Se120037bul Kit Kortisol

Cortisol ELISA
Catalog Number SE120037

Storage Temperature 2–8 °C
TECHNICAL BULLETIN
Product Description Components

Cortisol (hydrocortisone, compound F) is the most
potent glucocorticoid synthesized from cholesterol. Materials Provided 96 Tests
Cortisol is found in the blood either as free cortisol or Microwells coated with Cortisol MAb 12 × 8 × 1
bound to corticosteroid-binding globulin. Cortisol Cortisol Standard: 7 vials (ready to
production has an ACTH-dependent circadian rhythm 0.5 ml
use)
with peak levels in the early morning and a nadir at Enzyme Conjugate (20×) 1.2 ml
night. The factors controlling this circadian rhythm are TMB Substrate: 1 bottle (ready to use) 12 ml
not completely defined. Serum levels are highest in the
Stop Solution: 1 bottle (ready to use) 12 ml
early morning and decrease throughout the day. In the
20× Wash concentrate: 1 bottle 25 ml
metabolic aspect, cortisol promotes gluconeogenesis,
liver glycogen deposition, and the reduction of glucose Assay Diluent 24 ml
utilization. Immunologically, cortisol functions as an
important anti-inflammatory, and plays a role in Reagents and Equipment Required but Not
hypersensitivity, immunosuppression, and disease Provided.
resistance. It has also been shown that plasma cortisol • Distilled or deionized water
levels elevate in response to stress. Abnormal cortisol • Precision pipettes
levels are seen with a variety of different conditions: • Disposable pipette tips
adrenal tumors, prostate cancer, depression, and • ELISA reader capable of reading absorbance at
schizophrenia. Elevated cortisol levels and lack of 450 nm
diurnal variation have been identified in patients with • Absorbent paper or paper towel
Cushing's disease • Graph paper
This kit is a solid phase competitive ELISA. The Precautions and Disclaimer
samples and cortisol enzyme conjugate are added to This product is for R&D use only, not for drug,
the wells coated with anti-cortisol monoclonal antibody. household, or other uses. Please consult the Safety
Cortisol in the patient’s sample competes with a cortisol Data Sheet for information regarding hazards and safe
enzyme conjugate for binding sites. Unbound cortisol handling practices.
and cortisol enzyme conjugate are washed off by
washing buffer. Upon the addition of the substrate, the Preparation Instructions
intensity of color is inversely proportional to the Sample Preparation
concentration of cortisol in the samples. A standard 1. Collect blood specimens and separate the serum
curve is prepared relating color intensity to the immediately.
concentration of the cortisol. 2. Specimens may be stored refrigerated at 2–8 °C for
5 days. If storage time exceeds 5 days, store frozen
The Cortisol ELISA Kit is intended for the quantitative at –20 °C for up to one month.
measurement of cortisol in human serum or plasma. 3. Avoid multiple freeze-thaw cycles.
4. Prior to assay, frozen sera should be completely
thawed and mixed well.
5. Do not use grossly lipemic specimens.
2
20× Enzyme Conjugate Results

