DiaMetra Estradiol Elisa Kit

CLINICAL SIGNIFICANCE
Estradiol (also known as 17β-estradiol, oestradiol or E2) is one of the three main estrogens produced in the human body. The other two are Estrone (E1) and Estriol (E3) which are mainly produced in post-menopausal women and during pregnancy respectively. Estradiol is the predominant estrogen synthesised in pre-menopausal women and has a key role in the regulation of fertility in women.
Estradiol is synthesised in the adrenal glands, the ovaries (women) and tests (men). It is produced by the conversion of testosterone to estradiol by the action of the enzyme aromatase. The enzyme aromatase is also responsible for production of estrone by the conversion of androstenedione.
Estrogens regulate the development of secondary sex characteristics; estradiol is also involved in other physiological functions in different organs and systems such as skin, blood vessels, bone, muscle, gastrointestinal tract, brain, lung and pancreas2 etc. Quantification od estrogens (and androgens) are useful in the assessment and management of different sex-hormone related disorders including hypogonadism, hirsutism, amenorrhea, fertility and ovarian tumours.

PRINCIPLE OF THE METHOD
The Estradiol ELISA is a competitive enzyme immunometric assay (ELISA) where 17β-Estradiol (antigen) in the sample competes with the antigenic 17β-Estradiol conjugated with horseradish peroxidase (HRP) for binding to the limited number of antibodies anti 17β-Estradiol coated on the microplate (solid phase).
After the incubation, the bound/free separation is performed by a simple solid phase washing. Then, the enzyme HRP in the bound fraction reacts with the Substrate (H2O2) and the TMB Substrate and develops a blue colour that changes into yellow when the Stop Solution (H2SO4) is added. The colour intensity is inversely proportional to the 17β-Estradiol concentration in the sample.
17β-Estradiol concentration in the sample is calculated through a calibration curve.

REAGENT STORAGE AND STABILITY
Store the kit at 2 – 8°C in the dark.
The kit is stable at 2 – 8°C until the expiry date stated on the external kit label.
Once opened, the kit is stable at 2 – 8°C for 6 months.
The diluted wash solution is stable for 30 days at 2-8°C.

Important note: open the bag containing the Coated Microplate only when it is at room temperature and close it immediately after use.

SAMPLE COLLECTION AND STORAGE
The assay should be performed using serum (standard sampling tubes or tubes containing serum separating gel) or plasma (lithium heparin, sodium heparin or potassium EDTA) samples.

Preparation of Samples
The determination of 17β-Estradiol can be performed in human serum (standard sampling tubes or tubes containing serum separating gel) or plasma (lithium heparin, sodium heparin or potassium EDTA) samples.
Fasting samples are not necessary and no special sample preparations are required.
Collect blood by venepuncture into vacutainers and separate serum (after clot formation) or plasma from the cells by centrifugation. Testing of heat-inactivated sera is not recommended.
Store the sample at -20°C if the determination is not performed on the same day of the sample collection. Avoid repetitive freezing and thawing of samples. Before using, mix gently for 5 minutes with a rotating mixer.

QUALITY CONTROL
Good Laboratory Practice (GLP) requires the use of quality control specimens in each series of assays in order to check the performance of the assay. Controls should be treated as unknown samples, and the results analysed with appropriate statistical methods.
The kit control provided in the kit should be tested as unknown and is intended to assist in assessing the validity of results obtained with each assay plate.
The mean concentration of each control level is documented in the QC report included with each kit. These mean concentration levels are determined over several assays which are run in duplicate in multiple locations across each plate.
DiaMetra recommends the users to maintain graphic records of the control values generated with each assay run, including the running means, SDs and %CVs. This information will facilitate the controls trending analysis relating to the performance of current and historical control lots relative to the supplied Quality Control data. The trending will assist in the identification of assays which give control values significantly different from their average range.

CALCULATION OF RESULTS
A variety of data reduction software packages are available, which may be employed to generate the mean calibration curve and to calculate the mean concentrations of unknown samples and controls. A 4-parameter logistic (4PL) curve fit, including Calibrator 0 is recommended.
Alternatively, a calibration curve may be prepared on semilog graph paper by plotting mean absorbance on the Y-axis against concentration of analyte on the X-axis. Calibrator 0 should be included in the calibration curve. Read the mean absorbance value of each unknown sample off the curve.
In order for the assay results to be considered valid the kit calibrators and control must fall within the specifications detailed in the lot specific certificate of analysis.
If a control is out of its specified range, the associated test results are invalid and samples must be retested.