DBC Dehydroepiandrosterone (DHEA) ELISA

PRINCIPLE OF THE TEST 
The DHEA ELISA is a competitive immunoassay. Competition occurs between DHEA present in calibrators, controls, specimen samples and an enzyme-labelled antigen (HRP conjugate) for a limited number of antiDHEA antibody binding sites on the microplate wells. After a washing step that removes unbound materials, the TMB substrate (enzyme substrate) is added which reacts with HRP to form a blue-coloured product that is inversely proportional to the amount of DHEA present. Following an incubation, the enzymatic reaction is terminated by the addition of the stopping solution, converting the blue colour to a yellow colour. The absorbance is measured on a microplate reader at 450 nm. A set of calibrators is used to plot a calibrator curve from which the amount of DHEA in specimen samples and controls can be directly read.

Specimen Collection & Storage 
Approximately 0.1 mL of serum is required per duplicate determination. Collect 4–5 mL of venous blood into an appropriately labelled tube and allow it to clot. Centrifuge at room temperature and carefully transfer the serum into a new storage tube or container. Serum samples may be stored at 2-8°C for up to 24 hours or at -10°C or lower for up to 7 days. Specimens may be more stable than indicated. Consider all human specimens as possible biohazardous materials and take appropriate precautions when handling.

CALCULATIONS 
1. Calculate the mean optical density for each calibrator, control and specimen sample duplicate. 
2. Use a 4-parameter or 5-parameter curve fit with immunoassay software to generate a calibrator curve. 
3. The immunoassay software will calculate the concentrations of the controls and specimen samples using the mean optical density values and the calibrator curve. 
4. If a sample reads more than 40 ng/mL and needs to be diluted and retested, then dilute with DHEA Sample Diluent (see Reagents And Equipment Needed But Not Provided section) not more than 1:10. The result obtained must be multiplied by the dilution factor.