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response curve, an unknown specimen's activity can be correlated One (1) vial contains a strong acid (0.5M H2SO4). Store at 8.0 REAGENT PREPARATION
2-30°C.
1. Working Enzyme Reagent - Stable for 1 year.
3.0 PRINCIPLE
I. Product
Instructions
Measure 0.7 ml of ‘17-OH Progesterone Enzyme Reagent’ and add to the vial containing Steroid Conjugate Buffer. Store at 2- Competitive Enzyme Immunoassay (TYPE 7):
Note 1: Do not use reagents beyond the kit expiration date.
The essential reagents required for a enzyme immunoassay Note 2: Avoid extended exposure to heat and light. Opened
2. Wash Buffer
include antibody, enzyme-antigen conjugate and native antigen. reagents are stable for sixty (60) days when stored at
Dilute contents of wash solution to 1000ml with distilled or Upon mixing biotinylated antibody, enzyme-antigen conjugate and deionized water in a suitable storage container. Diluted buffer a serum containing the native antigen, a competition reaction °C. Kit and component stability are identified on the
can be stored at 2-30°C for up to 60 days. results between the native antigen and the enzyme-antigen Note 3: Above reagents are for a single 96-well microplate.
Note1 : Do not use the working substrate if it looks blue.
conjugate for a limited number of antibody binding sites. The Note 2: Do not use reagents that are contaminated or have
interaction is illustrated by the followed equation: 4.1 Required But Not Provided:
bacteria growth.
1. Pipette capable of delivering 25 μl and 50 μl with a precision of 9.0 TEST PROCEDURE
2. Dispenser(s) for repetitive deliveries of 0.100ml and 0.350ml Before proceeding with the assay, bring all reagents, serum volumes with a precision of better than 1.5%. 3. Adjustable volume (200-1000µl) dispenser(s) for conjugate. references and controls to room temperature (20 - 27°C). AbBtn = Biotinylated Antibody (Constant Quantity) 4. Microplate washer or a squeeze bottle (optional). **Test Procedure should be performed by a skilled individual
17α-OH Progesterone Test System
5. Microplate Reader with 450nm and 620nm wavelength or trained professional**
Product Code: 5225-300
Ag = Enzyme-antigen Conjugate (Constant Quantity) 6 Absorbent Paper for blotting the microplate wel s. 1. Format the microplates’ wel s for each serum reference, 7 Plastic wrap or microplate cover for incubation steps. control and patient specimen to be assayed in duplicate. 8 Vacuum aspirator (optional) for wash steps. Replace any unused microwell strips back into the
Btn = Enzyme-antigen Conjugate -Antibody Complex aluminum bag, seal and store at 2-8
1.0 INTRODUCTION
2. Pipette 0.025 ml (25 µL) of the appropriate serum reference, control or specimen into the assigned wel . Intended Use: The Quantitative Determination of 17-OH
5.0 PRECAUTIONS
3. Add 0.050 ml (50µl) of working 17α-OH Progesterone Enzyme Progesterone Concentration in Human Serum or Plasma by a
Microplate Enzyme Immunoassay
4. Swirl the microplate gently for 20-30 seconds to mix. A simultaneous reaction between the biotin attached to the For In Vitro Diagnostic Use
5. Add 0.050 ml (50µl) of the 17α-OH Progesterone Biotin antibody and the streptavidin immobilized on the microwell occurs. Not for Internal or External Use in Humans or Animals
2.0 SUMMARY AND EXPLANATION OF THE TEST
This effects the separation of the antibody bound fraction after 6. Swirl the microplate gently for 20-30 seconds to mix All products that contain human serum have been found to be Plasma/Serum concentrations of 17α-hydroxyprogesterone (17α- non-reactive for Hepatitis B Surface Antigen, HIV 1&2 and HCV 7. Cover and incubate for 60minutes at room temperature. OHP) are valuable in the initial diagnosis of congenital adrenal 8. Discard the contents of the microplate by decantation or Btn + EnzAgAbBtn + StreptavidinCW ⇒ immobilized complex Antibodies by FDA required tests. Since no known test can offer hyperplasia (CAH) 1, 2. This common inborn error of metabolism is aspiration. If decanting, blot the plate dry with absorbent complete assurance that infectious agents are absent, all human usually characterized by deficiency in the C21-hydroxylase serum products should be handled as potentially hazardous and enzyme system, and necessitates steroid replacement therapy. Immobilized complex = sandwich complex bound to the solid capable of transmitting disease. Good laboratory procedures for 9. Add 350µl of wash buffer (see Reagent Preparation Section), Adequacy of treatment has been monitored by determining handling blood products can be found in the Center for Disease decant (tap and blot) or aspirate. Repeat two (2) additional The enzyme activity in the antibody bound fraction is inversely Control / National Institute of Health, "Biosafety in Microbiological times for a total of three (3) washes. An automatic or manual
proportional to the native antigen concentration. By utilizing and Biomedical Laboratories," 2nd Edition, 1988, HHS Publication plate washer can be used. Follow the manufacturer’s
The incidence is roughly estimated to be 1 in 15,000 newborns several different serum references of known antigen con- instruction for proper usage. If a squeeze bottle is
and can reach as high as 1 in 1480 in native Alaskans. Early centration, a dose response curve can be generated from which employed, fill each well by depressing the container
diagnosis is valuable to detect CAH in newborns afflicted with the the antigen concentration of an unknown can be ascertained. Safe Disposal of kit components must be according to local
(avoiding air bubbles) to dispense the wash. Decant the
disease, not clinically recognizable but which will lead to life regulatory and statutory requirement.
