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Progesterone spec sheet pss-ps1113-2

PROGESTERONE ENZYME IMMUNOASSAY TEST KIT
Catalog Number: PS-1113
Enzyme Immunoassay for the
PRINCIPLE OF THE TEST
Quantitative Determination of
Progesterone Concentration in
The progesterone EIA is based on the principle of Human Serum
competitive binding between progesterone in the test specimen and progesterone-HRP conjugate for a constant amount of rabbit anti-progesterone. In the FOR RESEARCH USE ONLY
incubation, goat anti-rabbit IgG-coated wells are incubated with 25 µl progesterone standards, controls, NOT FOR DIAGNOSTIC USE
samples, 100 µl progesterone-HRP Conjugate Reagent and 50 µl rabbit anti-progesterone reagent at room temperature (18-25°C) for 90 minutes. During the PROPRIETARY AND COMMON NAMES
incubation, a fixed amount of HRP-labeled progesterone competes with the endogenous progesterone in the standard, sample, or quality control serum for a fixed number of binding sites of the specific progesterone NTENDED USE
antibody. Thus, the amount of progesterone peroxidase For the quantitative determination of Progesterone conjugate immunologically bound to the well progressively decreases as the concentration of progesterone in the specimen increases.
INTRODUCTION
Unbound progesterone peroxidase conjugate is then Progesterone is a C21 steroid which is synthesized from removed and the wells washed. Next, a solution of TMB both tissue and circulating cholesterol. Cholesterol is Reagent is then added and incubated at room transformed to pregnenolone which is then converted via temperature for 20 minutes, resulting in the development a combined dehydrogenase and isomerase to of blue color. The color development is stopped with the progesterone. The principle production sites are the addition of Stop Solution, and the absorbance is adrenals and ovaries and the placenta during pregnancy. measured spectrophotometrically at 450 nm. The The majority of this steroid is metabolized in the liver to intensity of the color formed is proportional to the pregnanediol and conjugated as a glucuronide prior to amount of enzyme present and is inversely related to the amount of unlabeled progesterone in the sample. A standard curve is obtained by plotting the concentration Progesterone exhibits a wide variety of end organ of the standard versus the absorbance. The effects. The primary role of progesterone is exhibited by progesterone concentration of the specimens and the reproductive organs. In males, progesterone is a controls run concurrently with the standards can be necessary intermediate for the production of corticosteroids and androgens. In females, progesterone remains relatively constant throughout the REAGENTS
follicular phase of the menstrual cycle. The concentration then increases rapidly following ovulation Materials provided with the kit:
and remains elevated for 4-6 days and decreases to the Goat Anti-Rabbit IgG-coated microtiter wells, 96 initial level 24 hours before the onset of menstruation. In pregnancy, placental progesterone production rises Progesterone Reference Standards: 0, 0.5, 3.0, 10, steadily to levels of 10 to 20 times those of the luteal 25, and 50 ng/ml. Liquids, 0.5 ml each, ready to Progesterone measurements are thus performed to Rabbit Anti-Progesterone Reagent (pink color), 7 ml.
determine ovulation as well as to characterize luteal Progesterone-HRP Conjugate Concentrate (11x),
phase defects. Monitoring of progesterone therapy and early stage pregnancy evaluations comprise the Progesterone-HRP Conjugate Diluent, 13 ml
remaining uses of progesterone assays.
Progesterone Control 1, Liquid, 0.5 ml, Ready to Progesterone Control 2, Liquid, 0.5 ml, Ready to For research use only. Not for diagnostic or therapeutic use. Prolias Technologies products may not be sold or modified for resale or used to manufacture commercial products without prior written approval by Prolias Technologies. If you are not satisfied with the product please contact us. 3. Samples with expected progesterone concentrations over 50 ng/ml may be quantitated by dilution with Materials required but not provided:
• Precision pipettes: 25 µl, 50 µl, 100 µl, 200 µl, and ASSAY PROCEDURE
1. Secure the desired number of coated wells in 2. Dispense 25 µl of standards, specimens and 3. Dispense 100 µl of Working Progesterone-
HRP Conjugate Reagent into each well.
