rev: January 18, 1999

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

Price: $390 - 96 Tests Catalogue No : RDI-RE52231 Product group : Gynaecology 12 x 8 Product name : Progesterone **510K** ELISA 96 Method : ELISA Incubation time: 1 h, 15 min Standard curve : 0.05-40 ng/ml Sample/Prep. : 25 µl Serum, Plasma Isotope/Substr.: TMB 450 nm


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 Progesterone Enzyme Immunoassay Kit provides materials for the quantitative determination of progesterone in serum and plasma. This assay is intended for in vitro diagnostic use. Progesterone (pregn-4-ene-3, 20-dione) is a C21 steroid hormone containing a keto-group (at C-3) and a double bond between C-4 and C-5. This steroid hormone is a female sex hormone which, in conjunction with estrogens, regulates the accessory organs during the menstrual cycle and it is particularly important in preparing the endometrium for the implantation of the blastocyte and in maintaining pregnancy. In non pregnant women progesterone is mainly secreted by the corpus luteum whereas in pregnancy the placenta becomes the major source. Minor sources are the adrenal cortex for both sexes and the testes for males. Progesterone circulates in blood mainly bound to corticosteroid binding globulin (CBG), sex hormone binding globulin (SHBG) and albumin. Only 2 - 10% of the total concentration circulates as free hormone. Blood progesterone concentrations vary widely according to the phases of menstrual cycle; they are lower than 1 ng/ml (3.2 nmol/l) in the follicular phase and around 10 - 20 ng/ml (32 - 64 nmol/l) in the luteal phase. The maximal levels are achieved 4 - 7 days after ovulation and remain elevated for 4 - 6 additional days prior to falling to the preovulatory levels 24 hours before the onset of menstruation. Since the rise and fall of progesterone parallel the activity of ovarian follicle and corpus luteum, measurements of plasma progesterone is clinically used to confirm ovulation and normal function of the corpus luteum in non pregnant women. If ovulation does not occur the corpus luteum is not formed and no cyclical rise of progesterone in plasma is observed. Abnormal progesterone secretion has been implicated in premenstrual tension, irregular shedding of endometrium, dysmenorrhoea, and luteal insufficiency. Progesterone concentration can vary not only from subject to subject but also in the same person from day to day or even from hour to hour. Consequently, in gynaecological disorders or abnormal pregnancies serial measurements rather than single ones are recommended for a proper interpretation of results. During pregnancy progesterone is widely produced by placenta, and plasma levels rise steadily achieving values as high as 200 ng/ml at term.

Contents of the Kit

Back to Contents 1. Microtiter Strips, 12 x 8 wells break apart strips coated with anti progesterone antiserum (rabbit polyclonal) in foilbag with desiccant. 2. Standard A (Zero Standard) or Specimen Diluent 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 progesterone in ng/ml Standard B C D E F G Concentration 0.3 1.25 2.5 5 15 40 in ng/ml 4. Enzyme Conjugate 1 vial 22 ml, ready to use Progesterone conjugated to the enzyme horseradish peroxidase 5. TMB Substrate Solution 1 vial 22 ml, ready to use containing a solution of tetramethylbenzidine (TMB) in citrate buffer with hydrogen peroxide 6. TMB Stop Solution 1 vial 11 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 reagents beyond this date. Enzyme conjugate, standard solution, substrate solution, wash solution and zero standard must be stored at 2 - 8 °C. Microtiter wells 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 wells is stable for approx. 6 weeks in the broken, but tightly closed bag containing the desiccant. 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. Do not use grossly hemolyzed or grossly lipemic specimens. Samples suspected to contain progesterone concentration higher than 40 ng/ml are to be diluted with zero standard. Please note: Samples containing sodium azide should not be used in the assay.

