Fecal/Tissue Cortisol ELISA Assay
The Fecal/Tissue Cortisol ELISA Assay is For Research Use Only
Size: 1×96 wells
Sensitivity: 17.3 pg/ml
Dynamic Range: 100-3200 pg/mL
Incubation Time: 1.5 hours
Sample Type: Dried Fecal Samples, Serum, Plasma (EDTA and Heparin, Tissue Culture Media, and Urine
Sample Size: 50 µL
Assay Background
Cortisol is a steroid hormone, in the glucocorticoid class of hormones. When used as a medication, it is known as hydrocortisone. It is produced in humans by the zona fasciculata of the adrenal cortex within the adrenal gland. It is released in response to stress and low blood-glucose concentration. It functions to increase blood sugar through gluconeogenesis, to suppress the immune system, and to aid in the metabolism of fat, protein, and carbohydrates. It also decreases bone formation. Cortisol is often referred to as the “stress hormone” as it is involved in the response to stress and it affects blood pressure, blood sugar levels, and other actions of stress adaptation. Immunologically, cortisol functions as an important anti-inflammatory and plays a role in hypersensitivity, immunosuppression, and disease resistance. In animals, cortisol is often used as an indicator of stress and can be measured in blood, saliva, urine, hair, and faeces.
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Assay Principle
This assay employs the competitive enzyme immunoassay technique. A specific anti-mouse-IgG antibody has been pre-coated onto a microtiter plate. Cortisol containing samples or standards and a Cortisol -HRP conjugate are given into the wells of the microtiter plate. Then a mouse monoclonal antibody to Cortisol is added into the wells. Cortisol in samples or standards and a fixed amount of Cortisol -HRP conjugate are competing for a limited amount of Cortisol mouse monoclonal antibody. The Cortisol-antibody complex binds tothe goat polyclonal anti-mouse IgG that has been previously bound to the well. After incubation, the wells are washed with wash buffer to remove unbound material. A substrate solution is then added and incubated, resulting in the development of a blue color. The color development is inhibited by the addition of a stop solution, and the color turns yellow. The yellow color is measured at 450 nm. The concentration of Cortisol is indirectly proportional to the color intensity of the test sample.
Assay Procedure
1. Remove excess microtiter strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal it. Standards and samples should be assayed in duplicates.
2. Add 50 μl of the Standards and diluted samples into the appropriate wells.
3. Add 75 μl of Assay Buffer into the non-specific binding (NSB) wells.
4. Add 25 µL of the Cortisol Conjugate to each well using a repeater pipet.
5. Add 25 µL of the Cortisol Antibody to each well, except the NSB wells, using a repeater pipet
6. Gently tap the sides of the plate to ensure adequate mixing of the reagents. Cover the plate with the plate sealer and shake at room temperature for 1 hour.
7. Aspirate each well and wash, repeating the process 3 times for a total 4 washes. Wash by filling each well with 1× Wash Buffer (300 μl) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating, decanting or blotting.
8. Add 100 μl of TMB substrate solution into each well. Incubate for 30 mins at RT without shaking. Avoid exposure to light.
9. Add 50 μl of Stop Solution to each well and shake lightly to ensure homogeneous mixing.
10. Read the OD with a microplate reader at 450nm immediately.
Package Inserts
Please note: All documents above are for reference use only and should not be used in place of the documents included with this physical product. If digital copies are needed, please contact us.
