DBC Estrone ELISA

PRINCIPLE OF THE TEST 
The Estrone ELISA is a competitive immunoassay. Competition occurs between estrone present in calibrators, controls, specimen samples and an enzyme-labelled antigen (HRP conjugate) for a limited number of antiestrone 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 estrone present. Following an incubation, the enzymatic reaction is terminated by the addition of the stopping solution, converting the colour from blue to yellow. 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 estrone in specimen samples and controls can be directly read.

Specimen Collection & Storage 
Approximately 0.15 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 room temperature for up to 3 days, at 2-8°C for up to 7 days or at -20°C or lower for up to 1 month.

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 2000 pg/mL and needs to be diluted and retested, then dilute with a known low estrone value (<60 pg/mL) serum sample not more than 1:10. The result obtained must be multiplied by the dilution factor.