Cortisol ELISA Kit

Antigen: Cortisol

Reactivity: Mouse (Murine)

Application: ELISA

Certificates: ISO 9001:2008

Catalog no.: ABIN367750

Additional Information

Characteristics: This immunoassay kit allows for the in vitro rapid detection of Mouse Cortisol concentrations in serum, plasma and other biological fluids. 

Description: Cortisol is a corticosteroid hormone or glucocorticoid produced by the adrenal cortex, which is part of the adrenal gland (in the zona fasciculata and the zona reticularis of the adrenal cortex). It is usually referred to as the "stress hormone" as it is involved in response to stress and anxiety, controlled by CRH. It increases blood pressure and blood sugar, and reduces immune responses. Various synthetic forms of cortisol are used to treat a variety of different illnesses. The most well-known of these is a natural metabolic intermediary of cortisol called hydrocortisone. When first introduced as a treatment for rheumatoid arthritis, hydrocortisone was referred to as Compound E.

Specificity: This assay recognizes cortisol. No significant cross-reactivity or interference was observed.

Application Details

Principle: This assay employs the competitive inhibition enzyme immunoassay technique. A anti-antibody specific to the antibody of cortisol has been pre-coated onto a microplate. Standards or samples are added to the appropriate microtiter plate wells with an antibody specific for cortisol 3 and Horseradish Peroxidase (HRP) conjugated cortisol, then incubated. A competitive inhibition reaction is launched between cortisol (Standards or samples) and Horseradish Peroxidase (HRP) conjugated cortisol with the antibody specific for cortisol. The more the amount of cortisol in samples, the less antibody bound by Horseradish Peroxidase (HRP) conjugated cortisol. The substrate solutions are added to the wells, respectively. And the color develops in opposite to the amount of cortisol in the sample. The color development is stopped and the intensity of the color is measured.

Protocol: Preparation of Reagents: Sample Collection and Storage: Serum Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at 1000 x g. Remove serum and assay immediately or aliquot and store samples at -20° C. Avoid repeated freeze-thaw cycles. Plasma Collect plasma using citrate, EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 x g within 30 minutes of collection. Assay immediately or aliquot and store samples at -20° C. Avoid repeated freeze-thaw cycles. Note: Grossly hemolyzed samples are not suitable for use in this assay. Assay Procedure: Calculation of Results: Average the duplicate readings for each standard, Blank, and sample and subtract the optical density of Blank. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a 9 standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the cortisol concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor. Limitations of the Procedure: The kit should not be used beyond the expiration date on the kit label. Do not mix or substitute reagents with those from other lots or sources. It is important that the Calibrator Diluent selected for the standard curve be consistent with the samples being assayed. If samples generate values higher than the highest standard, dilute the samples with the appropriate Calibrator Diluent and repeat the assay. 10 Any variation in Standard Diluent, operator, pipetting technique, washing technique, incubation time or temperature, and kit age can cause variation in binding. This assay is designed to eliminate interference by soluble receptors, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded. 11.

Application Notes: 1. Bring all reagents and plate to room temperature for at least 30 minutes before use. Unused wells need store at 2-8℃and avoid sunlight. 2. Wash Buffer If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Dilute 15 ml of Wash Buffer Concentrate into deionized or distilled water to prepare 300 ml of Wash Buffer. 3. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent. 6 4. When using an automated plate washer, adding a 30 second soak period following the addition of wash buffer, and/or rotating the plate 180 degrees between wash steps may improve assay precision. 5. Substrate Solution should remain colorless until added to the plate. Keep Substrate Solution protected from light. Substrate Solution should change from colorless to gradations of blue. 6. Stop Solution should be added to the plate in the same order as the Substrate Solution. The color developed in the wells will turn from blue to yellow upon addition of the Stop Solution. Wells that are green in color indicate that the Stop Solution has not mixed thoroughly with the Substrate Solution. Precaution: The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.

Components: Reagent Quantity Assay plate 1 Standards (S1-S5) 5 Antibody HRP-conjugated 1 x 6 ml 1 x 6 ml Wash Buffer 1 x 15 ml (20×concentrate) Substrate A 1 x 7 ml Substrate B 1 x 7 ml Stop Solution 1 x 7 ml Standard S1 S2 S3 S4 S5 Concentration(ng/ml) 0.08 0.31 1.25 5 20 STORAGE 1. Unopened test kits should be stored at 2-8°C upon receipt and the microtiter plate should be kept in a sealed bag to minimize exposure to damp air. The test kit may be used throughout the expiration date of the kit. Refer to the package label for the expiration date. 5 2. Opened test kits will remain stable until the expiring date shown, provided it is stored as prescribed above. 3. A microtiter plate reader with a bandwidth of 10 nm or less and an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement.

Storage: 1. Unopened test kits should be stored at 2-8°C upon receipt and the microtiter plate should be kept in a sealed bag to minimize exposure to damp air. The test kit may be used throughout the expiration date of the kit. Refer to the package label for the expiration date. 5 2. Opened test kits will remain stable until the expiring date shown, provided it is stored as prescribed above. 3. A microtiter plate reader with a bandwidth of 10 nm or less and an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement. Expiration date: six months from the date of manufacture.

Research Area: Hormones

Restrictions: For Research Use only