NEOGEN's Cortisol ELISA is used for the quantitative analysis of Cortisol levels in biological fluid.

Cortisol, or hydrocortisone, is the primary corticosteroid secreted by the adrenal cortex. Cortisol is synthesized from cholesterol and may be found in the blood as free Cortisol or bound to corticosteroid-binding globulin. The release of Cortisol is controlled by ACTH, which is produced in the anterior pituitary. Plasma Cortisol levels are highest in the morning and decrease throughout the day. Cortisol concentration in the plasma also elevates in response to stress. Cortisol has an anti-inflammatory effect and aids in carbohydrate metabolism, renal function and the promotion of glucogenesis.

Measurement of plasma Cortisol levels is useful in diagnosing conditions related to functions of the adrenal cortex, including Cushing's syndrome (hypercortisolism), Addison's disease (hypocortisolism) and adrenal tumors. Abnormal Cortisol levels may also possibly be linked to prostate cancer, depression, and schizophrenia.

Specifications
Analyte Cortisol
Application Urine, Oral Fluid, Tissue Culture Supernatant, Plasma, Serum
Assay Range 0.04 - 10 ng/mL
Assay Sample Size 50 µL
Brand NEOGEN®
Drug Panel Hormones & Steroids
Intended Use For the determination of Cortisol in urine, oral fluid, tissue culture supernatant, plasma, or serum samples. Contact a NEOGEN representative for information on other matrices.
Microplate Size 96 well microplate
Platform ELISA
Result Type Quantitative
Species Non-Species Specific
Storage Conditions 2-8°C
Total Assay Incubation Time 90 minutes
Wavelength 650 nm or 450 nm with acid stop

Analytes Cross-Reactivity
Cortisol 100.0%
Prednisolone 47.4%
Cortisone 15.7%
11-Deoxycortisol 15.0%
Prednisone 7.83%
Corticosterone 4.81%
6β-Hydroxycortisol 1.37%
17-Hydroxyprogesterone 1.36%
Deoxycorticosterone 0.94%
Progesterone 0.06%
Bethamethasone 0.05%
Dehydroepiandrosterone 0.03%
Dexamethasone 0.03%
Beclomethasone 0.01%
d-Aldosterone 0.01%
Testosterone 0.01%
17α-Hydroxypregnenolone < 0.01%
Androstenedione < 0.01%
Cholesterol < 0.01%
Estradiol < 0.01%
Estriol < 0.01%
Estrone < 0.01%
Pregnenolone < 0.01%
  1. EIA Buffer
  2. Wash Buffer (10X)
  3. K-Blue Substrate
  4. Extraction Buffer (5X)
  5. Cortisol Enzyme Conjugate
  6. Cortisol Standard
  7. Cortisol Antibody Coated Microplate
  1. Deionized water
  2. Precision pipettes that range from 10 μL-1000 μL and disposable tips. NOTE: If all or several strips are to be used at one time, it is suggested that a multichannel pipette be used.
  3. Clean test tubes used to dilute the standards and conjugate.
  4. Graduated cylinders to dilute and mix wash buffer and extraction buffer.
  5. Microplate reader with 650 nm filter.
  6. Plate cover or plastic film to cover plate during incubation.

Additional Information

Calculation of Results

NEOGEN has partnered with MyAssays.com to provide an online tool for data calculations.

Follow the simple step-by-step guide to generate your data.

Calculate Results

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Technology

The sample or standard solution is first added to the microplate. Next, the diluted enzyme conjugate is added, and the mixture is shaken and incubated at room temperature for one hour. During the incubation, competition for binding sites is taking place. The plate is then washed removing all the unbound material. The bound enzyme conjugate is detected by the addition of substrate which generates an optimal color after 30 minutes. Quantitative test results may be obtained by measuring and comparing the absorbance reading of the wells of the samples against the standards with a microplate reader at 650 nm. The extent of color development is inversely proportional to the amount of Cortisol in the sample or standard. For example, the absence of Cortisol in the sample will result in a bright blue color, whereas the presence of Cortisol will result in decreased or no color development.
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Training

Our customers’ success is our shared success. Our experts are ready to train you and your team on our solutions, so you can rest easy knowing procedures are performed properly and yield accurate results. In addition, we provide certificates upon completion of training.


Support

The following are common examples of unexpected results when running NEOGEN's ELISA kits with possible explanations.

  1. Extreme deep blue color development with samples and standards.
    1. The plate was not properly washed (3 x 300 µL) with the diluted wash buffer. If using an automated washer, ensure the machine is working correctly and increase wash cycle to 5 x 300 µL.
    2. The enzyme conjugate concentrate was incorrectly diluted.

  2. Extremely low color development with samples and standards.
    1. The wash buffer was not diluted correctly before use.
    2. A poor quality of water was used to dilute the wash buffer. Deionized water should be used for dilutions.
    3. The enzyme conjugate concentrate was incorrectly diluted.
    4. The kit prematurely deteriorated, possibly from adverse shipping and storage conditions. Investigate the condition of the kit when it was received and how it was stored prior to use. Proper storage conditions are listed in the package insert.
    5. The kit has expired. Check the expiration dates on the test kit and reagents. No component of the kit should be used past the expiration date. Do not mix any reagents or components of one kit with the reagents or components of another kit.
    6. Contamination. Always use aseptic techniques when opening and removing reagents from vials and bottles. Keep the plate covered except when adding reagents, washing or reading. Always use different pipette tips for each reagent. When pipetting, do not allow the pipette tip to touch any of the reagents already in the well.

  3. No color development with samples and standards.
    1. Improper dilution of enzyme conjugate concentrate.
    2. The kit has expired. Check the expiration dates on the test kits and reagents. No components of the kit should be used past the expiration date. Do not mix any reagents or components of one kit with the reagents or components of another kit.
    3. Incorrect addition of kit reagents. Review assay procedure.

  4. Little to no displacement with the standard curve.
    1. Incorrect dilution of standards. Refer to the dilution scheme in the package insert.
    2. Contamination.
    3. The plate was not properly washed (3 x 300 µL) with the diluted wash buffer. If using an automated washer, ensure the machine is working correctly and increase wash cycle to 5 x 300 µL.
    4. Standard has deteriorated prematurely. Contact your NEOGEN representative and provide them with the kit name, lot number, expiration date and your OD readings for further investigation.

  5. The standard curve performed correctly, but the known negative samples gave low color development.
    1. Samples need to be diluted or extracted to eliminate interference. Refer to the extraction procedure in the kit insert.

  6. The standard curve performed correctly, but the extracted samples gave low color development.
    1. The concentration of the analyte in the extracted sample is too high. The extracted sample will need to be diluted before running it in the assay so the sample OD reading will fit in the standard curve. When a dilution is used, the concentration determined from the standard curve must be multiplied by the dilution factor.
    2. Inadequate extraction of samples resulting in the presence of a solvent. Refer to the recommended extraction procedure in the package insert.

  7. Variability with duplicates.
    1. Inconsistent and/or inadequate pipetting technique when adding reagents. Improve pipetting technique.
    2. Inconsistent washing. The wells should be washed 3 x 300 µL with the diluted wash buffer. If using an automated washer, ensure the machine is working correctly and increase wash cycle to 5 x 300 µL.
    3. Inadequate aspiration during washing. The wells should be dumped and tapped between each washing. Tap out excess liquid in wells before adding substrate. Add substrate immediately to the wells once the excess wash buffer has been removed. If using an automated washer, ensure the machine is aspirating correctly.
    4. Interruption during assay set-up. Have all reagents prepared before assay set-up commences. Reagent addition should be performed in a timely and accurate manner.