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 1. General Remarks: All reagents and specimens must be allowed to come to room temperature (18 - 24°C) 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. Pipet standards and samples onto the bottom of the well. For pipetting of enzyme conjugate and stop solution it is recommended to hold the pipette in a vertical position above the well and dispense the correspondent solution into the centre of the well so that a complete mixing of enzyme conjugate with sample or standard and of the stop solution with the substrate solution is achieved. 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. As a general rule the enzymatic reaction is linearly proportional to time and temperature. This makes interpolation possible for fixed physico-chemical conditions. If in a test run the absorbance of zero standard is lower than 1.0 or above the upper performance limit of your microtiterplate spectrophotomter you can extend or reduce the incubation time of the final enzymatic formation of color to 30 or 10 minutes accordingly. Since calibrators are assayed in each run, absorbance fluctuations do not affect the result. The substrate solution should be colourless or slightly blue or green. If the solution is dark blue the reagent is unusable and must be discarded. During incubation with substrate solution avoid direct sunlight on the microtiter plate. 2. Reagent Preparation Wash Buffer: Add distilled water to the 20x concentrated wash buffer (content: 25 ml) to a final volume of 500 ml. The diluted wash buffer is stable for 2 weeks at room temperature (18 - 24°C). 3. Test Protocol 3.1. Secure the desired number of coated strips in the holder. 3.2. Dispense 25 µl of standards into appropriate wells. 3.3. Dispense 25 µl of sample into selected wells. Time between distribution of first standard and last sample can be up to 10 minutes without affecting the results. 3.4. Incubate plate for 5 minutes at room temperature (18 - 24°C). 3.5. Dispense 200 µl of enzyme conjugate into each well. 3.6. Thoroughly mix the plate for 10 seconds. It is important to have complete mixing in this step. 3.7. Incubate for 60 minutes at room temperature (18 - 24°C). 3.8. Briskly shake out the contents of the wells. 3.9. Rinse the wells 3 times with diluted wash buffer (300 µl per well). Strike the wells sharply on absorbent paper to remove residual droplets. 3.10. Add 200 µl of TMB substrate solution to each well, at timed intervals. 3.11. Incubate for 15 minutes at room temperature (18 - 24°C). 3.12. Stop the enzymatic reaction by adding 100 µl of TMB stop solution to each well at the same timed intervals as in step 10 and determine the absorbance of each well at 450 ñ 10 nm. Final Reaction Stability It is recommended that the wells be read within 10 minutes following step 12. Calculation of Results Any microwell reader capable of determining the absorbance at 450 ñ 10 nm may be used. The Progesterone value of each sample is obtained as follows: 1. 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. For construction of the standard curve we recommend a four parameter logistic function. 2. Use the average absorbance of each sample to determine the corresponding progesterone value by simple interpolation from this standard curve, multiplying by the initial sample dilution, if necessary. EXAMPLE OF TYPICAL STANDARD CURVE The following data is for demonstration only and cannot be used in place of data generations at the time of assay. Standard ng/ml Optical Units Standard 0 0 1.52 Standard 1 0.3 1.17 Standard 2 1.25 0.88 Standard 3 2.5 0.69 Standard 4 5.0 0.55 Standard 5 15 0.35 Standard 6 40 0.13

