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Dnov006-progesterone-02122010

Progesterone
determination of Progesterone in human serum or plasma ________________________________________________________________ Product Number: DNOV006 (96 Determinations) ________________________________________________________________ CONTENTS
1. INTRODUCTION
2. INTENDED USE
3. PRINCIPLE OF THE ASSAY
4. MATERIALS
5. STABILITY AND STORAGE
6. REAGENT PREPARATION
7. SPECIMEN COLLECTION AND PREPARATION
8. ASSAY PROCEDURE
9. RESULTS
10. QUALITY CONTROL
11. SPECIFIC PERFORMANCE CHARACTERISTICS
12. LIMITATIONS OF THE PROCEDURE
13. PRECAUTIONS AND WARNINGS
14. LITERATURE
15. ORDERING INFORMATION
1. INTRODUCTION
Progesterone is a C-21 steroid hormone involved in the female menstrual cycle, pregnancy (supports gestation) and embryogenesis of humans and other species. Progesterone is the major naturally occurring human progestagen. Progesterone is important for aldosterone (mineralocorticoid) synthesis, as 17-hydroxyprogesterone is for cortisol (glucocorticoid). Progesterone levels are relatively low in children and postmenopausal women. Adult males have levels similar to those in women during the follicular phase of the menstrual cycle. In women, progesterone levels are relatively low during the preovulatory phase of the menstrual cycle, rise after ovulation, and are elevated during the luteal phase. If pregnancy occurs, progesterone levels are maintained at luteal levels initially. After delivery of the placenta and during lactation, progesterone levels are very low. The fall in progesterone levels following delivery is one of the triggers for milk production . Progesterone is produced in the adrenal glands, the gonads (specifically after ovulation in the corpus luteum), the brain, and, during pregnancy, in the placenta. Progesterone converts the endometrium to its secretory stage to prepare the uterus for implantation. If pregnancy does not occur, progesterone levels will decrease, leading, in the human, to menstruation. Progesterone belongs to the group of neurosteroids that are found in high concentrations in certain areas in the brain and are synthesized there. Neurosteroids affect synaptic functioning, are neuroprotective, and affect myelinization. Progesterone has multiple effects outside of the reproductive system. Progesterone is thermogenic, it reduces spasm and relaxes smooth muscle. Bronchi are widened and mucus regulated. Progesterone acts as an antiinflammatory agent and regulates the immune response. Progesterone also assists in thyroid function, in bone building by osteoblasts. Measurement of serum progesterone concentrations have been used in evaluating ovarian function. 2. INTENDED USE
Competitive immunoenzymatic colorimetric method for quantitative determination of Progesterone in serum or plasma. 3. PRINCIPLE OF THE ASSAY
Microtiter strip wells are precoated with anti-Progesterone antibodies (solid-phase). Progesterone in the sample competes
with added horseradish peroxidase labelled Progesterone (enzyme-labelled antigen) for antibody binding. After incubation
a bound/free separation is performed by solid-phase washing. The immune complex formed by enzyme-labelled antigen
is visualized by adding Tetramethylbenzidine (TMB) substrate which gives a blue reaction product. The intensity of this
product is inversely proportional to the amount of Progesterone in the sample. Sulphuric acid is added to stop the
reaction. This produces a yellow endpoint colour. Absorption at 450 nm is read using an ELISA microwell plate reader.
4. MATERIALS
4.1. Reagents supplied
Anti-Progesterone IgG Coated Wells: 12 breakapart 8-well snap-off strips coated with anti-Progesterone IgG; in
resealable aluminium foil.
Stop Solution: 1 bottle containing 15 ml sulphuric acid, 0.15 mol/l (avoid any skin contact).
Progesterone-HRP Conjugate: 1 bottle containing 22 ml of horseradish peroxidase labelled Progesterone.
TMB Substrate Solution: 1 bottle containing 15 ml 3, 3´, 5, 5´-tetramethylbenzidine (H2O2-TMB 0.26 g/l) (avoid any
skin contact).
Wash solution 10x conc.: 1 bottle containing 50 ml of a 10x concentrated solution of phosphate buffer 20 mM,
Proclin < 0.002%
Progesterone Standards: 5 bottles, 1 ml each
4.2. Materials supplied
4.3. Materials and Equipment needed
ELISA microwell plate reader, equipped for the measurement of absorbance at 450 nm Manual or automatic equipment for rinsing wells Pipettes to deliver volumes between 10 and 1000 µl 5. STABILITY AND STORAGE
The reagents are stable up to the expiry date stated on the label when stored at 2.8 °C in the dark.
6. REAGENT PREPARATION
It is very important to bring all reagents, samples and standards to room temperature (22…28°C) before starting the test run! 6.1. Coated snap-off Strips
The ready to use break apart snap-off strips are coated with anti-Progesterone IgG antibodies. Store at 2…8°C. Open the bag only when it is at room temperature. Immediately after removal of strips, the remaining strips should be resealed in the aluminium foil along with the desiccant supplied and stored at 2…8 °C; stability until expiry date . Do not remove the adhesive sheets on the unused strips. 6.2. Progesterone-HRP Conjugate
