TECO® Cortisol Saliva ELISA

Solid phase enzyme‐linked immunosorbent assay (ELISA) based on the competition principle. An unknown amount of antigen present in the sample and a fixed amount of enzyme labelled antigen compete for the binding sites of the antibodies coated onto the wells. After incubation the wells are washed to stop the competition reaction. After the substrate reaction the intensity of the developed color is inversely proportional to the amount of the antigen in the sample. Results of samples can be determined directly using the standard curve.

Performing the Cortisol Saliva ELISA in epidermal mucus samples of fish

The TECO® Cortisol Saliva ELISA has been developed to measure free cortisol in human saliva. For epidermal mucus collected with a swab, the sample has to be extracted using a specially designed Extraction Buffer, which is included in the TECO® Mucus Collection Set (TE1034). 50μl of the extracted mucus samples will directly be used in the TECO® Cortisol Saliva ELISA. The effect of this specific extraction buffer has been tested for the TECO® Cortisol Saliva ELISA.

Preparation of Mucus Samples

Collect mucus as described in the package insert of the TECO® Mucus Collection Set (TE1034). For assay, add 500μl Extraction Buffer (TECO® Mucus Collection Set) to the swab 15‐30min before vortex. For more determinations (e.g. total protein) the swab should be removed from each vial and discarded before cortisol measurement. Before pipetting sample into wells repeat vortexing the sample.

Cortisol in mucus from Tilapia

As in all Perciformes the classification of fish into dominants and subalterns is possible by observing typical behavior. Mucus samples from fish of both groups were obtained by using the TECO® Mucus Collection set (TE1034). The dominant fish were then placed into a bucket containing 30l fresh water and swirled for one minute to induce stress ‐ afterwards mucus samples were obtained for a second time. Free cortisol in mucus was determined using the TECO® Cortisol Saliva ELISA. There was a clear difference in the cortisol levels between dominant (mean: 0.05ng cortisol/mg total protein) and subaltern fish (mean: 0.22ng cortisol/mg total protein) as shown in Figure 1. While dominant fish had cortisol level close to the detection level (mean: 0.05ng cortisol/mg total protein), the values rose after induction of stress (mean: 1.44ng cortisol/mg total protein) as shown in Figure 2.

Fig.2: Mucus cortisol level (ng cortisol/mg total protein) in non‐stressed and stressed Tilapia fish

Fig.1: Mucus cortisol level (ng cortisol/mg total protein) in dominant and subaltern Tilapia fish

Calculations of Results

The obtained OD of the standards (y‐axis, linear) are plotted against their concentration (x‐axis, logarithmic) either on semi‐logarithmic graph paper or using an automated method. A good fit is provided with cubic spline, 4 Parameter Logistics or Logit‐Log.

For the calculation of the standard curve, apply each signal of the standards (one obvious outlier of duplicates might be omitted and the more plausible single value might be used). The concentration of the samples can be read directly from the standard curve. Saliva samples with remarkably elevated values should be reviewed for blood contamination.

Conversion:

• Cortisol (ng/ml) x 2.76 = nmol/l
• Cortisol (μg/dl) x 27.6 = nmol/l

Reportable range:

• Saliva: 0.015 – 3μg/dl Cortisol

(Example. Do not use for calculation!)

Standards and Controls are calibrated by use of an isotope dilution‐LCMS as reference method.

Cortisol (hydrocortisone, compound F) is the main glucocorticoid in humans and is produced in the zona fasciculata of the adrenal cortex. 90% of the circulating cortisol are bound to corticoid binding globulin (CBG, Transcortin), ca. 7% are bound to albumin and only 1–3% are unbound. Only the latter part represents the active form of cortisol.

The free cortisol is released in saliva and is excreted via the kidneys as a small part among the metabolites of cortisol. The level of free cortisol in blood regulates mainly its secretion in the adrenal cortex in a negative feedback mechanism via CRH (corticotropin releasing hormone) in the hypothalamic region and the ACTH in the pituitary gland, but it is also affected by different situations above all by stress.

In humans there is a physiological fluctuation of cortisol achieving the highest level in the morning and the lowest during midnight. This fluctuation of cortisol plasma level is reflected in saliva normally with a peak in the first 90 minutes after wake up.

The cortisol measurement is indicated in diseases with abnormal gluco‐corticoid production e.g. Cushing Syndrome and Addison’s Disease. Because of the diurnal fluctuation of cortisol levels it is necessary to take several samples for an individual cortisol profile or during dynamic tests like dexamethasone‐suppression‐ or ACTH‐stimulation‐test. Therefore a salivary sample collection is an easy method without the stress of repeated venipunctures. The measurement of cortisol in saliva is advisable in subjects with abnormal CBG levels such as women in pregnancy, people with hypothyroidism, nephrotic syndrome or marked adipositas and during the application of different drugs, including oral contraceptives.