Microsoft word - pathozyme progesterone ce v5

Enzyme Immunoassay for the quantitative determination of Progesterone
in human serum or plasma.
Store at 2oC to 8oC. DO NOT FREEZE.
For in-vitro use only.

Reference Standard: Progesterone diluted in human Progesterone is a C21 steroid which is synthesised from both tissue and circulating cholesterol. Cholesterol is transformed to pregnenolone which is Level as stated on vial
then converted via a combined dehydrogenase and isomerase to Known level of Progesterone diluted in human progesterone. The principle production sites are the adrenals and ovaries and the placenta during pregnancy. The majority of this steroid is Level as stated on vial
metabolised in the liver to pregnanediol and conjugated as a glucuronide Known level of Progesterone diluted in human Progesterone exhibits a wide variety of end organ effects. The primary role Progesterone
of progesterone is exhibited by the reproductive organs. In males, Rabbit anti Progesterone reagent. Ready to use progesterone is a necessary intermediate for the production of cortcosteroids and androgens. In females, progesterone remains relatively constant throughout the follicular phase of the menstrual cycle. The Progesterone conjugated to Horseradish Peroxidase. concentration then increases rapidly following ovulation and remains elevated for 4-6 days and decreases to the initial level 24 hours before the onset of menstruation. In pregnancy, placental progesterone production Phosphate based buffer containing stabilising rises steadily to levels of 10 to 20 times those of the luteal phase peak. Progesterone measurements are thus performed to determine ovulation as well as to characterise luteal phase defects. Monitoring of progesterone Substrate Solution: 3,3’, 5,5’ Tetramethyl Benzidine therapy and early stage pregnancy evaluations comprise the remaining uses in a citrate buffer. Ready to use. (Colourless) INTENDED USE
Stop Solution: Hydrochloric Acid diluted in purified Instruction Leaflet and EIA Data Recording Sheet PATHOZYME PROGESTERONE is an Enzyme Immunoassay (EIA) for the
quantitative determination of total Progesterone in human serum or plasma. MATERIAL REQUIRED BUT NOT PROVIDED
Micropipettes: 100μl, 200μl, 1000μl and 5000μl Disposable pipette tips The PATHOZYME PROGESTERONE is based on the principle of
Microplate reader fitted with a 450nm filter competitive binding between Progesterone in the test specimen and Progesterone-HRP Conjugate for a constant amount of rabbit anti- Progesterone. In the incubation, goat anti-rabbit IgG-coated wells are incubated with Progesterone standards, controls, patient samples, Progesterone-HRP Conjugate Reagent and rabbit anti-Progesterone PRECAUTIONS
Reagent. During the incubation, a fixed amount of HRP-labelled Progesterone competes with the endogenous Progesterone in the standard PATHOZYME PROGESTERONE contains materials of human origin
and sample or quality control serum for a fixed number of binding sites of which have been tested and confirmed negative for HCV, HIV I and II the specific Progesterone antibody. Thus, the amount of Progesterone antibodies and HBsAg by FDA approved methods at single donor peroxidase conjugate immunologically bound to the well progressively level. Because no test can offer complete assurance that products decreases as the concentration of Progesterone in the specimen increases. derived from human source will not transmit infectious agents it is Unbound Progesterone peroxidase conjugate is then removed and the wells recommended that the reagents within this kit be handled with due washed. The Substrate (TMB) is then added, resulting in the development of care and attention during use and disposal. All reagents should, blue colour. The colour development is stopped with the addition of stop however, be treated as potential Biohazards in use and for disposal. solution, and the absorbance is measured spectrophotometrically at 450nm. The intensity of the colour formed is proportional to the amount of enzyme present and is inversely related to the amount of unlabelled Progesterone in PATHOZYME PROGESTERONE Reagents do not contain dangerous
the sample. A standard curve is obtained by plotting the concentration of substances as defined by current UK Chemicals (Hazardous the standard versus the absorbance. The Progesterone concentration of Information and Packaging for Supply) regulations. All reagents the specimens and controls run concurrently with the standards can be should, however, be treated as potential biohazards in use and disposal. Final disposal must be in accordance with local legislation. This test has been calibrated against in house standards. There is no PATHOZYME PROGESTERONE Stop Solution is dilute Hydrochloric
Acid and is therefore corrosive. Handle with care. In case of contact,
PATHOZYME PROGESTERONE reagents contain 1.0% Proclin™
