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limited number of antibody binding sites. The interaction is illustrated by D. Progesterone Biotin Reagent - 6.0ml
One (1) bottle of reagent contains anti-progesterone biotinylated purified rabbit IgG conjugate in buffer, yellow dye and preservative. Summary:
E. Streptavidin Coated Plate - 96 wells
Measurement of progesterone in serum or plasma is considered to be One 96-well microplate coated with 1.0µg/ml streptavidin and Btn = Biotinylated Antibody (Constant Quantity) the most reliable way to assess its rate of production.
packaged in an aluminum bag with a drying agent. Store at 2-8ºC.
F. Wash Concentrate Solution - 10ml
Progesterone is a steroid hormone, which plays an important role in the One (1) vial containing a surfactant in buffered saline. A preservative preparation for and maintenance of pregnancy. It is synthesized from EnzAg = Enzyme-antigen Conjugate (Constant Quantity) cholesterol via pregnenolone - then rapidly metabolized to G.Substrate Reagent - 12ml/vial
pregnanediol primarily in the liver. The ovary and placenta are the major One (1) bottle containing tetramethylbenzidine (TMB) and hydrogen production sites; but a small amount is also produced by the adrenal peroxide (H2O2) in buffer. Store at 2-8ºC.
cortex in both men and women. Circulating progesterone levels, which Btn = Enzyme-antigen Conjugate - Antibody Complex H. Stop Solution - 8ml/vial
are characteristically low during the follicular phase, increase sharply One (1) vial containing a strong acid (H2SO4). Store at 2-30ºC.
during the luteal phase of menstrual cycles, reaching a maximum I. Production Instructions
approximately 5 to 10 days after the midcycle LH peak. Unless Note 1: Do not use reagents beyond the kit expiration date.
pregnancy occurs, a steep decline to follicular levels sets in about 4 Note 2: Opened reagents are stable for sixty (60) days when stored at 2-
days before the next menstrual period. This pattern constitutes the rationale behind the well established use of serum progesterone A simultaneous reaction between the biotin attached to the antibody Note 3: Above reagents are for a single 96-well microplate.
measurements as a simple and reliable method for ovulation detection.
and the streptavidin immobilized on the microwell occurs. This effects Required But Not Provided:
For routine measurements, immunoassays using steroid specific the separation of the antibody bound fraction after decantation or 1. Pipette capable of delivering 25µl and 50µl volumes with a precision antibodies are preferred. Initial immunoassays, for serum progesterone, used organic solvents to remove the steroid from endogenous binding 2. Dispenser(s) for repetitive deliveries of 0.100ml and 0.300ml volumes proteins such as corticosteroid binding globulin (CBG) and albumin.
Direct measurement of progesterone in serum or plasma is considered to StreptavidinCW = Streptavidin immobilized on well 3. Adjustable volume (200-1000µl) dispenser(s) for conjugate be the method of choice for routine applications. Both RIA and EIA (and 4. Microplate washer or a squeeze bottle (optional) some FIA) are available in the market. Since RIA involves handling Immobilized complex = sandwich complex bound to the solid surface 5. Microplate Reader with 450nm and 620nm wavelength absorbance radioactivity and causes radioactive waste disposal issues, various non- isotopic methods have replaced the RIA. These methods use very The enzyme activity activity in the antibody-bound fraction is inversely 6. Absorbent paper for blotting the microplate wells specific antibodies to determine levels of progesterone in circulation.
proportional to the native antigen concentration. By utilizing several different serum references of known antigen concentration, a dose 7. Plastic wrap or microplate cover for incubation steps The Progesterone ELISA Kit uses a specific anti-progesterone antibody, response curve can be generated from which the antigen 8. Vacuum aspirator (optional) for wash steps and does not require sample extraction of serum or plasma. Cross- concentration of an unknown can be ascertained.
reactivity to other naturally occuring and structurally related steroids is Reagent Materials Provided:
A. Progesterone Calibrators - 0.5ml/vial
The employment of several serum references of known progesterone Seven (7) vials of serum reference for progesterone at concentrations Not for Internal or External Use in Humans or Animals concentration permits construction of a graph of activity and of 0 (A), 0.3 (B), 2.0 (C), 5.0 (D), 15 (E), 30 (F) and 60.0 (G) in ng/ml. concentration. From comparison to the dose response curve, an Store at 2-8ºC. A preservative has been added. The calibrators can Reagent Preparation:
unknown specimen’s activity can be correlated with progesterone be expressed in molar concentrations (nM/L) by multiplying by 3.18 1. Working Enzyme Reagent - Stable for 1 year.
