SUMMARY AND EXPLANATION OFTHE TEST
Estradiol E2 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 is secreted into the blood stream where 98% bound to sex hormone binding globulin (SHBG). Estrogenic activity is effected via estradiol-receptor complexes which trigger the appropriate response at 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. 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.

PRINCIPLE OF THE TEST
The E2 EIA 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 and rabbit anti-Estradiol reagent at room temperature 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. 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 stop solution, and the absorbance is measured
spectrophotometrically at 450 nm. A standard curve is obtained by plotting the concentration of the standard versus the absorbance.

             Materials provided                                            96 tests
1.  Microwell coated withGoat Anti-Rabbit IgG             12x8x1
2.  Estradiol Reference Standards:6 vials (ready to use)   0.5 ml
3.  Rabbit Anti-Estradiol Reagent (pink color)                   7 ml
4.  Estradiol-HRP Conjugate Reagent (blue color)           12 ml
5.  Estradiol Control 1, Liquid. (ready to use)                 0.5 ml
6.  Estradiol Control 2, Liquid. (ready to use)                 0.5 ml
7.  TMB Reagent                                                            11 ml
8.  Stop Solution (1N HCL)                                            11 ml

CALCULATION OF RESULTS
1.  Calculate the mean absorbance value (A450) for each set of reference standards, controls and samples.
2.  Construct a standard curve by plotting the mean absorbance obtained for each reference standard against its concentration in pg/ml on a linear-linear graph paper, with absorbance values on the vertical or Y axis, and concentrations on the horizontal or X axis.
3.  Use the mean absorbance values for each specimen to determine the corresponding concentration of Estradiol in pg/ml from the standard curve.
4.  Any values obtained for diluted samples must be further converted by applying the appropriate dilution factor in the calculations.

Example of A Standard Curve
Results of a typical standard run with optical density readings at 450 nm shown in the Y-axis against Estradiol concentrations shown in the X axis. Note: This standard curve is for the purpose of illustration only, and should not be used to calculate unknowns. Each laboratory must provide its own data and standard curve in each experiment.