Dsnov23-17 oh progesterone saliva-engl,dt-2704201

17 OH Progesterone Saliva
Enzyme immunoassay for the quantitative determination of 17 α OH progesterone in human saliva Enzymimmunoassay zur quantitativen Bestimmung von 17 α OH Progesteron in humanem Speichel
Only for in-vitro diagnostic use
English:
Summary of Test Procedure/ Kurzanleitung ________________________________________________________________ Product Number: DSNOV23 (96 Determinations) ________________________________________________________________ 1. INTRODUCTION
Progesterone (17α-OHP, 17-hydroxy-4-Pregene-3, 20-dione) is a C-21 steroid hormone involved in the female menstrual cycle, pregnancy (supports gestation) and embryogenesis of humans. As with cortisol, serum α-OHP levels normally have an ACTH-dependent diurnal variation, with peak levels in the morning and a nadir at night. In addition, ovarian production of 17 α-OHP increase during the luteal phase of the menstrual cycle, leading to serum levels which are several-fold higher than during the follicular phase. The level of 17 α-OHP in saliva (pg/mL) is significantly lower than levels in the general circulation (ng/mL). Progesterone appears to prevent endometrial cancer by regulating the effects of estrogen. Measurement of circulating 17 α-OHP levels is a standard tool for clinical assessment of 21-hydroxylase deficiency, the most common cause of congential adrenal hyperplasia (CAH). Due to the decreased activity of 21-hydroxylase, 17 α-OHP cannot be effectively converted to cortisol and, instead, accumulates in large amounts and is shunted into the androgen biosynthetic pathway. 17 α-OHP levels are useful in monitoring steroid replacement therapy. 2. INTENDED USE
Competitive immunoenzymatic colorimetric method for quantitative determination of 17 α OH Progesterone in saliva. 3. PRINCIPLE OF THE ASSAY
Microtiter strip wells are precoated with anti-17 α OH Progesterone antibodies (solid-phase). 17 α OH Progesterone in the
sample competes with added horseradish peroxidase labelled 17 α OH 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 17 α OH 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-17 α OH Progesterone IgG Coated Wells: 12 breakapart 8-well snap-off strips coated with anti-17 α OH
Progesterone IgG; in resealable aluminium foil.
Stop Solution: 1 bottle containing 15 ml sulphuric acid, 0.15 mol/l (avoid any skin contact).
17 α OH Progesterone-HRP Conjugate (conc.): 1 bottle containing 1 ml of horseradish peroxidase labelled
17 α OH Progesterone.
TMB Substrate Solution: 1 bottle containing 15 ml 3, 3´, 5, 5´-tetramethylbenzidine (H2O2-TMB 0.26 g/l) (avoid any
skin contact).
Incubation buffer: 1 bottle containing 30 ml of phosphate buffer, pH 7.5, BSA 1 g/L, stabilizer
Wash solution 50x conc.: 1 bottle containing 20 ml (NaCl 45g/l, Tween-20 55 g/l)
L Control: 1 bottle containing 1 ml of a ready to use control solution with low 17 α-OHP concentration. The exact
value is given on the label and the certificate of analysis. M Control: 1 bottle containing 1 ml of a ready to use control solution with medium 17 α-OHP concentration. The
exact value is given on the label and the certificate of analysis. 17 α OH 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-17 α OH 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 of the kit. Do not remove the adhesive sheets on the unused strips. 6.2. 17 α OH Progesterone -HRP Conjugate
The bottle contains 1 ml of a concentrated solution with 17 α OH Progesterone conjugated with horseradish peroxidase. Dilute immediately before use: Add 10 µl concentrated conjugate to 1.0 ml of Incubation Buffer. Mix gently. Stable for 3 hours at room temperature (22…28°C). 6.3. 17 α OH Progesterone Standards
Before use, mix 5 min. with a rotating mixer. The standard solutions are ready to use. After first use the standards are stabile until the expiry date of the kit if stored at 2…8°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 contents of concentrated Wash solution to 1l with distilled water. For smaller volumes respect the 1:50 ratio. The diluted wash solution is stable for 30 days at 2…8°C. 7. SPECIMEN COLLECTION AND PREPARATION
7.1. Method and Limitations
The determination of 17 α OH Progesterone can be performed in saliva. It is recommended to collect saliva samples with a centrifuge glass tube and a plastic straw. Do not use sample collectors commercially available like “SALIVETTE”. Other equipment of sample collection commercially available has not been tested. Collect saliva samples at the times indicated. If no specific instructions have been given, saliva samples may be collected at any time, paying attention to the following indications: a) If saliva collection has to be carried out in the morning ensure that this is carried out prior to brushing teeth. During the day saliva should be collected at least 1 hour after any food or drink. It is very important that a good clear sample is received – i.e. no contamination with food, lipstick, blood (bleeding gums) or other such extraneous materials. 7.2. Processing
Let the saliva flow down through the straw into the centrifuge glass tube Centrifuge the sample for 15 minutes at 3000 rpm Centrifuge again for 15 minutes at 3000 rpm The saliva sample is now ready to be tested. Store the sample at 2…8°C for one week or at – 2 0°C for longer time. 7.3. Precaution
Maximum precision is required for reconstitution and dispensation of the reagents. This method allows the determination of Progesterone from 20 – 1600 pg/ml. Treatment of the patient with cortisone, natural or synthetic steroids can impair Progesterone determination. Avoid the exposure of TMB substrate solution to direct sunlight, metals or oxidants. Samples with concentrations higher than 1600 pg/ml should be diluted 1:1 with standard 0. 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. 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. Please allocate at least:
It is necessary 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 50 µl standards, controls and samples into their respective wells. Add 150 µl 17 α OH 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 of the absorbance (OD) for each point of the standard curve and of each sample. Plot the mean value of absorbance of the standards (OD) against concentration. Draw the best-fit curve through the plotted points. (es: Four Parameter Logistic). Interpolate the values of the samples on the standard curve to obtain the corresponding values of the concentrations expressed in pg/ml. 9.2. Reference Value
We recommend this assay as reliable and powerful technique to monitor metabolic control in patient with CAH (congenital
adrenal hyperplasia).
As the values of salivary 17 OH Progesterone have a circadian pattern we suggest collecting the samples at the same
hour (8 A.M.):
The following values can be used as preliminary guideline until each laboratory established its own normal range.
Age
Median (pg/mL)
Range (pg/mL)

