DBC Cortisol Elisa

PRINCIPLE OF THE TEST 
The Cortisol ELISA is a competitive immunoassay. Competition occurs between cortisol present in calibrators, controls, specimen samples and an enzyme-labelled antigen (HRP conjugate) for a limited number of anticortisol 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 cortisol 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 cortisol in specimen samples and controls can be directly read.

SPECIMEN COLLECTION, STORAGE AND PRETREATMENT 
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 room temperature for up to 24 hours, 2-8°C for up to 5 days or at -20°C or lower for up to 30 days. Specimens may be more stable than indicated. Consider all human specimens as possible biohazardous materials and take appropriate precautions when handling.
Specimen Pre-Treatment Specimen pre-treatment is not required.

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 If a sample reads more than 40 μg/dL and needs to be diluted and retested, dilute it with calibrator A not more than 1:10. The result obtained must be multiplied by the dilution factor.