DBC 5α-Androstane-3α, 17β Diol Glucuronide (3α-DIOL G) 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 3α-Diol G in the sample. A set of standards is used to plot a standard curve from which the amount of 3α-Diol G in patient samples and controls can be directly read.

CLINICAL APPLICATIONS 
5α-Androstane-3α, 17β-diol glucuronide is a C19 steroid and is either abbreviated as 3α-Diol G, 5α-Diol G or simply, α-Diol G. It is produced mainly as a metabolite of testosterone and dihydrotestosterone (DHT). It is largely produced in target peripheral tissues such as the skin, especially around hair follicles. The stimulation by large amounts of 3α-Diol G leads to excessive hair formation, notably where hair is not normally present in women. In recent years the interest in the measurement of this steroid has increased among clinical investigators studying women suffering from idiopathic hirsutism. Among the steroids known to be precursors for 3α-Diol G are dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulphate (DHEAS), dihydrotestosterone (DHT), androstenedione and testosterone. Only 3α-Diol G has been shown to increase with hirsutism and decrease with treatment. This correlation has also been demonstrated in patients with polycystic ovarian syndrome (PCO). 3α-Diol G determinations have therefore proved to be a useful indicator in a variety of ways including monitoring the progress of treatment of idiopathic hirsutism and women with PCO. Furthermore, diabetic patients (both men and women) under cyclosporine A therapy have shown increased 3α-Diol G levels, a side effect resulting in the appearance of hair in previously hairless areas

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 50 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.