wash and repeat two (2) additional times.
threatening adrenal crisis in the neonatal period and to determine 4.0 REAGENTS
10. Add 0.100 ml (100µl) of substrate solution to all wel s (see the cause of infants with ambiguous genitalia. Delayed diagnosis 6.0 SPECIMEN COLLECTION AND PREPARATION
Reagent Preparation Section). Always add reagents in the
may also lead to further virilization in female children, acceleration Materials Provided:
same order to minimize reaction time differences between
of skeletal maturation and premature development of secondary A. 17α-OH Progesterone Calibrators – 1ml/vial - Icons A-F
The specimens shall be blood; serum or heparanised plasma in sex characteristics in male children. Prompt treatment can save DO NOT SHAKE THE PLATE AFTER SUBSTRATE ADDITION
Six (6) vials of serum reference for 17-OH Progesterone at type and taken with the usual precautions in the col ection of the life of infants and allow afflicted children to attain normal concentrations of 0 (A), 0.1 (B), 0.5 (C), 1.0 (D), 2.5 (E), and
venipuncture samples. For accurate comparison to establish 11. Incubate at room temperature for twenty (20) minutes. 10 (F) ng/ml. Store at 2-8°C. A preservative has been added. normal values, a fasting morning serum sample should be 12. Add 0.050ml (50µl) of stop solution to each wel and gently mix The calibrators can be expressed in molar concentrations obtained. The blood should be collected in a redtop (with or for 15-20 seconds. Always add reagents in the same order
17P is a steroid produced in the adrenal cortex and the gonads. It (nM/L) by multiplying by 3.03.For example: 1ng/ml x 3.03 = without gel additives) venipuncture tube or for plasma use to minimize reaction time differences between wells.
is the immediate precursor to 11-desoxycortisol (CpS) which is evacuated tube(s) containing heparin. Allow the blood to clot for 13. Read the absorbance in each wel at 450nm (using a reference converted to cortisol. Because CpS is produced by 21- serum samples. Centrifuge the specimen to separate the serum or wavelength of 620-630nm. The results should be read
hydroxylation of 17P, measurement of 17P is an indirect indicator B. 17α-OH Progesterone Enzyme Reagent – 1.0 ml/vial E
within fifteen (15) minutes of adding the stop solution.
of 21-hydroxylase activity. CAH occurs where there is a deficiency One (1) vial of 17-OH Progesterone (Analog)-horseradish of this enzyme. The result is a decrease in the conversion of 17P peroxides (HRP) conjugate in a protein stabilizing matrix with Samples may be refrigerated at 2-8oC for a maximum period of Note: Dilute the samples suspected of concentrations higher than
to CpS which blocks the normal synthesis of cortisol. Due to the five (5) days. If the specimen(s) cannot be assayed within this 20ng/ml 1:1 and 1:5 with 17-OH Progesterone ‘0’ ng/ml feed back mechanism, a decrease in cortisol causes an increase time, the sample(s) may be stored at temperatures of -20oC for up calibrator or male patient serum pools with a known low value in ACTH secretion resulting in adrenal hyperplasia. As 17P is not C. Steroid Conjugate Buffer – 7.0 ml/vial - Icon B
to 30 days. Avoid use of contaminated devices. Avoid repetitive being converted, increased concentrations of this steroid will be One (1) vial of reagent contains buffer, red dye, preservative, freezing and thawing. When assayed in duplicate, 0.050ml of the and binding protein inhibitors. Store at 2-8°C. 10.0 CALCULATION OF RESULTS
D. 17α-OH Progesterone Biotin Reagent – 6.0 ml - Icon
17P concentration increases during pregnancy in the maternal One (1) bottle of reagent contains anti-17α-OH Progesterone 7.0 QUALITY CONTROL
A dose response curve is used to ascertain the concentration of and fetal blood. After birth, values decline rapidly to reach normal biotinylated purified rabbit IgG conjugate in buffer, blue dye 17-OH Progesterone in unknown specimens. adult values in 2 to 7 days. Thus it is advisable not to col ect Each laboratory should assay controls at levels in the low, normal 1. Record the absorbance obtained from the printout of the samples before the 3rd day of life. Premature and sick term and high range for monitoring assay performance. These controls microplate reader as outlined in Example 1. infants exhibit 2 to 3 fold 17P values with no CAH disorder. It is E. Streptavidin Coated Plate – 96 wells –Icon