10. QUALITY CONTROL
Each laboratory should assay controls at normal, high and low levels range of 17 OH-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 (16x) of two different saliva controls in one assay. The within assay variability is ≤ 6.1%. Inter Assay Variation Between run variations was determined by replicate measurements (12x) of two different saliva controls in different lots. The between assay variability is ≤ 12.1%. 11.2. Specificity
The cross reaction of the antibody calculated at 50% according to Abraham is: 11.3. Accuracy
The recovery of 100 – 200 - 400 pg/ml 17 α OH Progesterone added to saliva sample gave an average value (±SD) of 99.95% ± 4.97% with reference to the original concentrations. 11.4. Analytical Sensitivity
The lowest detectable concentration of 17 OH Progesterone that can be distinguished from standard 0 is 4.9 pg/ml at the 95% confidence limit. 11.5. Correlation
The NovaTec 17OH Progesterone saliva ELISA kit was compared to another commercially available 17OH Progesterone saliva assay. 27 saliva samples were analysed according in both test systems. The linear regression curve was calculated: y = 0.87x + 15.25 r2 = 0.835 y = 17OH Progesterone saliva NovaTec Elisa kit x = 17OH Progesterone saliva Salimetrics Elisa kit 12. LIMITATIONS OF THE PROCEDURE
Sample(s), which are contaminated microbiologically, should not be used in the assay. 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 it lasts longer then ten minutes, follow the same order of dispensation. 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 test kits 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. 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. 13. ORDERING INFORMATION
17 α OH Progesterone Saliva (96 Determinations) 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 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 : [email protected] Internet: www.NovaTec-ID.com

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