CORTISOL ENZYME IMMUNOASSAY TEST; Page 1
Atlas Link
12720 Dogwood Hills Lane
Fairfax, VA 22033 USA
Phone: (703) 266-5667, FAX: (703) 266-5664
http://www.atlaslink-inc.com,
CORTISOL ENZYME IMMUNOASSAY TEST KIT
CATALOG NUMBER: 6101
ENZYME IMMUNOASSAY FOR THE QUANTITATIVE DETERMINATION OF CORTISOL CONCENTRATION IN HUMAN SERUM AND URINE
FOR IN VITRO USE ONLY.
Store at 2 to 8 °C
PROPRIETARY AND COMMON NAMES
Atlas Link Cortisol Enzyme Immunoassay
INTENDED USE
For the quantitative determination of Cortisol concentration in human serum and urine.
PRINCIPLE OF TEST
The Atlas Link Cortisol Quantitative Test is based on a widely used immunoassay technique. A sample containing an unknown amount of the substance to be assayed (unlabeled antigen) is added to a standard amount of a labelled derivative of the same substance (labelled antigen). The labelled and unlabeled antigens are then allowed to compete for high affinity binding sites on a limited number of antibodies. After washing away the free antigen, the amount of labelled antigen in the sample is reversely proportional to the concentration of the unlabeled antigen. The actual concentrations in unknown samples are obtained by means of a standard curve based on known concentrations of unlabeled antigen analyzed in parallel with the unknowns.
In this kit an enzyme label is used. The biospecific reaction takes place during a 2 hour room temperature incubation. After washing, substrate solution is added and the enzyme allowed to work for a fixed time before the reaction is terminated. Absorbances are measured at 405 nm using a plate reader. A standard curve is produced using values for 6 serum standards from which absorbance values for blank tubes have been subtracted. Results for unknowns may be read directly from this standard curve. As an alternative to the manual calculation of results, the data may be analyzed by a suitable computer program.
This kit is suitable for the direct measurement of cortisol in serum samples. It may also be used, following an extraction procedure, for assaying urinary cortisol.
REAGENTS
1. Antibody-Coated Wells.
Microwells coated with secondary antibody.
2. Cortisol Enzyme Conjugate.
Contains enzyme-labeled Cortisol stock solution.
3. Cortisol Antibody.
Contains Cortisol antibody (rabbit) stock solution.
4. Cortisol Assay Buffer.
5. Cortisol Standard Set.
Contains 0, 20, 50, 150, 500 and 2000 nmol/l.
6. Substrate Concentrate.
7. Substrate Buffer.
8. Stop Solution.
9. Wash Concentrate.
WARNINGS AND RECAUTIONS FOR USERS
1. CAUTION: This kit contains reagents manufactured from human blood components. The source materials have been tested by immunoassay for hepatitis B surface antigen and antibodies to HIV virus and found to be negative. Nevertheless, all human blood products and samples should be considered potentially infectious. Handling and disposal should be in accordance with the procedures defined by an appropriate national biohazard safety guideline or regulation, where it exists (e.g. U.S. Department of Health and Human Services, Bethesda, MD.) Publication number (CDC) 88-8395 on laboratory safety procedures or any other local or national regulation.
2. The contents of this kit, and their residues, must not come into contact with ruminating animals or swine.
3. Avoid contact with the Stopping Solution. It may cause skin irritation and burns.
4. Do not use reagents after expiration date.
5. Do not mix or use components from kits with different lot numbers.
6. Replace caps on reagents immediately. Do not switch caps.
7. Reagents contain sodium azide (NaN3) as a preservative. Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. On disposal, flush with a large volume of water to prevent azide build-up.
8. Do not pipette reagents by mouth.
9. For research use only. Not for use in diagnostic procedures.
STORAGE CONDITIONS
1. Store the kit at 2 to 8 ° C upon receipt and when it is not in use.
2. Keep microwells in a sealed bag with desiccants to minimize exposure to damp air.
INSTRUMENTATION
A microwell reader with a bandwidth of 10 nm or less and an optical density range of 0 to 2 OD or greater at 405 nm wavelength is acceptable for use in absorbance measurement.
SPECIMEN COLLECTION AND PREPARATION
1. This kit is suitable for use with serum or heparin plasma samples.
The use of hemolytic or lipemic samples will not affect results, but icteric samples with bilirubin may interfere with the assay.