should be treated as unknowns and values determined in every 2. Plot the absorbance for each duplicate serum reference versus suggested that a different cut off be adopted to pre-term and sick One 96-well microplate coated with 1.0 µg/ml streptavidin and test procedure performed. Quality control charts should be the corresponding 17-OH Progesterone concentration in ng/ml packaged in an aluminum bag with a drying agent. Store at maintained to fol ow the performance of the supplied reagents. on linear graph paper (do not average the duplicates of the Pertinent statistical methods should be employed to ascertain In this method, a sample containing 17-OH progesterone is F. Wash Solution Concentrate – 20ml – Icon
trends. The individual laboratory should set acceptable assay 3. Connect the points with a best-fit curve. dispensed into a microplate wel . An enzyme labeled 17OH One (1) vial contains a surfactant in buffered saline. A performance limits. In addition, maximum absorbance should be 4. To determine the concentration of 17-OH Progesterone for an progesterone derivative and biotinylated anti-17OH-progesterone consistent with past experience. Significant deviation from unknown, locate the average absorbance of the duplicates for are than added. After a suitable incubation, the antibody fraction is preservative has been added. Store at 2-30°C. established performance can indicate unnoticed change in each unknown on the vertical axis of the graph, find the G. Substrate Solution – 12ml/vial - Icon S
experimental conditions or degradation of kit reagents. Fresh intersecting point on the curve, and read the concentration (in One (1) bottle contains tetramethylbenzidine (TMB) and reagents should be used to determine the reason for the ng/ml) from the horizontal axis of the graph (the duplicates of The employment of several serum references of known 17-OH hydrogen peroxide (H2O2) in buffer. Store at 2-8°C. the unknown may be averaged as indicated). In the following Progesterone concentration permits construction of a graph of example, the average absorbance (0.880) intersects the dose activity and concentration. From comparison to the dose H. Stop Solution – 8ml/vial - Icon STOP
response curve at (1.41ng/ml) 17-OH Progesterone the same sequence to eliminate any time-deviation during Within Assay Precision (Values in ng/ml ) 15.0 REFERENCES
6. Plate readers measure vertically. Do not touch the bottom of Note: Computer data reduction software designed for ELISA
1. Strott, C.A., Yoshimi, T., and Lipsett, M.B: Plasma progesterone and assays may also be used for the data reduction. If such 7. Failure to remove adhering solution adequately in the 17α-hydroxyprogesterone in normal men and children with congenital software is utilized, the validation of the software should be aspiration or decantation wash step(s) may result in poor adrenal hyperplasia (CAH). J. Clin. Invest.48,930(1969). 2. Yousseff, N, David R. Early diagnosis of congenital adrenal 8. Use components from the same lot. No intermixing of hyperplasia by measurement of 17α-OH Progesterone. EXAMPLE 1
Between Assay Precision (Values in ng/ml ) 9. Accurate and precise pipetting, as wel as fol owing the exact 3. Lippe B.M, LaFranchi, S.H, Lavin, N. Serum 17α-OH progesterone, time and temperature requirements prescribed are essential. progesterone and testosterone and Estradiol in the diagnosis and Any deviation from Monobind’s IFU may yield inaccurate management of congenital adrenal Hyperplasia. J. Pediatrics 10. All applicable national standards, regulations and laws, 4. Abraham GE. The application of natural steroid radioimmunoassay to including, but not limited to, good laboratory procedures, must gynecologic endocrinology. In: Abraham GE, editor. Radioassay be strictly fol owed to ensure compliance and proper device *As measured in ten experiments in duplicate over a ten day Systems in Clinical Endocrinology, Basel: Marcel Dekker,: 475-529 11. It is important to calibrate all the equipment e.g. Pipettes, 5. Aufrere MB, Benson H. Progesterone: an overview and recent Readers, Washers and/or the automated instruments used advances. , 65:783-800 (1976).
