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Enzyme-immunoassay for the quantitative
determination of Estradiol in serum or plasma
Estradiol (17β-estradiol) is a sex hormone and represents
1. Reagent A – Microplate
the major estrogen in humans. Estradiol has not only a
critical impact on reproductive and sexual functioning, but
8 wells breakable strips, coated with anti-Estradiol
also affects other organs including bone structure.
antibody. The strips are assembled on a plastic frame
During the reproductive years most estradiol in women is
and contained in a sealed bag with desiccant. Bring the
produced by the ovaries, smaller amounts of estradiol are
strips to room temperature before use, to prevent any
also produced by the adrenal cortex. In men, it is produced
by the testes. In plasma estradiol is largely bound to sex
hormone binding globulin, also to albumin, only a fraction is
2. Reagent B – Enzymatic Tracer
Serum estradiol measurement in women reflect primarily
Estradiol, conjugated with Horseradish peroxidase
During pregnancy estrogen levels, including estradiol, rise
steadily towards term. Estradiol increases due to placental
3. Reagent C – Washing Solution 10x
production. In adult premenopausal women, ovarian
estradiol production is stimulated by the interactions of
Concentrated solution to be diluted 1:10 with distilled
luteinizing hormone (LH) and follicle-stimulating hormone
It contains Phosphate buffer 0.2 M, Proclin 0.002%.
In adult women, estradiol levels are measured in the
evaluation of fertility and menstrual irregularities, and to
4. Reagent D/E – Chromogen/Substrate
monitor ovarian follicular function during induction of
Ready to use solution containing Tetramethylbenzidine
In the female, estradiol acts as a growth hormone for tissue
of the reproductive organs and for the development of
Avoid any skin contact and light exposure.
Estradiol is also involved in man fertility.
5. Reagent F – Stop Solution
Estradiol regulates the bone maintenance. Women who past
the menopause experience an accelerated loss of bone
Ready to use solution containing Sulphuric acid 0.15 M.
mass due to a relative estrogen deficiency.
Avoid any skin contact.
Estradiol affects the production of multiple proteins
including lipoproteins, binding proteins, and proteins
6. Estradiol Standards
Estrogens have been found to have neuroprotective
Ready to use liquids containing Estradiol approximately
function. Estrogen is considered an oncogene as it supports
certain cancers, notably breast cancer and cancer of the
: 0 pg/ml, S1
: 20 pg/ml, S2
: 120 pg/ml,
uterine lining. In addition there are several benign
: 300 pg/ml, S4
: 600 pg/ml, S5
: 2000 pg/ml.
gynecologic conditions that are dependent on estrogen such
Actual concentrations to be used for calculation are stated
PRINCIPLE OF THE ASSAY
7. Cardboard sealers
This test is based on “one step” competition enzyme
2 cardboard sealers to be used to cover the plate
immunoassay principle (ELISA). Tested specimen is placed
into the microwells coated by specific anti-Estradiol
antibodies simultaneously with Estradiol conjugated to
8. Package insert:
instruction for use GD7260 00 it/ing.
Horseradish peroxidase (HRP). Estradiol from the specimen
competes with the conjugated antigen for coated
antibodies. After washing procedure, the remaining
enzymatic activity bound to the microwell surface is
detected and quantified by addition of chromogen-substrate
solution. The developed colour, detected at 450 nm, is
inversely related to the quantity of Estradiol present in the
Estradiol concentration in the sample is calculated based on
a series of standards.
Serum or plasma (heparin, EDTA).Samples can be stored
1. All the materials of human origin resulted negative to
at 2–8 °C for a short time (max two days). For longer
HbsAg, HIV 1&2 and HCV FDA approved tests. Anyhow,
storage the specimen should be frozen. Avoid repeated
as no test can guarantee the absolute absence of
freezing and thawing. Highly lipemic, hemolysed or
infective agents, handle reagents as potentially
microbiologically contaminated samples should not be used
infected, especially standards, controls and samples. All
objects come in direct contact with samples and all
residuals of the assay should be treated or eliminated
as potentially infected. Best procedures for inactivation
WASHING SOLUTION: dilute 1:10 with distilled or
are treatments with autoclave at 121°C for 30 minutes
ELISA grade water (ex.: 20 ml of reagent C + 200 ml
or with sodium hypochlorite at a final concentration of
of distilled water) and mix carefully before use. The
diluted washing solution can be stored for one week
2. Avoid any contact with skin and mucous membrane, in
at room temperature or four weeks at +2-8°C. It is
recommended to store diluted washing solution at
3. Use protective disposable talk-free gloves.
room temperature for immediate use. In Reagent C it is
4. Avoid contaminating reagents when taking them from
possible to observe the presence of crystals, in this
the vials. We recommend to use automatic pipettes
case mix at room temperature until complete
with disposable tips. When dispensing reagents, do not
touch with tips the wall of wells in order to avoid cross-
5. For the washing step, follow carefully the indications
A good washing procedure is essential to obtain correct and
reported in "WASHING INSTRUCTION".
6. Avoid the substrate/chromogen to come in contact with
We therefore recommend to use a good quality ELISA
oxidizing agents or metallic surfaces; avoid intense
microplate washer, maintained at a good level of washing
Generally, 2-3 automatic washing cycles of 0.3 ml/well are
sufficient to avoid false positive reactions and remove high
STORAGE AND STABILITY OF THE KIT
background. Anyhow we recommend to calibrate the
1. The kit has to be stored at 2-8 °C and used before the
washing system on the kit itself so to match the declared
2. Unused strips have to be placed in the bag
In case of manual washing, we suggest to perform 3
containing the desiccant and firmly sealed before
washing cycles, dispensing and aspirating 0.3 ml/well per
restore at 2-8 °C. After opening the strips are stable up
In any case the liquid washed out from the plates must be
3. All other reagents can be repeatedly used up to
inactivated with a sodium hypochlorite solution at a final
exhaustion if stored at 2-8 °C, provided that they are
concentration of 2.5%, before being thrown away or
autoclaved, as it must be considered as potentially infected.
contamination. Under these conditions the reagents are
stable up to the expiry date stated on the labels.