4. Dispense 50 µl of rabbit anti-progesterone WARNINGS AND PRECAUTIONS FOR USERS
Test methods are not available which can offer complete 5. Thoroughly mix for 30 seconds. It is very
a s s u r a n c e t h a t H e p a t i t i s B v i r u s , H u m a n important to mix them completely.
Immunodeficiency Virus (HIV/HTLV-III/LAV), or other 6. Incubate at room temperature (18-25°C) for 90 infectious agents are absent from the reagents in this kit. Therefore, all human blood products, including samples, 7. Rinse and flick the microwells 5 times with should be considered potentially infectious. Handling distilled or deionized water. (Please do not use and disposal should be in accordance with the procedures defined by an appropriate national biohazard 8. Dispense 100 µl of TMB Reagent into each well. safety guideline or regulation, where it exists (e.g., USA Center for Disease Control/National Institute of Health 9. Incubate at room temperature (18-25°C) for 20 Manual, “Biosafety in Microbiological and Biomedical 10. Stop the reaction by adding 100 µl of Stop AMPLE PREPARATION
11. Gently mix 30 seconds. It is important to make sure that all the blue color changes to yellow 2. No special pretreatment of sample is necessary.
3. Serum samples may be stored at 2-8°C for up to 24 12. Read absorbance at 450 nm with a microtiter hours, and should be frozen at −10°C or lower for well reader within 15 minutes.
longer periods. Do not use grossly hemolyzed or CALCULATION OF RESULTS
4. Please note: Samples containing sodium azide
1. Calculate the mean absorbance value (A
each set of reference standards, controls and STORAGE OF TEST KIT AND
INSTRUMENTATION
2. Construct a standard curve by plotting the mean
absorbance obtained for each reference standard Unopened test kits should be stored at 2-8°C upon against its concentration in ng/ml on a linear-linear
receipt and the microtiter plate should be kept in a graph paper, with absorbance values on the vertical
sealed bag with desiccants to minimize exposure to or Y axis, and concentrations on the horizontal or X damp air. Opened test kits will remain stable until the expiration date shown, provided it is stored as described 3. Use the mean absorbance values for each specimen
above. A microtiter plate reader with a bandwidth of 10 to determine the corresponding concentration of nm or less and an optical density range of 0-3 O.D. at Progesterone in ng/ml from the standard curve.
450 nm wavelength is acceptable for use in absorbance 4. Any values obtained for diluted samples must be
further converted by applying the appropriate dilution REAGENT PREPARATION
1. All reagents should be brought to room temperature EXAMPLE OF STANDARD CURVE
Results of a typical standard run with optical density 2. To prepare Working Progesterone-HRP
readings at 450 nm shown in the Y axis against Conjugate Reagent, add 0.1 ml of Progesterone-
Progesterone concentrations shown in the X axis. Note:
HRP Conjugate Concentrate (11x) to 1.0 ml of
This standard curve is for the purpose of illustration only, Progesterone-HRP Conjugate Diluent (1:10
and should not be used to calculate unknowns. Each dilution) and mix well. The amount of conjugate
laboratory must provide its own data and standard curve diluted depends on your assay size. Discard the
excess after use.
For research use only. Not for diagnostic or therapeutic use. Prolias Technologies products may not be sold or modified for resale or used to manufacture commercial products without prior written approval by Prolias 2Technologies. If you are not satisfied with the product please contact replicate measurements of six different serum samples over a series of individually calibrated assays. Between-assay variability is shown Absorbance (450 nm)
Progesterone Conc. (ng/ml)
3. Linearity Study
Four samples were serially diluted to determine linearity. The mean linearity was 105.9%.
EXPECTED VALUES
Dilution
Expected Conc. Observed Conc.