with this device, and to perform routine preventative 14.2 Sensitivity
6. Walker R.F, Read GF., and Fahmy D.R. Adrenal status assessed by The 17-OH Progesterone AccuBind™ Microplate EIA Test System has a sensitivity of 0.077ng/ml. The sensitivity was ascertained by direct radioimmunassay of Cortisol in whole saliva or parotid fluid. Clin. 12. Risk Analysis- as required by CE Mark IVD Directive 98/79/EC - for this and other devices, made by Monobind, can be determining the variability of the 0 ng/ml serum calibrator and 7. Bacon G.E, Spencer M.L., and Kelch R.P. Effect of Cortisol therapy on requested via email from Monobind@monobind.com. using the 2σ (95% certainty) statistic to calculate the minimum hormonal relationships in congenital adrenal hyperplasia. Clin. 12.2 Interpretation
8. David M, Foresr M.G., Prenatal treatment of congenital adrenal 1. Measurements and interpretation of results must be
14.3 Accuracy
The 17-OH Progesterone AccuBind™ Microplate ELISA Test
hyperplasia resulting from 21-hydroxylase deficiency. J. performed by a skilled individual or trained professional.
2. Laboratory results alone are only one aspect for determining System was compared with a chemiluminescence immunoassay 9. August G.P: Growth and development in the normal infant and child. patient care and should not be the sole basis for therapy, method. Biological specimens from low, normal and high 17-OH particularly if the results conflict with other determinants. Progesterone level populations were used (The values ranged from < 0.15 ng/ml – 128 ng/ml). The total number of such 10. BIO-ED slide/seminar educational program, Rochester: 3. For valid test results, adequate controls and other parameters must be within the listed ranges and assay requirements. specimens was 66. The least square regression equation and the 11. Tietz, Textbook of clinical chemistry,2nd ed. Philadelphia: 4. If test kits are altered, such as by mixing parts of different kits, correlation coefficient were computed for this method in comparison with the reference method. The data obtained is which could produce false test results, or if results are incorrectly interpreted, Monobind shall have no liability. Revision: 3
Date: 061312 DCO: 0649
5. If computer control ed data reduction is used to interpret the Cat #: 5225-300
results of the test, it is imperative that the predicted values for Least Square
Correlation
*The presented in Example 1 and Figure is for illustration only and the calibrators fall within 10% of the assigned concentrations. Regression
Coefficient
should NOT be used in lieu of a dose response curve prepared
Analysis
13.0 EXPECTED RANGES OF VALUES
In agreement with established reference intervals for a “normal“ adult population and females during gestation the expected Reference
ranges for the 17α-OH Progesterone AccuBind ELISA Test
Only slight amounts of bias between this method and the reference method are indicated by the closeness of the mean Expected Values for the 17α-OH Progesterone Test System
values. The least square regression equation and correlation sorbance
(nmol/L)
coefficient indicates excellent method agreement. Reagent (fill)
14.4 Specificity
The % cross reactivity of the 17OH-progesterone antibody to Adult woman
selected substances was evaluated by adding the interfering substance to a serum matrix at various concentrations. The cross- reactivity was calculated by deriving a ratio between dose of 11.0 Q.C. PARAMETERS
interfering substance to dose of 17-OH Progesterone needed to displace the same amount of labeled analog. Postmenopausal woman
In order for the assay results to be considered valid the fol owing It is important to keep in mind that establishment of a range of Substance
1. The absorbance (OD) of calibrator 0 ng/ml should be > 1.3. values which can be expected to be found by a given method for a Reactivity
2. Four out of six quality control pools should be within the population of "normal” persons is dependent upon a multiplicity of factors: the specificity of the method, the population tested and the precision of the method in the hands of the analyst. For these 12.0 RISK ANALYSIS
reasons each laboratory should depend upon the range of expected values established by the manufacturer only until an in- house range can be determined by the analysts using the method The MSDS and Risk Analysis Form for this product is available on with a population indigenous to the area in which the laboratory is 12.1 Assay Performance
14.0 PERFORMANCE CHARACTERISTICS
1. It is important that the time of reaction in each well is held constant to achieve reproducible results. 2. Pipetting of samples should not extend beyond ten (10) 14.1 Precision
The within and between assay precision of the 17-OH 3. Highly lipemic, hemolyzed or grossly contaminated Progesterone AccuBind™ Microplate EIA Test System were determined by analyses on three different levels of pool control 4. If more than one (1) plate is used, it is recommended to sera. The number, mean values, standard deviation and coefficient of variation for each of these control sera are presented 5. The addition of substrate solution initiates a kinetic reaction, which is terminated by the addition of the stop solution. Therefore, the substrate and stop solution should be added in

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