300* as a preservative which may be toxic if ingested. In case of contact, rinse thoroughly with water and seek medical advice. CONTENTS
* Proclin™ 300 is a Trade Mark of ROHM and HAAS Ltd. Microtitre Plate
12 x 8 wells x 1
Reagents must be stored at temperatures between 2oC to 8oC. Breakable wells coated with Goat anti Rabbit IgG contained in a resealable foil bag with a desiccant. Expiry date is the last day of the month on the bottle and the kit label. The kit will perform within specification until the stated expiry date as determined from date of product manufacture and stated on kit and Progesterone. Ready to use. (Colourless) components. Do not use reagents after the expiry date. 0.5 ng / ml
Reference Standard: Progesterone diluted in human Exposure of reagents to excessive temperatures should be avoided. 3.0 ng / ml
Reference Standard: Progesterone diluted in human DO NOT FREEZE ANY OF THE REAGENTS as this will cause Reference Standard: Progesterone diluted in human Reference Standard: Progesterone diluted in human SPECIMEN COLLECTION AND PREPARATION
For use by operatives with at least a minimum of basic laboratory Obtain a sample of venous blood from the patient and allow a clot to form and retract. Centrifuge clotted blood sample and collect clear serum. Fresh Do not use damaged or contaminated kit components. Use a separate disposable tip for each sample to prevent cross Obtain a sample of venous blood from the patient and add to EDTA blood collection vial. Centrifuge sample and collect clear plasma. Fresh plasma Duplication of all standards and specimens, although not required, is Do not use haemolysed, contaminated or lipaemic serum or plasma for testing as this will adversly affect the results. Specimens and standards should be run at the same time to keep Serum or plasma may be stored at 2oC to 8oC for up to 48 hours prior to testing. If longer storage is required, store at –20oC for up to 1 year. It is recommended that no more than 32 wells be used for each assay Thawed samples must be mixed prior to testing. run if manual pipetting is used, since pipetting of all Standards and specimens should be completed within 3 minutes. A full plate of 96 Do not use Sodium Azide as a preservative as this may inhibit the wells may be used if automated pipetting is available. Replace caps on all reagents immediately after use. Do not repeatedly freeze-thaw the specimens as this will cause false results. Avoid repeated pipetting from stock reagents as this is likely to cause REAGENT PREPARATION
Do not mix reagents or antibody coated strips from different kits. All reagents should be brought to room temperature (20oC to 25oC) and When dispensing, care should be taken not to touch the surface of the mixed gently prior to use. Do not induce foaming. Working Solution: Dilute the concentrated conjugate using 1 part conjugate Do not allow reagent to run down the sides of the well. Prior to the to 10 parts conjugate diluent ( 1/11 dilution ) 100μl is required per well. start of the assay bring all reagents to room temperature (20oC to Diluted reagent is stable at 2oC to 8oC for one month. 25oC). Gently mix all reagents by gentle inversion or swirling. Once an assay has been initiated, the wells should not be allowed to LIMITATIONS OF USE
The use of samples other than serum and EDTA plasma have not been Do not contaminate the Substrate Solution as this will render the whole validated in this test. There is no reuse protocol for this product. When making an interpretation of the test it is strongly advised to take all clinical data into consideration. Diagnosis should not be made solely on the findings of one Check the precision and accuracy of the laboratory equipment used during the procedure to ensure reproducible results. ASSAY PROCEDURE
The unused strips should be resealed in the foil bag, containing the desiccant, using the resealing zip-lock before being replaced at 2oC to Bring all the kit components and the test sample to room temperature (20°C to 25°C) prior to the start of the assy. One set of Standards should be run with each batch of test sample. CALCULATION OF RESULTS
Secure the desired number of coated wells in the holder. Record the position of the standards and the test samples on the EIA Data Calculate the mean absorbance value (A450) for each set of Standards, Unused strips should be resealed in the foil bag containing the Construct a point to point standard curve by plotting the mean desiccant, using the resealing zip-lock before being replaced at 2°C to absorbance obtained for each Standard against its concentration in ng/ml on graph paper, with absorbance values on the vertical or Y-axis 25μl of standards, test samples and controls into the and concentrations horizontal or the X-axis. Use the mean absorbance values for each specimen to determine the 100μl working solution of Progesterone-HRP Conjugate corresponding concentration of Progesterone in ng/ml from the 50μl of rabbit anti-Progesterone Reagent to each well. If levels of controls or users known samples do not give expected Thoroughly mix for 30 seconds. It is very important to mix completely. results, test results must be considered invalid. Incubate at room temperature (20oC to 25 oC) for 90 minutes. If using a software package choose a polygon with data extrapolation At the end of the incubation period, discard the contents of the wells by flicking plate contents into a Biohazard container. Then strike the wells sharply against absorbent paper. Ensure adequate disinfectant EXPECTED VALUES AND SENSITIVITY