% Expected
Each laboratory should establish its own normal range based on the sample population. The Progesterone EIA Undiluted
was performed on randomly selected laboratory samples. The following information is cited from Prepubertal (children) 0.07 − 0.52 ng/ml Females: follicular phase 0.15 – 0.70 ng/ml Mean = 113.8%
Undiluted
PERFORMANCE CHARACTERISTICS
Mean = 97.9%
1. Sensitivity
The minimum detectable concentration of the Progesterone ELISA assay as measured by 2 Undiluted
2. Precision
Within-run precision was determined by replicate determinations of four different serum samples Mean = 95.1%
in one assay. Within-assay variability is shown Undiluted
For research use only. Not for diagnostic or therapeutic use. Prolias Technologies products may not be sold or modified for resale or used to manufacture commercial products without prior written approval by Prolias 3Technologies. If you are not satisfied with the product please contact LIMITATIONS OF THE PROCEDURE
1. 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 2. The wash procedure is critical. Insufficient washing Mean = 116.9%
will result in poor precision and falsely elevated 3. Serum samples demonstrating gross lipemia, gross hemolysis, or turbidity should not be used with this 4. Recovery Study
Various serum samples of known Progesterone QUALITY CONTROL
levels were combined and assayed in duplicate. Good laboratory practice requires that controls are run with each calibration curve. A statistically significant number of controls should be assayed to establish mean EXPECTED
OBSERVED
% RECOVERY
values and acceptable ranges to assure proper [Progesterone]
[Progesterone]
We recommend using Bio-Rad Lyphochek Immunoassay Control Sera as controls. The Progesterone EIA kit also provides with internal controls, Level 1 and 2.
REFERENCES
Radwanska, E., Frankenberg, J., and Allen, E., Plasma progesterone levels in normal and abnormal early human pregnancy. Fertility and Sterility, 1978; 5. Specificity
2. Autrere,M.B., and Benson, H., Progesterone: An The following materials have been checked for cross overview and recent advances, J. Par. Sci., 1976; reactivity. The percentage indicates cross reactivity at 50% displacement compared to Progesterone.
3. March, C.M., Goebelsmann, U., Nakamura, R.M., and Mishell, D.R. Jr., Roles of estradiol and Data on the cross-reactivity for several endogenous progesterone in eliciting the midcycle luteinizing and pharmaceutical steroids are summarized in the hormone and follicle-stimulating hormone surges, J. Clin. Endo. Metab., 1979; 49, 507-513.
4. Ross, G.T., Vande Wiele, R.L., and Frantz, A.G., Cross-reactivity (%) = Observed Progesterone The Ovaries and the breasts. In: Williams, R.H., ed., Textbook of Endocrinology. Saunders Company, 5. Chattoraj, S.C., Endocrine function. In: Tietz, N.W., Cross-Reactivity
ed., Fundamentals of Clinical Chemistry. Saunders Company, Philadelphia; 1976: 699-823.
6. Shepard, M.K., and Senturia, Y.D., Comparison of serum progesterone and endometrial biopsy for confirmation of ovulation and evaluation of luteal function. Fertility and Sterility, 1977; 28: 541-548.
7. Johansson, E.D.B., and Jonasson, L.-E., Progesterone levels in amniotic fluid and plasma from women: I. Levels during normal pregnancy. Acta Obstet. Gynec. Scand., 1971; 50: 339-343.
8. USA Center for Disease Control/National Institute of Health Manual, “Biosafety in Microbiological and Biomedical Laboratories"” 1984.
9. Tietz, N.W. ed., Clinical Guide to Laboratory Tests, 3rd Edition, W.B. Saunders, Co., Philadelphia, 1995: 10. ICN Guide to Endocrine Testing. Diagnostic Division, For research use only. Not for diagnostic or therapeutic use. Prolias Technologies products may not be sold or modified for resale or used to manufacture commercial products without prior written approval by Prolias 4Technologies. If you are not satisfied with the product please contact

Source: http://www.proliastechnologies.com/pdfs/Progesterone-Spec-Sheet-PSS-PS1113-2.pdf

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