DBC Cortisol Saliva 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 cortisol in the sample. A set of standards is used to plot a standard curve from which the amount of cortisol in patient samples and controls can be directly read.

CLINICAL APPLICATIONS 
Cortisol is the most abundant circulating steroid and the major glucocorticoid secreted by the adrenal cortex. Cortisol is physiologically effective in blood pressure maintenance and anti-inflammatory activity. It is also involved in calcium absorption, gluconeogenesis as well as the secretion of gastric acid and pepsin. It is increased under stress situations, physical exercise and external administration of ACTH. Measurement of cortisol levels in general can be used as an indicator of adrenal function and the differential diagnosis of Addison’s and Cushing’s diseases as well as adrenal hyperplasia and carcinoma. 
Most circulating cortisol is bound to cortisol binding globulin or transcortin and albumin. The free cortisol, which is considered the active part of blood, is about 1–2%. In the absence of appreciable amounts of the cortisol binding proteins in saliva, salivary cortisol is considered to be free and shows a diurnal rhythm with the highest levels in the morning and the lowest levels at night.

SPECIMEN COLLECTION AND STORAGE 
Approximately 1 mL of saliva is required per duplicate determination. Collect 4–5 mL of saliva into a clean glass tube (Salivette by Sarstedt may be used) without force or inducement and before eating, drinking or brushing the teeth. Simply rinse the mouth with water before collection. Do not use blood-contaminated specimens. Store samples 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 100 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.