This Kit is an enzyme immunoassay for measurement of active free Estradiol, an estrogenic steroid, in saliva.
Contents of Kit
1. Microtiterwells, 12 x 8 (break apart) strips, 96 wells;
2. Standard (Standard 0-5), 6 vials, 1 mL each, ready to use;
3. Control Low & High, 2 vials, 1.0 mL each, ready to use;
4. Enzyme Conjugate, 1 vial, 26 mL, ready to use;
5. Substrate Solution, 1 vial, 25 mL, ready to use;
6. Stop Solution, 1 vial, 14 mL, ready to use;
7. Wash Solution, 1 vial, 30 mL;
Assay Dynamic Range
The range of the assay is between 0.8 - 93 pg/mL. Specificity The following materials have been evaluated for cross reactivity. The percentage indicates cross reactivity at 50% displacement compared to Estradiol. Sensitivity
The lowest detectable level of Estradiol that can be distinguished from the Zero Standard is 0.4 pg/mL at the 95 % confidence limit. Reproducibility Intra Assay
The intra-assay variation was determined by 20 replicate measurements of 4 saliva samples using this ELISA kit.
The within assay variability is shown below: Inter Assay
The inter-assay (between-run) variation was determined by duplicate measurements of five saliva samples over 10 different days runs.
The between assay variability is shown below: Inter-Lot
The Inter-Lot (between-lot) variation was determined by triplicate measurements of five saliva samples in three different kit lots.
The between lot variability is shown below: Recovery
Recovery of the CD ELISA was determined by adding increasing amounts of the analyte to three different saliva samples containing different amounts of endogenous analyte. Each sample (non-spiked and spiked) was assayed and analyte concentrations of the samples were calculated from the standard curve. The percentage recoveries were determined by comparing expected and measured values of the samples. Linearity
Four samples (saliva) containing different amounts of analyte were serially diluted up to 1:128 with 0.9% NaCl and assayed with the CD ELISA. An assay linearity of 0.8 - 93 pg/mL has been identified as the usable range. Samples above this range must be diluted and re-run.
Estradiol (1,3,5(10)-estratriene-3,17β-diol; 17β-estradiol; E21) is a C18 steroid hormone with a phenolic A ring. This steroid hormone has a molecular weight of 272.4. It is the most potent natural Estrogen, produced mainly by the Graffian follicle of the female ovary and the placenta, and in smaller amounts by the adrenals, and the male testes. Estradiol (E2) is secreted into the blood stream where 98% of it circulates bound to sex hormone binding globulin (SHBG) and to a lesser extent to other serum proteins such as albumin. Only a small fraction circulates as free hormone or in the conjugated form. Estrogenic activity is effected via estradiol-receptor complexes which trigger the appropriate response at the nuclear level in the target sites. These sites include the follicles, uterus, breast, vagine, 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. 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.
Standard Absorbance Units
Standard 0 (0 pg/mL) 2.08
Standard 1 (1 pg/mL) 1.90
Standard 2 (5 pg/mL) 1.62
Standard 3 (10 pg/mL) 1.40
Standard 4 (50 pg/mL) 0.58
Standard 5 (100 pg/mL) 0.21
Reconstitution And Storage
When stored at 2°C to 8°C unopened reagents will retain reactivity until expiration date. Do not use reagents beyond this date. Opened reagents must be stored at 2°C to 8°C. Microtiter wells must be stored at 2°C to 8°C. Once the foil bag has been opened, care should be taken to close it tightly again. Opened kits retain activity for two months if stored as described above.