Progesterone-HRP Conjugate is a ready to use solution. 6.3. Progesterone Standards
The standards are ready to use. After first opening stable for another 6 months at +4°C. 6.4. TMB Substrate Solution
The bottle contains 15 ml of a tetramethylbenzidine/hydrogen peroxide system. The reagent is ready to use and has to be stored at 2.8°C in the dark. The solution should be colourless or could have a slight blue tinge. If the substrate turns into blue, it may have become contaminated and should be thrown away. 6.5. Stop Solution
The bottle contains 15 ml 0.15 M sulphuric acid solution (R 36/38, S 26). This ready to use solution has to be stored at 2.8°C. 6.6. Wash Solution
Dilute the content of the concentrated Wash Solution with distilled water to a final volume of 500 ml prior to use. For smaller volumes respect the 1:10 dilution ratio. The diluted wash solution is stable for 30 days at 2…8°C. In concentrated wash solution it is possible to observe the presence of crystals, in this case mix at room temperature until complete dissolution of crystals, for greater accuracy dilute the whole bottle of concentrated wash solution to 500ml on taking care also to transfer crystals with washing of the bottle, then mix until crystals are completely dissolved. 7. SPECIMEN COLLECTION AND PREPARATION
The determination of Progesterone can be performed in plasma as well as in serum. Store the sample at -20°C if the determination is not performed on the same day as the sample collection. If samples are stored frozen, mix thawed samples well before testing. Avoid repeated freezing and thawing. 7.1. Precaution
The reagents contain Proclin 300® as preservative Maximum precision is required for dispensation of the reagents. Avoid the exposure of reagent TMB/H2O2 to directed sunlight, metals or oxidants. This method allows the determination of Progesterone from 0.2 – 40.0 ng/ml. For higher values, for example in pregnancy, dilute the sample; consider the dilution factor when calculating the result. Treatment of the patient with cortisone, natural or synthetic steroids can impair Progesterone determination.
8. ASSAY PROCEDURE
8.1. Test Preparation
Please read the test protocol carefully before performing the assay. Result reliability depends on strict adherence to the
test protocol as described. Prior to commencing the assay, the distribution and identification plan for all specimens and
standards should be carefully established on the result sheet supplied in the kit. Select the required number of microtiter
strips or wells and insert them into the holder. Please allocate at least:
It is recommended to determine standards and patient samples in duplicate. Perform all assay steps in the order given and without any appreciable delays between the steps. A clean, disposable tip should be used for dispensing each standard and each patient sample. Dispense 20 µl standards and samples into their respective wells. Add 200 µl Progesterone-HRP Conjugate to each well. Leave well A1 for substrate blank. Cover wells with the foil supplied in the kit. Incubate for 1 hour at 37 °C.
When incubation has been completed, remove the foil, aspirate the content of the wells and wash each well three times with 300 µl diluted wash solution. Avoid overflows from the reaction wells. The soak time between each wash cycle should be >5sec. At the end carefully remove remaining fluid by tapping strips on tissue paper prior to the next step! Note: Washing is critical! Insufficient washing results in poor precision and falsely elevated absorbance values. Dispense 100 µl TMB Substrate Solution into all wells. Incubate for exactly 15 min at room temperature (22…28°C) in the dark.
Dispense 100 µl Stop Solution into all wells in the same order and at the same rate as for the TMB Substrate Solution. Shake the microplate gently. Any blue colour developed during the incubation turns into yellow. Measure the absorbance of the specimen at 450 nm within 30 min after addition of the Stop Solution. 8.2. Measurement
Adjust the ELISA Microwell Plate Reader to zero using the substrate blank in well A1.
If - due to technical reasons - the ELISA reader cannot be adjusted to zero using the substrate blank in well A1, subtract the absorbance value of well A1 from all other absorbance values measured in order to obtain reliable results! Measure the absorbance of all wells at 450 nm and record the absorbance values for each standard and patient sample
in the distribution and identification plan.
Where applicable calculate the mean absorbance values of all duplicates.
9. RESULTS
9.1. Calculation of results
Calculate the mean absorbance for each point of the standard curve and each sample. Plot the mean value of absorbance of the standards against concentration. Draw the best-fit curve through the plotted points. (Four Parameter Logistic). Interpolate the values of the samples on the standard curve to obtain the corresponding values of the concentrations expressed in ng/ml. 9.2. Reference values
The following value for serum or plasma Progesterone should be considered as a guideline: 10. QUALITY CONTROL
