DBC Estradiol Elisa

PRINCIPLE OF THE TEST 
The principle of the following enzyme immunoassay test follows the typical competitive binding scenario. Competition occurs between an unlabelled estradiol (present in standards, controls and patient samples) and an enzyme-labelled estradiol (conjugate) for a limited number of antibody binding sites on the microplate. The washing and decanting procedures remove unbound materials. After the washing step, the enzyme substrate is added. The enzymatic reaction is terminated by addition of the stopping solution. The absorbance is measured on a microtiter plate reader. The intensity of the colour formed is inversely proportional to the concentration of unlabelled estradiol in the sample. A set of standards is used to plot a standard curve from which the amount of estradiol in patient samples and controls can be directly read.

CLINICAL APPLICATIONS 
Estradiol is one of the main components of naturally occurring estrogens and is the major estrogen secreted during the menstrual cycle. The serum levels of estradiol are low during the follicular phase rising gradually until about one day before ovulation when a marked rise in the estradiol level occurs (Ovulatory Peak). The estradiol level falls rapidly at, or right after ovulation and is again within the levels of the follicular phase. There is a second rise of estradiol around day 21 of the cycle (Luteal Peak). The levels then decline gradually to the lowest level at the onset of the next menstrual cycle.

SPECIMEN COLLECTION AND STORAGE 
Approximately 0.2 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 at a later date. Consider all human specimens as possible biohazardous materials and take appropriate precautions when handling.

CALCULATIONS 
1. Calculate the mean optical density of each calibrator duplicate. 
2. Draw a calibrator curve on semi-log paper with the mean optical densities on the Y-axis and the calibrator concentrations on the X-axis. If immunoassay software is being used, a 4-parameter or 5-parameter curve is recommended. 
3. Calculate the mean optical density of each unknown duplicate. 
4. Read the values of the unknowns directly off the calibrator curve. 5. If a sample reads more than 3200 pg/mL then dilute it with calibrator A at a dilution of no more than 1:8. The result obtained should be multiplied by the dilution factor.