DBC Androstenedione ELISA

PRINCIPLE OF THE TEST 
The principle of the following enzyme immunoassay test follows the typical competitive binding scenario. Competition occurs between an unlabelled antigen (present in standards, controls and patient samples) and an enzyme-labelled antigen (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 androstenedione in the sample. A set of standards is used to plot a standard curve from which the amount of androstenedione in patient samples and controls can be directly read. 

CLINICAL APPLICATIONS 
Androstenedione is produced by the adrenals and gonads. As a result, the determination of the level of androstenedione in serum is important in the evaluation of the functional state of these glands. Androstenedione is a precursor of testosterone and estrone. Besides the adrenals, in females, the ovaries have been shown to be an important source of androstenedione. It has been reported that there is a fluctuation day by day of androstenedione during the ovulatory cycle. The principle production of testosterone in females is from the conversion of other related androgens, especially androstenedione. An abnormal testosterone level in women should be accompanied by the estimation of serum androstenedione. The use of serum testosterone determination in conjunction with the enzyme immunoassay of androstenedione can be used to determine if the source of the excess androgen production is adrenal or ovarian.

SPECIMEN COLLECTION AND STORAGE 
Approximately 0.1 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 10 ng/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.