1. At least one hour before use, bring all reagents,
standards and samples to room temperature (18-
Semi automatic pipettes of 10, 200 and 1000 µl
30°C), mixing them carefully on vortex.
2. Do not mix reagents from different lots.
3. We recommend to distribute standards and samples in
4. Distribution and incubation times must be the same for
Photometric reader of microplates or microstrips, linear up to at least 2 OD and supplied with filter of 450 nm
5. Avoid long interruptions between each step of the
6. It is suggested to eliminate the excess of washing
Automatic microplates washing device or manual apparatus capable of aspirating and dispensing
solution from the microplate after washing by blotting
7. The colour developed in the last incubation is stable
for a maximum of one hour. Otherwise, in case of
reading after 10-15 min after dispensing stop solution,
immediately place the strips in the dark
8. We recommend to read the plate with an ELISA
automatic reader able to subtract the background at 620-630 nm and to read the absorbance of samples and standards at 450 nm. The "blanking" of the instrument is to be carried out in the blank reagent well (well A1).
1. Put the desidered number of microstrips into the frame.
2. If suggested analyte concentration in the sample exceeds 2000 pg/ml, dilute this sample accordingly, using Standard 0.
3. Follow the scheme:
Microplate wells coated with anti-Estradiol antibody
- Cover the strips with cardboard sealer
- Incubate 120 minutes at 37 ± 1 °C
- Peel out the cardboard sealer and aspirate the reaction solution from all wells
- Rinse 3 times with 300 µl of diluted washing solution, carefully aspirating off the remaining liquid
- Cover the strips with cardboard sealer
- Incubate 30 minutes at room temperature (22-28 °C)
, avoiding light exposure
Read the absorbance of each well against Blank at 450 nm (and 620-630 nm)
It is recommended, in each analytical run, to use control
From data obtained by AMS the following reference ranges
sera with known Estradiol values, to check the
are suggested. Otherwise, it is recommended that each
correspondence of the obtained results with those
laboratory establishes its own reference range.
expected and consequently validate the data.
CALCULATION OF RESULTS
1. Calculate the mean of the absorbance (Em) for each
point of the standard curve (S0 – S5) and of each
2. Plot the mean value of absorbance of the standards
(Em) against proper Estradiol concentrations. Draw
the best-fit curve through the plotted points. (Ex.:
3. Interpolate the values of the samples on the standard
curve to obtain the corresponding values of the
The clinical significance of Estradiol determination can be
4. If computer controlled data reduction is used to
invalidated if the patient was treated with cortisone or
calculate the results of the test, it is imperative that
the predicted values for the standards fall within 10% of the assigned concentrations.
PRECAUTIONS IN USE
The reagents contain inactive components such as
The lowest detectable concentration of Estradiol is
preservatives (Sodium azide or others), surfactants etc.
The total concentrations of these components is lower than
the limits reported by 67/548/EEC and 88/379/EEC
directives about classification, packaging and labelling of
a. Intra Assay Variation
dangerous substances. However, the reagents should be
Within run variation was determined by 16 replicate
handled with caution, avoiding swallowing and contact with
determination of two different control sera in the same
skin, eyes and mucous membranes. The use of laboratory
analytical run. %CV values found were < 7% according to
reagents according to good laboratory practice is
b. Inter Assay Variation
Between run variation was determined by replicate
Please refer to local legal requirements.
measurements of three different control sera in 2 different
lots. %CV values found were < 10.5% according to the
1. Joshi,U.M., Steroids 34 (1) 35 (1979)
2. D.Exley and R. Abuknesha Febs Letters 91,(2) 162
The recovery of 50 – 100 – 200 - 400 pg/ml of Estradiol
3. Ismail A.A, et al J.Clin.Endocrin.Metab. 34,177-184
added to sample gave an average value (± SD) of 98.4%
4. Rajkowski,K.M, et al Steroids 29-5 (1977)
5. Wisdom G.B. Clin. Chem. 22/8, 1243-1255 (1976)
Correlation with RIA
6. D. Sadem, et al J. of Immunolog. Methods 28 125-
The present kit was compared to a well-established RIA
method. sera were assayed with both methods.
The following linear regression curve was calculated:
The cross reaction studied and relative results are shown
The method shows no “Hook” Effect up to 20.000 pg/ml.
AMS S.p.A. – Registered Office and Plant
Ain Shams Journal of Anesthesiology Vol 5-1; Jan 2012 DEXAMETHASONE AS ADJUVANT TO CAUDAL ROPIVACAINE AS ANALGESIC FOR LABOR PAIN Ahmed Abdalla Mohammed1, Wael Ahmed Ibrahim2 , Tamer Fayez Safan1 1 Department of Anesthesiology, Cairo University , Cairo, Egypt 2 Department of Anesthesiology, NCI, Cairo University , Cairo, Egypt Abstract Objectives : To evaluate analges
TRANSLATION Page 1 of 2 Hormonally active substances in mineral water from PET bottles Information No. 006/2009 of the [German] Federal Institute for Risk Assessment [Bundesinstitut für Risikobewertung (BfR)] of March 18, 2009 with respect to a study carried out by the University of Frankfurt am MainIn a recently published study of mineral waters produced by various different manufa