Performance Characteristics

Back to Contents 1. Specificity The following materials have been checked for cross-reactivity. The percentage indicate cross reactivity at 50% displacement compared to Progesterone. Steroid % Cross-Reactivity Progesterone 100 17a OH Progesterone 0.3 Estriol < 0.1 Estradiol 17 < 0.1 Testosterone < 0.1 11-Desoxycorticosterone 1.1 DHEA-S < 0.02 Cortisol < 0.02 Corticosterone 0.2 Pregnenolone 0.35 Cortison < 0.1 11-Desoxycortisol 0.1 2. Sensitivity The lowest detectable level of progesterone that can be distinguished from the zero standard is 0.05 ng/ml at the 95 % confidence limit. 3. Precision 3.a. 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: 3.b. 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: Intra Assay Inter Assay Serum n Mean ñ SD CV n Mean ñ SD CV ng/ml % ng/ml % 1 10 1.21 ñ 0.1 8.3 10 1.31 ñ 0.13 9.9 2 10 2.62 ñ 0.12 4.6 10 2.71 ñ 0.13 4.8 3 10 12.3 ñ 0.64 5.2 10 11.9 ñ 0.77 6.5 4. Linearity Three patient samples were serially diluted with zero standard in a linearity study. Serum Dilution Measured conc. Recovery factor ng/ml % neat 6.95 1 1:2 3.52 101 1:4 1.81 104 1:8 0.89 102 neat 16.2 1:2 7.93 98 2 1:4 3.95 98 1:8 2.12 105 1:16 1.21 119 1:32 0.52 103 neat 10.1 1:2 4.98 99 3 1:4 2.49 99 1:8 1.31 104 1:16 0.64 101 1:32 0.34 108 5. Recovery Serum Endogenous Prog. Added Prog Recovery ng/ml ng/ml % 5 109 1 3.1 2.5 106 1.25 98 5 101 2 1.9 2.5 92 1.25 106 0.5 91

Expected Values

Back to Contents Each laboratory must establish its own normal ranges based on patient population. The results provided below are based on a limited number of healthy adult blood specimens. Females ng/ml nmol/l Follicular phase 0.2-1.4 0.64 - 4.45 Luteal phase 4 - 25 12.7 - 79.5 Menopause 0.1 - 1 0.32 - 3.18 Males 0.1 - 1 0.32 - 3.18 Conversion factor: 1 ng/ml = 3.18 nmol/l Limitations of use 1.Reliable and reproducable results will be obtained when the assay procedure is carried out with a complete understanding of the package insert instruction and with adherence to good laboratory practice. 2.The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbances. 3.Complete mixing of conjugate with standard or sample (step 5) and of stop solution with substrate solution (step 12) is critical. Insufficient mixing will result in poor precision.

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-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 ( 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. Documentation of ovulation: Plasma progesterone concentrations are useful in the documentation of recent ovulation. They may also be useful in the evaluation of patients with sterility due to luteal phase defects. In order for a progesterone measurement to be useful as a sign of ovulation, it must be obtained as closely as possible to mid-luteal phase of the cycle. Samples obtained too close to menses or within 3 - 4 days of the LH peak can lead to confusing results since progesterone is either rising or falling rapidly during this time. A random luteal phase progesterone concentration of > 4.0 ng/ml is strong presumptive evidence of ovulation and has approximately the same clinical meaning as a secretory phase endomentrial biopsy. There is no clear cut off value for diagnosis of luteal insufficiency. Progesterone can also be used to predict ovulation much as LH since it rises in parallel with LH. 2. Assessment of corpus luteum function in recurrent abortion: Spontaneous first trimester abortions are accompanied by falling progesterone concentrations to subnormal levels prior to expulsion of the pregnancy. Repeated investigations show that falling progesterone concentrations are of marginal value in prediction of pregnancy outcome since this phenomenon is nonspecifically related to failure of the corpus luteum and placentation. Measurement of progesterone does not provide useful clinical information as to the cause of the pregnancy loss. 3. Ectopic pregnancy: Progesterone concentrations are chronically < 20 ng/ml in patients with tubal pregnancy. The reasons for this subnormal value are probably due to corpus luteum disfuntion in association with the abnormal implantation. The diagnostic value of this observation is not established. 4. In vitro fertilization: Progesterone measurements can be used to identify premature ovulation when inducing ovulation for in vitro fertilization. Factors affecting normal values: 1. Combination oral contraceptives: Progesterone concentrations are suppressed to < 1 ng/ml since ovulation does not occur. 2. Superovulatory drugs: Progesterone concentrations reflect on development of multiple corpura lutei from multiple ovulations. Clomiphene citrate and human menopausal gonadotropin (HMG) therapy are both associated with superphysiologic progesterone concentrations in the 25 to 60 ng/ml range following successful ovulation induction. 3. Estrogen replacement therapy: Progesterone concentrations are in the < 1 ng/ml range in postmenopausal women under estrogen replacement and are not affected by the administration of estrogens. 4. GnRH Analogous: Progesterone concentrations are suppressed in women under chronic analogue suppression. 5. Surgical Oophorectomy: Progesterone concentrations remain in the < 1 ng/ml range. 6. Chemotherapy: Progesterone concentrations remain in the < 1 ng/ml range after destruction of ovarian function.