Measure 0.7ml of ‘Progesterone Enzyme Reagent’ and add to the vial containing Steroid Conjugate Buffer. Store at 2-8ºC.
Competitive Enzyme Immunoassay (TYPE 7):
B. Progesterone Enzyme Reagent - 1.0ml/vial
2. Wash Buffer
The essential reagents required for an enzyme immunoassay include One (1) vial of Progesterone (Analog) - horseradish peroxides (HRP) Dilute contents of wash solution to 1000ml with distilled or deionized antibody, enzyme-antigen conjugate and native antigen. Upon mixing conjugate in a protein-stabilizing matrix red with dye. Store at 2-8ºC.
water in a suitable storage container. Diluted buffer can be stored at biotinylated antibody, enzyme-antigen conjugate and a serum C. Steroid Conjugate Buffer - 7.0ml/vial
room temperature (20-27ºC) for up to 60 days.
containing the native antigen, a competition reaction reaction results One (1) vial of reagent contains buffer, red dye, preservative and Note: Do not use the working substrate if it looks blue.
between the native antigen and the enzyme-antigen conjugate for a binding protein inhibitors. Store at 2-8ºC.
Fortress Diagnostics Limited Unit 2C Antrim Technology Park, Antrim BT41 1QS (United Kingdom)
TEL: +44 (0) 2894 487676 FAX: +44 (0) 2894 469933 BXE0661A
Test Procedure:
change in experimental conditions or degradation of kit reagents. Fresh Before proceeding with assay, bring all reagents, serum references and reagents should be used to determine the reason for the variations.
controls to room temperature (20-27ºC).
Calculation of Results:
1. Format the micro plates’ wells for each serum reference, control and A dose response curve is used to ascertain the concentration of patient specimen to be assayed in duplicate. Replace any unused micro well strips back into the aluminium bag, seal and store at 2-8ºC.
1. Record the absorbance obtained from the printout of the microplate 2. Pipette 0.025ml (25µL) of the appropriate serum reference, control or 2. Plot the absorbance for each duplicate serum reference versus the bsorbance(s)
3. Add 0.050ml (50µl) of working Progesterone Enzyme Reagent to all the corresponding progesterone concentration in ng/ml on linear graph paper (do not average the duplicates of the serum references 4. Swirl the microplate gently for 10-20 seconds to mix.
5. Add 0.050ml (50µl) of working Progesterone Biotin Reagent to all 3. Connect the points with a best-fit curve.
4. To determine the concentration of progesterone for an unknown, 6. Swirl the microplate gently for 10-20 seconds to mix.
locate the average absorbance of the duplicates for each unknown QC Parameters:
7. Cover and incubate for 60 minutes at room temperature.
on the vertical axis of the graph, find the intersecting point on the In order for the assay results to be considered valid the following criteria 8. Discard the contents of the microplate by decantation or aspiration. curve, read the concentration (in ng/ml) from the horizontal axis of If decanting, blot the plate dry with absorbent paper.
the graph (the duplicates of the unknown may be averaged as 1. The absorbance (OD) of calibrator 0pg/ml should be ≥1.3.
