DBC Total Estrogens Elisa

PRINCIPLE OF THE TEST 
The total estrogens ELISA is a competitive immunoassay. Competition occurs between total estrogens (estrone, estradiol, and estriol) present in calibrators, controls and patient samples and an enzyme-labelled antigen (conjugate) for a limited number of anti-estrogen antibody binding sites on the microplate wells. After a washing step that removes unbound materials, the enzyme substrate is added, and approximately 30 minutes later the enzymatic reaction is terminated by addition of stopping solution. The resulting optical density (OD), measured with a microplate reader, is inversely proportional to the concentration of total estrogens in the sample. A calibrator curve is plotted with a provided set of calibrators to calculate directly the concentration of total estrogens in patient samples and controls.

CLINICAL APPLICATIONS 
Total estrogens comprise the total quantity of estrone, estradiol, and estriol. The estrogens are involved in the development of female sex organs and secondary sex characteristics. Before the ovum is fertilized, the main action of the estrogens is on the growth and function of the reproductive tract to prepare it for the fertilized ovum. 
During the follicular phase of the menstrual cycle, the total estrogens level shows a slight increase. The production of total estrogens then increases markedly to peak at around day 13. The peak is of short duration and by day 16 of the cycle levels will be low. A second peak occurs at around day 21 of the cycle. If fertilization does not occur, the production of total estrogens decreases. 
In post-menopausal women, the concentration of all estrogens decreases substantially and estrone becomes the predominant estrogen. In pregnant women, the concentration of all estrogens escalates and estriol becomes the predominant estrogen. 
A total estrogens test is commonly indicated to: 
• Aid in diagnosis of sex steroid metabolism related conditions, for example, premature or delayed puberty, and aromatase and 17 alpha-hydroxylase deficiencies. 
• Follow-up female hormone replacement therapy in postmenopausal women. 
• Prognose antiestrogen therapy, for example, aromatase inhibitor therapy.

SPECIMEN COLLECTION AND STORAGE 
Approximately 0.15 mL of serum is required per duplicate determination. Collect 4–5 mL of blood into an appropriately labelled tube and allow it to clot. Centrifuge and carefully remove the serum layer. Store at 4°C for up to 24 hours or at -10°C or lower if the analyses are to be done later. Consider all human specimens as possible biohazardous materials and take appropriate precautions when handling.

CALCULATIONS 
1. Calculate the mean optical density of each calibrator, control and sample duplicate. 
2. Use a 4-parameter or 5-parameter curve fit with immunoassay software to generate a calibrator curve. 
3. Read the values of the unknowns directly off the calibrator curve. 
4. If a sample reads more than 2,500 pg/mL dilute it with calibrator A not more than 10-fold. The result obtained must be multiplied by the dilution factor.