0.04

Expected Values

Back to Contents 1. Expected Values Females ng/ml Follicle phase 0.1 - 0.8 Luteal phase 0.3 - 2.9 after ACTH stimulation < 3 Pregnancy 2 - 12 Males ng /ml 0.3 - 2 Expected values may be influenced by: Drugs: Glucocorticoides suppress 17-OH-progesterone. Pregnancy: The level of 17-OH-progesterone increases significantly after the 32. week of pregnancy. Ovulation: The level of 17-OH-progesterone increases during the luteal phase and correlates with the level of progesterone.

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-OHP, 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 Clinical Applications 1. Congenital adrenal hyperplasia: The principal application of the 17-OHP is in the sreening for and diagnosis of CAH in newborn children with ambiguous genitalia and in girls who become virilized during adolescence. Since 17-OHP is the immediate precursor to 11-desoxycortisol, basal 17-OHP concentrations are sharply elevated in patients with 11-hydroxylase deficiency and to a lesser degree in patients with 11-hydroxylase deficiency. Because 17-OHP concentrations are so markedly elevated in newborns and adolescent girls afflicted with CAH, a single basal measurement is ordinarily all that is required to make the diagnosis. 2. Late-onset adrenal hyperplasia: Recent reports indicate that some 6% of women with adult hirsutism have varying degrees of 21-hydroxylase deficiency. It has thus become an increasingly common practise to measure 17-OHP concentrations in the cases. This condition presents as a classical case of polycystic ovarian disease and differs from it clinically only by the elevation in basal 17-OHP concentrations. In cases of late-onset 21-hydroxylase deficiency 17-OHP concentrations are usually in excess of 0.9 ng/ml (follicular phase). In doubtful cases, the diagnosis can be re-affirmed by administration of 0.25 mg of synthetic ACTH with measurement of 17-OHP concentrations 60 minutes later; however, this is probably rarely necessary. 3. Other applications: Measurement of 17-OHP concentrations is also utilized in the evaluation of both men and women with acne vulgaris, pattern baldness, and in some subtle forms of infertility. Factors affecting normal values: 1. Drugs: Adrenal corticosteroids: 17-OHP concentrations are suppressed in patients taking adrenal corticosteroids. 2. Pregnancy: 17-OHP concentrations increase in third trimester of pregnancy. 3. Ovulation: 17-OHP concentration are higher in luteal phase and rise in parallel with progesterone.

Sales Arguments

Back to Contents 17-OH-Progesterone ELISA Cat.-No. RDI-RE52071 Assay advantages for the IBL assay - 510k exempt from FDA - Also available to determine neonatal samples - Break apart strips for short assay runs - Reagents ready to use - Short incubation time - Suitable for automation - Suitable for the determination in rat samples, after extraction also for the determination in bovine and equine samples - Excellent correlation with RIA Potential customers University-labs, Private-labs, Gynaecologists, Endocrinologists, Neonatal-Center Clinical Applications - Differential diagnosis of patients with hirsutism - Screening for and diagnosis of congenital adrenal hyperplasia - Evaluation of some subtle forms of infertility in both males and females

Product Literature

Back to Contents References 1. Weil J, Bindlingmaier F, Sippell WG, Butenandt O, Knorr D. Comparsion of two tests for heterozygosity in congenital adrenal hyperplasia (CAH). Acta Endocrinol., 91: 109 (1979). 2. Schnakenburg K, Bindlingmaier F, Knorr D. 17-hydroxy-progesterone, androstendione, and testosterone in normal children and in prepubertal patients with congenital adrenal hyperplasia. Eur. J. Pediatr., 133: 259 (1980). 3. Chrousos GP, Loriaux DL, Mann DL, Cutler GB. Lateonset 21-Hydroxylase deficiency mimicking idiopathic hirsutism or polycystic ovarian disease. Ann. Int. Med., 96: 143 (1982).

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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