2. No special preparation of the sample is required. A venous blood sample (enough to produce about 0.5 ml serum) is collected aseptically.
PERFORMANCE OF THE ASSAY
Materials Provided With Test Kit
1. Antibody-Coated Wells, 96 wells.
2. Cortisol Enzyme Conjugate, 300 µl.
3. Cortisol Antibody, 300 µl.
4. Cortisol Assay Buffer, 33 ml.
5. Cortisol Standard Set, 0.5 ml per vial.
6. Substrate Concentrate, 0.5 ml.
7. Substrate Buffer, 25 ml.
8. Stop Solution, 10 ml.
9. Wash Concentrate, 40 ml.
Materials Required But Not Provided
1. Semiautomatic pipettes: 25, 50, 100 and 200 µl
2. Disposable pipette tips
3. Microwell plate shaker
4. Microtiter well reader
5. Plate washer
6. Absorbent paper
Reagent Preparation
1. Prepare Cortisol Standards by accurately pipetting 0.5 ml distilled water into each of the vials. Cap and mix well by gently swirling or inversion. Allow to stand 60 minutes at room temperature before use. Reconstituted standards are stable for up to 2 months at 2-8 °C.
2. Prepare Enzyme Conjugate Reagent by mixing Cortisol Enzyme Conjugate Stock Solution with Assay Buffer (1:50) to the required volume. The working solution thus prepared is stable for up to 2 months.
3. Prepare Cortisol Antibody Solution by mixing Antibody Stock Solution with Assay Buffer (1:50) to the required volume. The working solution is stable for up to 2 months at 2-8 °C.
4. Prepare Substrate Solution by mixing Substrate Concentrate with Substrate Buffer(1:50). The solution should be kept in the dark and should be used within 1 week.
5. Prepare Wash Solution by diluting the Wash Concentrate to 1000 ml with distilled water. The Wash Solution is stable until the expiration date on the Concentrate label.
Assay Procedure
1. All reagents should be allowed to reach room temperature (18 to 25 °C) before use.
2. Pipette 25 µl of standards, samples, and controls into appropriate wells. Two wells, e.g. A1 and B1 should be left empty for blanking.
3. Add 100 µl of diluted Cortisol Enzyme Conjugate Solution to each well (except those set aside for blanks).
4. Add 100 µl of diluted Cortisol Antibody Solution into each well (except those set aside for blanks).
5. Incubate for 2 hours at room temperature on a shaker (at about 1000 rpm).
6. Wash the plate 4 times with Wash Solution (250 - 300 µl per well). Invert plate and tap firmly against absorbent paper to remove residual moisture.
7. Add 200 µl Substrate Solution into each well (including the blanks). Remember the pipetting order.
8. Incubate the plate for 20 minutes without shaking.
9. Stop reaction by adding 50 µl of Stopping Solution to wells in the same sequence that the Substrate Solution was added. Gently mix for 1 - 2 minutes.
10. Read the absorbance at 405 nm with a microwell reader.
Note: The substrate incubation should be carried out within the temperature range 20 - 25 ° C. For temperatures outside this range, the duration of the incubation should be adjusted by approximately 1 minute/1° C.
Urinary Cortisol
As an alternative to the normal, direct assay method, urinary cortisol may be assayed using the following extraction method (standards should not be extracted):
1. Using an automatic reagent dispenser, add 4.0 ml dichloromethane to 400 µl of urine sample.
2. Vortex mix for 30 seconds in pulses of about 5 seconds. Allow layers to separate.
3. Discard the upper aqueous layer.
4. Transfer 1000 µl of dichloromethane phase with a positive displacement pipette to another tube and evaporate to dryness.
5. Redissolve the residue in 200 µl of zero standard. Vortex mix for 15 seconds, and again just before pipetting. Additional vials of zero standard, 3 ml in volume, are available from Medix.
6. Run the assay according to the details of the procedure given above, but with a modification at point 2. Use 25 µl aliquots of standards, but 50 µl aliquots of the extracted samples.