Sales Arguments

Back to Contents Assay advantages for the IBL assay: - 510k approved by FDA - Break apart strips for individual determinations - Reagents ready for use - Incubation at room temperature - Short incubation time - High sensitivity - Suitable for automation - Excellent correlation with RIA - Suitable for the determination in rat samples Potential customers University-labs, Private-labs, Gynaecologists, Endocrinologists, Clinical Applications - Documentation of ovulation - Assessment of corpus luteum function - Follow up in therapy of infertility

Product Literature

Back to Contents 1. Carson, S. A., Buster, J. E.: Ectopic pregnancy: evolution of a medical disease. N. Engl. J. Med. (in press, 1993). 2. Stovall, T. G., Ling, F.W., Gray, L. A., Carson, S. A., Buster, J. E.: Methotrexate treatment of unruptured ectopic pregnancy: a report of 100 cases. Obstet. Gynecol. 77: 749-753, 1991. 3. Stovall, T. G., Ling, F.W., Gray, L. A.: Single-dose methotrexate for treatment of ectopic pregnancy. Obstet. Gynecol. 77: 754-757, 1991. 4. Hahlin, M., Wallin, A., Sjoblom, P., Lindblom, B.: Single progesterone assay for early recognition of abnormal pregnancy. Hum. Reprod. 5: 622-626, 1990. 5. Buck, R. H., Joubert, R. J., Normal, R. J.: Serum progesterone in the diagnosis of ectopic pregnancy: A valuable diagnostic test. Fertil. Steril. 50: 752-755, 1988. 6. Yeko, T. R., Gorrill, M. J., Hughes, L. H., Rodi, I. A., Buster, J.E., Sauer, M. V.: Timely diagnosis of early ectopic pregnancy using a single blood progesterone measurement. Fertil. Steril. 48: 1048-1050, 1987. 7. Matthews, C. P., Coulson, P. B., Wild, R. A.: Serum progesterone levels as an aid in the diagnosis of ectopic pregnancy. Obstet. Gynecol. 68: 390, 1986. 8. Stovall, T. G., Ling, F.W., Cope, B. J., Buster, J. E.: Preventing ruptured ectopic pregnancy with a single serum progesterone. Am. J. Obstet. Gynecol. 160: 1425-1428, 1989. 9. Stovall, T. G., Ling, F.W., Cope, B. J., Buster, J. E.: Nonsurgical diagnosis and treatment of tubal pregnancy. Fertil. Steril. 54: 537-538, 1990. 10. Isreal, R., Mishell, D. R., Stone, S. C., Thorneycroft, I. H., Moyer, D. L.: Single luteal phase serum progesterone as an indicator of ovulation. Am. J. Obstet. Gynecl. 112: 1043, 1971. 11. Soules, M. R., Hughes, C. L., Askel, S., Tyrey, L., Hammond, C. B.: The function of the corpus luteum in pregnancy in ovulatory dysfunction, and in luteal phase deficiency. Fertil. Steril. 36: 31, 1981. 12. Maganiello, P. D., Nazian, S. J., Ellegood, J. O., McDonough, P. G., Mahash, V. B.: Serum progesterone, 17- hydroxyprogesterone, human chorionic gonadotropin, and prolactin in early pregnancy and a case of spontaneous abortion. Fertil. Steril. 36: 55, 1981. 13. Csapo, A. I., Pulkkinen, M. O., Wiest, W. G.: Affects of luteectomy and progesterone replacement therapy in early pregnant patients. Am. J. Obstet. Gynecol. 225: 759, 1973.


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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