Prepare 1× working solution by diluting 20-fold with The standard curve is constructed as follows:
assay diluent as needed (e.g., 0.1 mL of the 1. Check Cortisol Standard value on each standard
20× Enzyme Conjugate in 1.9 mL of Assay Diluent is vial. This value might vary from lot to lot. Make sure
sufficient for 20 wells). The diluted conjugate has to be the value is checked on every kit.
used the same day. 2. To construct the standard curve, plot the
absorbance for Cortisol standards (vertical axis)
20× Wash Buffer Concentrate versus Cortisol standard concentrations (horizontal
Prepare 1× Wash buffer by adding the contents of the axis) on a linear graph paper. Draw the best curve
bottle (25 ml, 20×) to 475 ml of distilled or deionized through the points.
water. Store at room temperature (18–26 °C).
Example of Standard Data
Storage/Stability
OD 450 nm Concentration (ng/ml)
Store the kit at 2–8 °C. Keep microwells sealed in a dry
bag with desiccants. The reagents are stable until Std 1 2.36 0
expiration of the kit. Do not expose test reagents to Std 2 1.76 20
heat, sun, or strong light. Std 3 1.10 50
Std 4 0.65 100
Procedure Std 5 0.29 200
Notes: The components in this kit are intended for use
Std 6 0.13 400
as an integral unit. The components of different lots
should not be mixed. Std 7 0.08 800
It is recommended that standards, control, and serum 3. Read the absorbance for controls and each
samples be run in duplicate. unknown sample from the curve. Record the value
for each control or unknown sample.
Optimal results will be obtained by strict adherence to
this protocol. Accurate and precise pipetting, as well as Expected Values
following the exact time and temperature requirements It is recommended each laboratory establish its own
prescribed are essential. Any deviation from this may normal ranges based on a representative sampling of
yield invalid data. the local population. The following values for cortisol
may be used as initial guideline ranges only:
All reagents must be brought to room temperature
(18–26 °C) before use and gently mix. Classification ng/ml
8:00 am – 10:00 am 50–230
1. Place the desired number of coated strips into the 4:00 pm 30–150
holder.
2. Pipette 25 µl of cortisol standards, control and Limitations of the Test
patient’s sera into selected wells. 1. The test results obtained using this kit serve only as
3. Add 200 µl of Enzyme Conjugate to all wells. an aid to diagnosis and should be interpreted in
4. Thoroughly mix for 10 seconds. relation to the patient’s history, physical findings,
5. Incubate for 60 minutes at room temperature and other diagnostic procedures.
(18–26 °C). 2. Do not use sodium azide as preservative. Sodium
6. Remove liquid from all wells. Wash wells three azide inhibits HRP enzyme activities.
times with 300 µl of 1× wash buffer. Blot on
absorbent paper towels.
7. Add 100 µl of TMB substrate to all wells.
8. Incubate for 15 minutes at room temperature
(18–26 °C).
9. Add 50 µl of Stop Solution to all wells. Shake the
plate gently to mix the solution.
10. Read absorbance on ELISA Reader at 450 nm
within 20 minutes after adding the Stop Solution.
3
References
1. Chernow, B. et al., Hormonal responses to graded 5. Stewart, P.M. et al., A rational approach for
surgical stress. Arch. Intern. Med., 147, 1273-1278 assessing the hypothalamo-pituitary-adrenal axis.
(1987). Lancet, 5, 1208-1210 (1988).
2. Crapo, L., Cushing's syndrome: A review of 6. Watts, N.B., and Tindall, G.T:, Rapid assessment of
diagnostic tests. Metabolism, 28, 955-977 (1979). corticotropin reserve after pituitary surgery. JAMA,
3. Lee, P.D.K. et al., Virilizing adrenocortical tumors in 259, 708-711 (1988).
childhood. Eight cases and a review of the 7. Schlaghecke, R. et al., The effect of long-term
literature. Pediatrics, 76, 437-444 (1985). glucocorticoid on pituitary-adrenal responses to
4. Leisti, S. et al., The diagnosis and staging of exogenous corticotropin-releasing hormone. New
hypocortisolism in progressing autoimmune Engl. J. Med., 326, 226-230 (1992).
adrenalitis. Pediatr. Res., 17, 861-867 (1983). 8. Tiertz, N.W., in Text book of Clinical Chemistry,
Saunders, 1968.
SG,CH,MAM 10/14-1
2014 Sigma-Aldrich Co. LLC. All rights reserved. SIGMA-ALDRICH is a trademark of Sigma-Aldrich Co. LLC, registered in the US and other
countries. Sigma brand products are sold through Sigma-Aldrich, Inc. Purchaser must determine the suitability of the product(s) for their
particular use. Additional terms and conditions may apply. Please see product information on the Sigma-Aldrich website at
www.sigmaaldrich.com and/or on the reverse side of the invoice or packing slip.

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Se120037bul Kit Kortisol

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Cortisol ELISA

Catalog Number SE120037

Product Description Components

20× Enzyme Conjugate Results

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