DBC Pregnenolone 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 pregnenolone in the sample. A set of standards is used to plot a standard curve from which the concentration of pregnenolone in patient samples and controls can be directly read.

CLINICAL APPLICATIONS 
Pregnenolone (3β-hydroxypregn-5-en-20-one) is the first steroid to be derived from cholesterol in the pathway of steroidogenesis, and it is the common precursor for all of the adrenal and gonadal steroids. Its production occurs in the mitochondrion by cleavage of the C-20 side chain of cholesterol by the P-450SCC enzyme. Once produced, pregnenolone may be utilized by two pathways of steroidogenesis. Pregnenolone may either be converted to 17-OH pregnenolone via the enzymatic action of 17α-hydroxylase or to progesterone via the enzymatic action of 3β-hydroxysteroid dehydrogenase. 
Elevated pregnenolone levels occur in forms of congenital adrenal hyperplasia (CAH), due to 3β-hydroxysteroid dehydrogenase deficiency or 17α-hydroxylase deficiencies. Higher levels have also been reported in women with idiopathic hirsutism. Studies on pregnenolone levels in regard to sex and age differences indicate that maximum levels occur at approximately 17 and 16 years of age for women and men, while minimum levels occur at approximately 37 and 38 years of age for women and men, respectively. In general, women were found to have slightly higher values when compared to men. Many areas of pregnenolone physiology remain to be investigated.
Current research indicates that the determination of pregnenolone in serum may be useful for studying its metabolite, pregnenolone sulfate, which has been reported to have various effects in the mammalian brain and central nervous system.

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 25.6 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.