Each laboratory should assay controls at normal, high and low levels range of Progesterone for monitoring assay performance. These controls should be treated as unknowns and values determined in every test procedure performed. Quality control charts should be maintained to follow the performance of the supplied reagents. Pertinent statistical methods should be employed to ascertain trends. The individual laboratory should set acceptable assay performance limits. Other parameters that should be monitored include the 80, 50 and 20% intercepts of the standard curve for run-to-run reproducibility. In addition, maximum absorbance should be consistent with past experience. Significant deviation from established performance can indicate unnoticed change in experimental conditions or degradation of kit reagents. Fresh reagents should be used to determine the reason for the variations. If computer controlled data reduction is used to calculate the results of the test, it is imperative that the predicted values for the calibrators fall within 10% of the assigned concentrations. 11. SPECIFIC PERFORMANCE CHARACTERISTICS
11.1. Precision
Intra Assay Variation Within run variation was determined by replicate determination (20x) of three different control sera in one assay. The within assay variability is ≤ 4%. Inter Assay Variation Between run variation was determined by replicate measurements (10x) of three different control sera in different lots. The between assay variability is ≤ 9.3 %. 11.2. Cross Reactivity
The cross reaction of the antibody calculated at 50% according to Abraham is: 11.3. Analytic Sensitivity
The lowest detectable concentration of Progesterone that can be distinguished from the zero standard is 0.05 ng/ml at the 95 % confidence limit. 11.4. Accuracy
The recovery of 1.0 – 2.0 – 4.0 – 8.0 ng/ml Progesterone added to sample gave an average value (±SE) of 100.88% ± 8.29 % with reference to the original concentrations. 11.5. Method comparison
The NovaTec Progesterone ELISA was compared to Adaltis Progesterone EIAGen. Serum samples of 31 serum samples were analysed according in both test systems. The linear regression curve was calculated. Progesterone NovaTec = 0.97 * Progesterone Adaltis + 0.04 r2 = 0.887 12. LIMITATIONS OF THE PROCEDURE
Sample(s), which are contaminated microbiologically, should not be used in the assay. Highly lipemeic or haemolysed specimen(s) should similarly not be used. It is important that the time of reaction in each well is held constant for reproducible results. Pipetting of samples should not extend beyond ten minutes to avoid assay drift. If more than one plate is used, it is recommended to repeat the dose response curve. Addition of the substrate solution initiates a kinetic reaction, which is terminated by the addition of the stop solution. Therefore, the addition of the substrate and the stopping solution should be added in the same sequence to eliminate any time deviation during reaction. Plate readers measure vertically. Do not touch the bottom of the wells. Failure to remove adhering solution adequately in the aspiration or decantation wash step(s) may result in poor replication and spurious results. 13. PRECAUTIONS AND WARNINGS
In compliance with article 1 paragraph 2b European directive 98/79/EC the use of the in vitro diagnostic medical devices is intended by the manufacturer to secure suitability, performances and safety of the product. Therefore the test procedure, the information, the precautions and warnings in the instructions for use have to be strictly followed. The use of the testkits with analyzers and similar equipment has to be validated. Any change in design, composition and test procedure as well as for any use in combination with other products not approved by the manufacturer is not authorized; the user himself is responsible for such changes. The manufacturer is not liable for false results and incidents for these reasons. The manufacturer is not liable for any results by visual analysis of the patient samples. All components of human origin used for the production of these reagents have been tested for anti-HIV antibodies, anti-HCV antibodies and HBsAg and have been found to be non-reactive. Nevertheless, all materials should still be regarded and handled as potentially infectious. Do not interchange reagents or strips of different production lots. No reagents of other manufacturers should be used along with reagents of this test kit. Do not use reagents after expiry date stated on the label. Use only clean pipette tips, dispensers, and lab ware. Do not interchange screw caps of reagent vials to avoid cross-contamination. Close reagent vials tightly immediately after use to avoid evaporation and microbial contamination. After first opening and subsequent storage check conjugate and control vials for microbial contamination prior to further use. To avoid cross-contamination and falsely elevated results pipette patient samples and dispense conjugate without splashing accurately to the bottom of wells. The ELISA is only designed for qualified personnel who are familiar with good laboratory practice. In the used concentration Proclin 300R has hardly any toxicological risk upon contact with skin and mucous membranes! Sulphuric acid irritates eyes and skin. Keep out of the reach of children. Upon contact with the eyes, rinse thoroughly with water and consult a doctor!
13.1. Disposal Considerations

