17ß-Estradiol Saliva ELISA

Improved Saliva Testing of hormone levels

Enzyme immunoassay for the quantitative measurement of 17beta-Estradiol in human saliva.

The 17-beta-Estradiol Saliva ELISA is intended for the measurement of 17-beta-Estradiol in human saliva. Estradiol is the most important natural estrogen and present in both women and men. The measurement of the hormone level is useful for detection of estrogen deficiency. Estradiol has an impact on puberty, primary and secondary amenorrhea and menopause. This test is not intended for assessing placental function in complicated pregnancy. Additional hormone assays are recommended for an interpretation of the estradiol level.

The 17-beta-Estradiol Saliva ELISA is based on the competition principle and measured on an absorbance reader. The assay is semi-automated requiring general purpose laboratory instruments and consumables such as absorbance microplate reader/washer, vortexer and pipettes to execute the test. Test results may be calculated manually from a standard curve and compared to laboratory established reference ranges from healthy adults (i.e. normal ranges). The device can be adapted to different ELISA processors and can be applied to open automated platforms like EVOlyzer®.

The test kit is intended for professional laboratory use by trained personnel. The test kit is not for home or lay person use. The test kit is intended for manual use.

Benefits of the 17ß-Estradiol Saliva ELISA

• Calibration to the LC-MS/MS;
• Only 50 µL Sample volume needed;
• Same sample diluent within the salivary steroid ELISA line;
• Defined reference values: monthly profiles for menstruating women and normal values for postmenopausal women and men

ELISA vs Mass Spectronomy

Mass spectrometry (MS) is considered as the reference for steroid hormone quantification. This methodology does however represent a large investment and as such is not available to all laboratories. The main Tecan saliva assays have been developed to correlate to this reference method, and even more importantly, only require low sample volume for quantification of the steroid hormones.

Consistent and reliable processing within Saliva product line

Our Saliva ELISA product line (Cortisol, Testosterone, Progesterone, 17beta Estradiol, Estriol, DHEA) can be processed in a similar way and be easily performed in combination:

• The TMB, stop solution and wash solution have the same design within Saliva ELISA product line *
• The kits contain 6 standards and 2 controls.
• Only 50 µL (max 100 µL) sample volume are required;
• All kits use the same sample diluent buffer.

*Provided the same lot number for each component is used. Valid lot numbers of TMB, stop solution and wash solution are stated on the kits outer labels and Quality Control certificates.

Automation

The 17beta Estradiol Saliva ELISA (Cat. No. 30121045) can be adapted on different ELISA processors. Open automated platforms like the EVOlyzer® from Tecan have been tested and show good correlation to the manual runs:

Physiology and Time Dependent Concentration

Estradiol is secreted into circulation by the ovaries, placenta, adrenal gland, and testes or is produced by extra-glandular conversion of secreted androgen precursors. Estradiol is biologically the most active of the naturally produced human estrogens. In the postmenopausal women, Estradiol originates from extra-glandular conversion of androgens and circulates in low, non-cyclic concentrations.

In prepubescent children and in males, salivary Estradiol concentrations are low and non-cyclic. The changes in the hormone levels are registered during the different the phases of the menstrual cycle.

Similar to Testosterone and Progesterone, 17ß-Estradiol also shows short time pulsating dynamics, especially in females. Therefore single saliva determinations will result in arbitrary values. We recommend collecting 3 to 5 saliva samples within 2 hours. In the laboratory, equal volumes of the individual saliva samples can be mixed. This mixed sample results in a mean 17ß-Estradiol value, which represents the active hormone concentration in a reproducible way.

Applications of 17ß-Estradiol determination

17beta-Estradiol [1, 3, 5 (10) -estratriene-3, 17beta-diol; E2] is a C18 steroid hormone and an important natural estrogen, which derives from the precursor cholesterol and belongs to the group steroid hormones called estrogens, which is present in women and men [1; 2]. Estradiol is the major estrogen in the body involving different functions within the human physiology throughout life [3]. Estradiol, the most potent bioactive estrogen, is primarily synthesized from testosterone in the ovarian follicles in females, whereas in males it is produced by the testes and extraglandular conversion of androgens [4; 5]. Estradiol is synthesized from testosterone or estrone, a derivative of cholesterol [6].
The main hormones responsible for advancing secondary sexual characteristics are two androgens: testosterone and dehydroepiandrosterone (DHEA), which facilitate masculine development, and estradiol, an estrogen which facilitates feminine development [7]. Estradiol is the primary estrogen released from the gonads and other peripheral tissues [8].
Levels of 17beta-Estradiol secretion increased with pubertal progression. Estradiol is an important hormone present in both women and men. Estrogen deficiency in boys and in girls but especially in young girls it has an impact on puberty. Girls with delayed puberty show advancing pubertal maturation when administered estradiol. The measurement of estradiol levels is useful regarding puberty to investigate if early puberty (precocious puberty) occurs in girls. Estradiol is 4-9 times higher in late adolescent girls as compared to childhood [9]. Ovarian volumes increase with pubertal breast stage and correlate positively with circulating estradiol levels [10].
Amenorrhea is classified into primary and secondary amenorrhea. Primary amenorrhea is defined as the absence of menarche by the age of 14 years, while secondary amenorrhea describes the absence of menses for more than six months in previously menstruating women [11]. In older women estrogens level should be observed in regards to menopause [12]. Additionally, estradiol plays a role in ovulation, menstrual cycle and menopause and in this regard the function of ovaries and placenta [13-17].
In addition to its role as a natural hormone, estradiol is used in the realm of hormone replacement therapy during menopause [18].
Naturally estradiol levels both in women and men decline with age, however, the estradiol level in older men are higher than in postmenopausal women and serve different functions in the male body (bone maturation and sex interest). Estradiol levels are related to body fat mass, bone health, affect skin metabolism and sex interest in men. Estradiol levels in men are higher than in postmenopausal women and comparable to estradiol levels in the early follicular phase. It is also worth mentioning that a correlation between testosterone and estradiol levels exists due to testosterone being the major precursor of estradiol [19].
Additional parameters should be measured for an interpretation of the results e.g. DHEA or testosterone [7]. Other physiological aspects where estradiol is involved are amenorrhea (primary and secondary) and menopause, which are indicated by low estradiol levels [11; 21].

Measurement of 17ß-Estradiol in saliva

In serum steroid hormones are bound to sex hormone binding globulin (SHBG) and to serum albumin, only 1-3% of estradiol circulates in plasma and is present in its free form. Only this portion represents the bioactive form of the endocrine regulation. However, in saliva only the free hormone is present as the protein bound hormones cannot pass the membranes into the oral cavity.
A good correlation between hormones in saliva with the free fraction in serum [20]. Proportions of estradiol levels in saliva are similar, between 0.2% and 7.9% of total serum concentrations [22]. Therefore, the measurement of estradiol in saliva is very attractive for clinicians due to its manifold advantages over venipuncture—being non-invasive, less stressful for the patient, multiple samples can be collected by a layperson after a short introduction.
Steroid hormones have distinct circadian rhythms, especially estradiol and progesterone in females are strongly correlated with the menstrual cycle [23; 24].