Estradiol ELISA kit
|Category Name||Steroid ELISA kits|
|Method||ELISA method: Enzyme Linked Immunosorbent Assay|
|Detection Range||0-1000 pg/ml|
|Total Time||~ 110 min|
|Shelf Life||12-14 Months from the manufacturing date|
SUMMARY AND EXPLANATION
Estradiol is the most potent natural Estrogen, produced mainly by the ovary, placenta, and in smaller amounts by the adrenal cortex, and the male testes.
Estradiol (E2) is secreted into the blood stream where 98% of it circulates bound to sex hormone binding globulin (SHBG).
To a lesser extent it is bound to other serum proteins such as albumin. Only a tiny fraction circulates as free hormone or in the conjugated form.
Estrogenic activity is affected via estradiol-receptor complexes which trigger the appropriate response at the nuclear level in the target sites.
These sites include the follicles, uterus, breast, vagina, urethra, hypothalamus, pituitary and to a lesser extent the liver and skin.
In non-pregnant women with normal menstrual cycles, estradiol secretion follows a cyclic, biphasic pattern with the highest concentration found immediately prior to ovulation.
The rising estradiol concentration is understood to exert a positive feedback influence at the level of the pituitary where it influences the secretion of the gonadotropins,
follicle stimulating hormone (FSH), and luteinizing hormone (LH), which are essential for follicular maturation and ovulation, respectively.
Estradiol is the major estrogen secreted by the premenopausal ovary. Estrogens direct the development of the female genotype in embryogenesis and at puberty.
In addition, estradiol is an important luteolytic agent in humans. The conversion of testosterone to estradiol is achieved by the aromatase system,
which consists of three enzyme activities localized to the endoplasmic reticulum of the cells in these tissues.
Within the Estradiol ELISA kit, it can be determined that the plasma estradiol levels increase gradually between days 1-7 of the menstrual cycle
followed by a sharp increase to a peak value at about 300 pg/ml on day 12, prior to ovulation.
Following ovulation, estradiol levels fall rapidly until the luteal cells become active resulting in a secondary gentle rise and plateau of estradiol in the luteal phase.
During pregnancy, maternal serum Estradiol levels increase considerably, to well above the pre-ovulatory peak levels and high levels are sustained throughout pregnancy.
Serum Estradiol measurements are a valuable index in evaluating a variety of menstrual dysfunctions such as precocious or delayed puberty in girls and primary and secondary amenorrhea and menopause.
Estradiol levels have been reported to be increased in patients with feminizing syndromes, gynaecomastia and testicular tumors.
In cases of infertility, serum Estradiol measurements are useful for monitoring induction of ovulation following treatment with, for example, clomiphene citrate, LH-releasing hormone (LH-RH), or exogenous gonadotropins.
During ovarian hyperstimulation for in vitro fertilization (IVF), serum estradiol concentrations are usually monitored daily for optimal timing of human chorionic gonadotropin (HCG) administration and oocyte collection.
ESTRADIOL EIA TEST PRINCIPLE
The Estradiol ELISA test kit is based on the principle of competitive binding between E2 in the test specimen and E2-HRP conjugate for a constant amount of rabbit anti-Estradiol.
In the incubation, goat anti-rabbit IgG-coated wells are incubated with E2 standards, controls, patient samples, Estradiol-HRP Conjugate Reagent anti-Estradiol reagent at room temperature (18-25?C) for 90 minutes.
During the incubation, a fixed amount of HRP-labeled E2 competes with the endogenous E2 in the standard, sample, or quality control serum for a fixed number of binding sites of the specific E2 antibody.
Thus, the amount of E2 peroxidase conjugate immunologically bound to the well progressively decreases as the concentration of E2 in the specimen increases.
Unbound E2 peroxidase conjugate is then removed and the wells washed. Next, a solution of TMB Reagent is added and incubated at room temperature for 20 minutes, resulting in the development of blue color.
The color development is stopped with the addition of 1N HCl, and the absorbance is measured spectrophotometrically at 450 nm.
The intensity of the color formed is proportional to the amount of enzyme present and is inversely related to the amount of unlabeled E2 in the sample.
A standard curve is obtained by plotting the concentration of the standard versus the absorbance.
The E2 concentration of the specimens and controls in the Estradiol ELISA kit are concurrently run with the standards, in order to calculate the standard curve.
ESTRADIOL ELISA KIT PERFORMANCE CHARACTERISTICS
The minimum detectable concentration of the Estradiol ELISA assay as measured by 2 SD from
the mean of a zero standard is estimated to be 10 pg/ml.
Within-run precision was determined by replicate determinations of four different serum samples in one
assay. Within-assay variability is shown in assay insert:
Between-run precision was determined by replicate measurements of six different serum samples over
a series of individually calibrated assays. Between-assay variability is shown in assay insert:
Various patient serum samples of known Estradiol levels were combined and assayed in duplicate.
The mean recovery was 101.3%.
The following materials have been checked for cross reactivity.
The percentage indicates cross reactivity at 50% displacement compared to Estradiol.
Data on the cross-reactivity for several endogenous and pharmaceutical steroids are summarized in the
following table in assay insert:
Cross-reactivity (%) = ((Observed Estradiol Concentration)/ Steroid Concentration) X 100