CALCULATION OF RESULTS
1. Calculate the mean absorbance values (A405) for each set of reference standards, controls, samples and blanks.
2. Subtract the value for blanks from those for standards, controls and unknown samples.
3. Calculate the B/B0 % values by dividing each value by the value for the zero-standard.
4. For the standards, plot a graph on semi-log graph paper with B/B0 % values on the ordinate and the Cortisol concentrations (nmol/l) on the abscissa.
5. Using the graph, read off the Cortisol concentrations for the unknown samples.
Cortisol values obtained are in the SI units, nmol/l. Conversion to ng/ml may be accomplished by using the following equation.
Cortisol (ng/ml) = Cortisol (nmol/l) x 0.3625
INTERPRETATION OF RESULTS
1. Results of a typical standard run are shown below:
Mean B/B0 Cortisol
Well A405 A405-Abl x 100% (nmol/l)
Blank 0.165
0.169
0 Std. 1.948
1.934 1.77 100
20 Std. 1.688 1.677 1.516 85.5
50 Std. 1.432
1.433 1.266 71.4
150 Std. 1.003
1.018 0.844 47.6
500 Std. 0.598
0.588 0.426 24.0
2000 Std. 0.319
0.332 0.159 9.0
Unknown 1 1.048
1.076 0.895 50.5 132
Unknown 2 0.616
0.612 0.447 25.2 464
2. Standard Curve
NOTE: This standard curve is for illustration only, and should not be used to calculate unknowns. Each laboratory must provide its own data and standard curve.
EXPECTED VALUES
The serum Cortisol concentrations of apparently healthy male and female subjects were measured at two different times of day. The results are shown below:
Sample ___ n x nmol/l Range nmol/l
Serum (7.40 - 9.00) 127 350 137-630
Serum (15.00 - 17.00) 46 213 121 - 454
It is recommended that each laboratory establish its own normal values.
PERFORMANCE CHARACTERISTICS
1. Sensitivity
The sensitivity of the method defined as the detectable concentration equivalent to two times the standard deviation of the zero-binding value is better than 3.5 nmol/l.
2. Precision
Intra-assay variation was determined for serum samples. Results are shown below.
Intra-Assay Precision
Serum Sample Mean (nmol/l) CV(%)
1 23.1 7.5
2 72.0 5.0
3 404 2.5
4 680 2.8
5 1241 3.1
Inter-Assay Precision
Serum Sample Mean (nmol/l) CV(%)
1 25.4 8.4
2 78.8 4.1
3 420 2.6
4 700 3.2
5 1300 2.2
3. Recovery
4 different amounts of Cortisol were added to 6 different serum samples. Mean recovery was 96% and the range was from 80 to 107%.
3 different amounts of Cortisol were added to 6 urine samples. Mean recovery was 101% and the recovery range was 94 to 108%.
4. Specificity
Cross-reactivity (at the 50% inhibition level) of the Cortisol antibody used in the kit is shown below:
Cortisol 100
6a-Methylprednisolone 93.0
Prednisolone 56.0
5b-Dihydroxycortisol 14.8
20a-Dihydroxycortisol 7.2
11-Deoxycortisol 1.7
Prednisone 1.5
17a-Hydroxyprogesterone 1.2
6ß-Hydroxycortisol 1.0
Tetrahydrocortisol 0.9
Cortisone 0.8
20b-Dihydrocortisol 0.8
Dexamethasone 0.3
Corticosterone 0.2
Beclomethasone <0.1
Betamethasone <0.1
Deoxycorticosterone <0.1
16ß-Methylprednisone <0.1
Tetrahydrocortisone <0.1
QUALITY CONTROL
Good laboratory practice requires that low, medium and high controls be run with each calibration curve. A statistically significant number of controls should be assayed to establish mean values and acceptable ranges to assure proper performance.
LIMITATIONS OF PROCEDURE
1. Reliable and reproducible results will be obtained when the assay procedure is carried out with a complete understanding of the package insert instructions and with adherence to good laboratory practice.
2. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.
Technical Consultation
Call or Write:
Atlas Link
12720 Dogwood Hills Lane
Fairfax, VA 22033 USA
Phone: (703) 266-5667, FAX: (703) 266-5664
http://www.atlaslink-inc.com,
Atlas Link, 12720 Dogwood Hills Lane, Fairfax, VA 22033 USA
Phone: (703) 266-5667, FAX: (703) 266-5664
http://www.atlaslink-inc.com,