is contained in the Biohazard container. Hand Washing: Fill the wells with a minimum of 300μl of distilled The graph produced by the calibrators should be Hyperbolic in shape water per well. Flick plate contents into a Biohazard container. Then with the OD450 of the calibrators inversely proportional to their strike the wells sharply against absorbent paper. Wash the empty concentration. The OD of calibrator A should be greater than 1.5 and the OD of calibrator F less than 0.75 for the assay results to be valid. 10. Strike the wells sharply onto absorbent paper or paper towel to Each laboratory should establish its own normal range based on the 11. Machine Washing: Ensure that 300μl of distilled water is dispensed patient population. PATHOZYME PROGESTERONE was performed
per well and that an appropriate disinfectant is added to the waste on randomly selected outpatient clinical laboratory samples. The collection bottle. Wash the empty wells 5 times. After washing results of these determinations are as follows: remove excess fluid by striking the wells sharply onto absorbent paper or paper towel to remove all residual water droplets. 100μl of Substrate solution into each well. Gently mix for 5 13. Incubate in the dark at room temperature (20°C to 25°C) for 20 14. Stop the reaction by adding 100μl of Stop Solution to each well. 15. Gently mix for 30 seconds. It is important to make sure that all the blue colour changes to yellow colour immediately. 16. Read the absorbance at 450nm with a microtitre well reader within 10 SENSITIVITY
The lowest detectable level of Progesterone in this test is approximately 25μl of standards, test samples, controls. 100μl working solution Progesterone HRP conjugate SPECIFICITY
The following materials have been checked for cross reactivity. The 50μl of Rabbit anti-Progesterone into each well. percentage indicates cross reactivity at 50% displacement compared to Incubate for 90 minutes at room temperature (20oC to Data on the cross-reactivity for several endogenous and pharmaceutical Discard the well contents and wash 5 times with distilled steroids are summarised in the following table: 100μl of substrate solution into each well and gently Cross-reactivity(%) = Observed Progesterone Concentrationx100 Incubate in the dark for 20 minutes at room temperature Cross-reactivity
100μl Stop Solution to each well and gently mix for 30 Read the Optical Densities immediately (no later than 10 minutes) using a microplate reader with a 450nm filter. <0.08% 8098 ISSUE 5 Revised March 2010 Oestradiol <0.01% Omega Diagnostics Ltd., 2010 Estrone EVALUATION DATA

Calibrated to major competitors and in house standards.
The co-efficient of variation of PATHOZYME PROGESTERONE is less than
or equal to 10%
In an evaluation between the Omega Pathozyme Progesterone kit and the
DRG BIOC Kit for samples with levels between 0.35 ng/ml and 73.83 ng/ml
the following data was generated.
These kits were shown to give good correlation. REFERENCES
Radwanska, E., Frankenberg, J., and Allen. E., Plasma
Progesterone Levels in Normal and Abnormal Early Pregnancy,
Fertility and Sterility 30, 398-402 (1978).
Autrere, M.B., and Benson, H., Progesterone: An Overview and
Recent Advances, J. Par. Sci. 65, 783-800 (1976).
March, C.M., Goebelsmann, U., Nakamura, R.M., and Mischell,
Roles of Estradiol and Progesterone in Eliciting the Midcycle
Luteinising Hormone and Follicle-Stimulating Hormone Surges. J.
Clin. Endo. And Metab. 48: 507-513, (1979).
Ross, G.T., Van De Wiele, R.L., and Frantz, A.G. The Ovaries and
the Breasts in Textbook of Endocrinology, R.H. Williams ed. P355-
407, W.B. Saunders, Phil. (1981).
Chattoraj, S.C., Endocrine Function in Fundamentals of Clinical
Chemistry, N.W. Tietz ed. p699-823, W.B. Saunders, Phil. Chap. 13
Shepard, M.K., and Fainstat, T. Comparison of Serum Progesterone
and Endometrial Biopsy for Confirmation of Ovulation and Evaluation
of Luteal Function. Fertility and Sterility, 28: 541; (1977).
Johansson, E.D., and Johansson, L.E. Progesterone Levels in
Amniotic Fluid and Plasma from Women. I. Levels During Normal
Pregnancy. Acta. Obstet. Gynaecol. Scand. 50: 339; (1971).
USA Centre for Disease Control/National Institute of Health Manual “Biosafety in Microbiological and Biomedical Laboratories” 1984. OMEGA DIAGNOSTICS LTD.
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