9. Add 350µl of wash buffer (see Reagent Preparation Section), decant indicated). In the following example, the average absorbance 2. Four out of six quality control pools should be within the established (tap and blot) or aspirate. Repeat two (2) additional times for a total (2.000) intersects the dose response curve at (0.68ng/ml) of three (3) washes. An automatic or manual plate washer can be progesterone concentration (see Figure 1).
used. Follow the manufacturer’s instruction for proper usage. If a A. Assay Performance
squeeze bottle is employed, fill each well by depressing the Example 1
1. It is important that the time of reaction in each well is held constant container (avoiding air bubbles) to dispense the wash. Decant the wash and repeat two (2) additional times.
2. Pipetting of samples should not extend beyond ten (10) minutes to 10. Add 0.100ml (100µl) of working substrate solution to all wells. Always add reagents in the same order to minimize reaction time differences 3. If more than one (1) plate is used, it is recommended to repeat the DO NOT SHAKE THE PLATE AFTER SUBSTRATE ADDITION.
4. Addition of the substrate solution initiates a kinetic reaction, which is 11. Incubate at room temperature for twenty (20) minutes.
terminated by the addition of the stop solution. Therefore, the 12. Add 0.050ml (50µl) of stop solution to each well and gently mix for 15- addition of the substrate and the stopping solution should be added 20 seconds. Always add reagents in the same order to minimize in the same sequence to eliminate any time-deviation during reaction time differences between wells.
13. Read the absorbance in each well at 450nm (using a reference 5. Plate readers measure vertically. Do not touch the bottom of the wavelength of 620-630nm). The results should be read within thirty (30) minutes of adding the stop solution.
6. Failure to remove adhering solution adequately in the aspiration or Note: Dilute the samples suspected of concentrations higher than decantation wash step(s) may result in poor replication and spurious 60ng/ml 1:5 and 1:10 with progesterone ‘0’ng/ml calibrator or male patient serum pools with a known low value for progesterone.
7. Use components from the same lot. No intermixing of reagents from Quality Control:
8. Accurate and precise pipetting, as well as following the exact time Each laboratory should assay controls at levels in the low, normal and and temperature requirements prescribed are essential. Any high range for monitoring assay performance. These controls should be deviation from this may yield inaccurate results.
treated as unknowns and values determined in every test procedure 9. It is important to calibrate all the equipment e.g. Pipettes, Readers, performed. Quality control charts should be maintained to follow the Washers and/or the automated instruments used for using this device.
performance of the supplied reagents. Pertinent statistical methods The above data and table is for example only. Do not use it for
should be employed to ascertain trends. The individual laboratory should B. Interpretation
calculating your results.
set acceptable assay performance limits. In addition, maximum 1. If computer controlled data reduction is used to interpret the results absorbance should be consistent with past experience. Significant of the test, it is imperative that the predicted values for the deviation from established performance can indicate unnoticed calibrators fall within 10% of the assigned concentrations. Fortress Diagnostics Limited Unit 2C Antrim Technology Park, Antrim BT41 1QS (United Kingdom)
TEL: +44 (0) 2894 487676 FAX: +44 (0) 2894 469933 BXE0661A
Expected Ranges of Values:
In agreement with established reference intervals for a “normal” adult Between Assay Precision (Values in ng/ml)
1. Abraham GE. The application of natural steroid radioimmunoassay to population and females during gestation the expected ranges for the gynecologic endocrinology. In: Abraham GE, editor. Radioassay Systems Progesterone ELISA Test Kit are detailed in Table 1.
in Clinical Endocrinology, Basel: Marcel Dekker,: 475-529 (1981) 2. Aufrere MB, Benson H. Progesterone: an overview and recent advances., Table 1: Expected Values for the Progesterone Test Kit
3. Bauman J, “Basal body temperature: unreliable method of ovulation detection”, Fertility Sterility, 36: 729-33 (1981) * As measured in ten experiments in duplicate over a ten day period
4. Brown JB, “Timing of Ovulation”, Med J Austral, 2:780-3 (1977) Accuracy
5. Gautray JP, et al, “Clinical investigation of the menstrual cycle: clinical, Adult Woman
The Progesterone ELISA Test Kit was compared with a endometrial and endocrine aspects of luteal defects”, Fertility Sterility, chemiluminescence immunoassay method. Biological specimens from low, normal and high progesterone level populations were used (The 6. Hensleigh PA, Fainstat T, “Corpus luteum dysfunction: serum progesterone values ranged from <0.15ng/ml-128ng/ml). The total number of such levels in diagnosis and assessment of therapy for recurrent and threatened Pregnant Woman
specimens was 60. The least square regression equation and the abortion”, Fertility Sterility, 32: 396-9 (1979) correlation coefficient were computed for this method in comparison 7. Hernandez JL, et al, “Direct evidence of luteal insufficiency in women with with the reference method. The data obtained in displayed in table 4.
