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Gd7260 00_ams_0_eng

Enzyme-immunoassay for the quantitative
determination of Estradiol in serum or plasma
GD7260 00

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 S0: 0 pg/ml, S1: 20 pg/ml, S2: 120 pg/ml,
uterine lining. In addition there are several benign S3: 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.

GD7260 00
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 REAGENTS PREPARATION
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- WASHING INSTRUCTION
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. ASSAY PROCEDURE
1. At least one hour before use, bring all reagents, AUXILIARY MATERIALS
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). GD7260 00

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 REAGENTS
- 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) QUALITY CONTROL
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. Subjects
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. GD7260 00


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 Precision
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
Waste Management
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 REFERENCES
1. Joshi,U.M., Steroids 34 (1) 35 (1979) 2. D.Exley and R. Abuknesha Febs Letters 91,(2) 162 Recovery
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: Specificity
The cross reaction studied and relative results are shown “Hook” Effect
The method shows no “Hook” Effect up to 20.000 pg/ml. AMS S.p.A. – Registered Office and Plant
Diagnostics Manufacturing


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