17beta-Estradiol is considered the true ovarian hormone in women. It is the most potent estrogen, including Estrone and Estriol. 17beta-Estradiol is responsible for the reproductive epithelia, breast growth, maturation of long bones and development of the secondary female sex characteristics.
The 17beta-Estradiol level in plasma is used in assaying for fertility, amenorrhea and precocious puberty in girls.
Principle of the Assay:
This is an ELISA for the quantitative analysis of Estradiol levels in biological fluid. This test kit operates on the basis of competition between the enzyme conjugate and the Estradiol in the sample for a limited number of binding sites on the antibody coated plate.
The sample or standard solution is first added to the microplate. Next, the diluted enzyme conjugate is added and the mixture is shaken and incubated at room temperature for one hour. During the incubation, competition for binding sites is taking place. The plate is then washed removing all the unbound material. The bound enzyme conjugate is detected by the addition of substrate, which generates an optimal color after 30 minutes.
Quantitative test results may be obtained by measuring and comparing the absorbance reading of the wells of the samples against the standards with a microplate reader at 650nm. The extent of color development is inversely proportional to the amount of Estradiol in the sample or standard. For example, the absence of Estradiol in the sample will result in a bright blue color, whereas the presence of Estradiol will result in decreased or no color development.
Usually, urine and tissue culture supernatant can be assayed directly by diluting them with the diluted extraction buffer. Plasma and most other mediums will need to be extracted.
E3550-02A: ELISA Buffer: 1x30ml. To be used to dilute enzyme conjugate and Estradiol standards.
E3550-02B: Wash Buffer (10X): 1x20ml. To be diluted 1X with deionized water. This is used to wash all unbound enzyme conjugate, samples and standards from the plate after the one hour incubation.
E3550-02C: Substrate: 1x20ml. Stabilized 3,3', 5,5' Tetramethylbenzidine (TMB) plus hydrogen peroxide (H2O2) in a single bottle. It is used to develop the color in the wells after they have been washed. Light Sensitive.
E3550-02D: Extraction Buffer (5X): 1x30ml. To be diluted 1X with deionized water. This is used for diluting extracted and non-extracted samples.
E3550-02E: Estradiol (HRP): 1x150ul. Estradiol HRP concentrate.
E3550-02F: Estradiol Standard: 1x100ul. Estradiol standard at the concentration of 1ug/ml.
E3550-02G: Estradiol Antibody-Coated Plate: 1x96 well microplate with anti-Estradiol rabbit antibody precoated on each well. The plate is ready for use as is. Do not wash.
Storage and Stability:
Store kit at 4C. Stable for at least 6 months.