Residues of chemicals and preparations are generally considered as hazardous waste. The disposal of this kind of waste
is regulated through national and regional laws and regulations. Contact your local authorities or waste management
companies which will give advice on how to dispose hazardous waste.

14. LITERATURE

Wisdom G.B. (1976) Clin. Chem. 22 (8), 1243 – 1255. De Villa G.O. et al. (1972) J. Clin. Endoc. Metab. 35, 458. Joyce,B.G. et al. Steroids 29, no 6, 761, (1977) Winkel P. et al. Clin. Chem. 22 (4), 422 (1976) Rajkowski K.N et al. Steroids 29, no 5 (1977)
15. ORDERING INFORMATION
SCHEME OF THE ASSAY
Test Preparation
Prepare reagents and samples as described. Establish the distribution and identification plan for all specimens and controls on the Select the required number of microtiter strips or wells and insert them into the holder. Assay Procedure
Cover wells with foil supplied in the kit Incubate for 1 h at 37 °C
Wash each well three times with 300 µl diluted wash solution Incubate for exactly 15 min at room temperature (22…28°C) in the dark
NovaTec Immundiagnostica GmbH
Technologie & Waldpark

Waldstr. 23 A6 D-63128 Dietzenbach, Germany Tel.: +49 (0) 6074-48760 Fax: +49 (0) 6074-487629 Email : info@NovaTec-ID.com Internet: www.NovaTec-ID.com

Source: http://www.novatec-id.com/fileadmin/user_upload/Hormones/Product_Insert/DNOV006-Progesterone-02122010.pdf

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