habitual abortion”, Obstetric Gynecology, 49:705-8 (1977) 8. Jones G. Luteal phase defects. In: Behman SJ, Kistner RW, editors. Progress in Infertility. Boston: Little, Brown and Company, 2nd ed., 1975: 299-324 Postmenopausal Woman
Mean (x) Least Square Regression
9. Klopper A, Fuchs F. Progestagens. In Fuchs F, Klopper A, editors.
Endocrinology of Pregnancy. Hagerstown: Harper & Row, 99-122 (1977) During pregnancy the progesterone serum levels rise rapidly till the end Analysis
10. Lehmann F, Bettendorf G, “The endocrine shift from a normal cycle to anovulation”, Insler V, Bettendorf G, editors. Advances in Diagnosis and Treatment of Infertility. Amsterdam: Elseveir/North Holland, 105-13 (1981) It is important to keep in mind that establishment of a range of values 11. March CM. Luteal phase defects. In: Mishell DR, Davajan V, editors. which can be expected to be found by a given method for a Only slight amounts of bias between this method and the reference Reproductive Endocrinology, Infertility and Contraception. Philadelphia: population of “normal” persons is dependent upon a multiplicity of method are indicated by the closeness of the mean values. The least factors: the specificity of the method, the population tested and the square regression equation and correlation coefficient indicates 12. March CM, Goebelsmann U, Nakamura RM, Mishell Dr. Roles of estradiol precision of the method in the hands of the analyst. For these reasons and progesterone in eliciting the midcycle luteinizing hormone and follicle each laboratory should depend upon the range of expected values Sensitivity:
stimulating hormone surges. J Clin Endocrinol Metab, 49: 507-13 (1979) established by the manufacturer only until an in-house range can be The Progesterone ELISA Test Kit has a sensitivity of 0.105ng/ml. The 13. BIO-ED slide/seminar eductional program. Rochester: Bioeducational determined by the analysts using the method with a population sensitivity was ascertained by determining the variability of the 0ng/ml indigenous to the area in which the laboratory is located.
serum calibrator and using the 2 (95% certainty) statistic to calculate 14. Radwanska E, et al, “Plasma progesterone and estradiol estimations in the diagnosis and treatment of luteal insufficiency in menstruating infertile Performance Characteristics:
women”, Acta Eur Fertility, 7:39-47 (1976) Precision
15. Radwanska E, et al, “Plasma progesterone levels in normal and abnormal The within and between assay precision of the Progesterone ELISA Test The % cross-reactivity of the progesterone antibody to selected early human pregnancy”, Fertility Sterility, 37: 398-402 (1978).
Kit were determined by analyses on three different levels of pool control substances was evaluated by adding the interfering substance to a 16. Tietz, Textbook of Clinical Chemistry, 2nd ed. Philadelphia: W.B. Saunders, sera. The number, mean values, standard deviation and coefficient of serum matrix at various concentrations. The cross-reactivity was variation for each of these control sera are presented in Table 2 and calculated by deriving a ratio between dose of interfering substance to dose of progesterone needed to displace the same amount of labelled Within Assay Precision (Values in ng/ml)
Cross Reactivity Substance
Cross Reactivity
Fortress Diagnostics Limited Unit 2C Antrim Technology Park, Antrim BT41 1QS (United Kingdom)
TEL: +44 (0) 2894 487676 FAX: +44 (0) 2894 469933


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