Instructions for use

Cortisol Urine ELISAMS E-5100R

Cortisol Urine ELISA
1. INTENDED USE
Competitive immunoenzymatic colorimetric method forconcentration in urine.
Cortisol Urine ELISA kit is intended for research use only.

quantitative

determinationof

freeCortisol

2. PRINCIPLEThe Cortisol (antigen) in the sample competes with the antigenic Cortisol conjugated with horseradish
peroxidase (HRP) for binding to the limited number of antibodies anti Cortisol coated on the microplate (solidphase).
After incubation, the bound/free separation is performed by a simple solid-phase washing.
Then, the enzyme HRP in the bound-fraction reacts with the Substrate (H2O2) and the TMB Substrate anddevelops a blue color that changes into yellow when the Stop Solution (H2SO4) is added.
The colour intensity is inversely proportional to the Cortisol concentration of in the sample.
Cortisol concentration in the sample is calculated through a standard curve.
3. REAGENTS, MATERIALS AND INSTRUMENTATION
3.1 Reagents and materials supplied in the kit
Standards and Controls
Cat. no.

Component

MS E-5101

Standard
Standard A

Concentration ng/ml
0Volume / vial
4 ml

MS E-5102
MS E-5103
MS E-5104
MS E-5105
MS E-5151
MS E-5152

Standard B
Standard C
Standard D
Standard E
Control 1 *Control 2 *1530200
Refer to vial labels or QCReport for expected value
and acceptable range!

1 ml
1 ml
1 ml
1 ml
1 ml
1 ml

* Control: ready to use
Conjugate

MS E-5140
Content:

Cortisol conjugated with horseradish peroxidase (HRP)

Volume:1 x 33 ml

MS E-5131
Content:

Microtiterwells
1 breakable microplate; Anti-Cortisol-antibody adsorbed on the microplate.

MS E-0030

Wash Solution 10x

Content:

Phosphate buffer 0.2 M, Proclin < 0.0015%.

Volume:1 x 50 ml

MS E-0055

Substrate Solution

Content:

H2O2-TMB, 0.26 g/l (avoid any skin contact).

Volume:1 x 15 ml

MS E-0080

Stop Solution

Content:

Sulphuric acid, 0.15 mol/l (avoid any skin contact).

Volume:1 x 15 ml

Hazards
identification:
H290 May be corrosive to metals.
H314 Causes severe skin burns and eye damage.

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3.2 Reagents necessary not supplied
Distilled water
3.3 Auxiliary materials and instrumentation
Automatic dispenser
Microplate reader (450 nm, 620-630 nm)
Note
Store all reagents at 2 °C - 8 °C in the dark.
Open the bag of Coated Microplate only when it is at room temperature and close immediately after use.
Once opened, the microplate is stable until expiry date of the kit.
4. WARNINGS--This kit is intended for research use only.
Use appropriate personal protective equipment while working with the reagents provided.
Follow Good Laboratory Practice (GLP) for handling blood products.
Material of animal origin used in the preparation of the kit has been obtained from animals certified ashealthy and the bovine protein has been obtained from countries not infected by BSE, but these materials
should be handled as potentially infectious
Some reagents contain small amounts of Proclin 300 as preservative. Avoid the contact with skin ormucosa.
The TMB Substrate contains an irritant, which may be harmful if inhaled, ingested or absorbed throughthe skin. To prevent injury, avoid inhalation, ingestion or contact with skin and eyes.
The Stop Solution consists of a diluted sulphuric acid solution. Sulphuric acid is poisonous and corrosiveand can be toxic if ingested. To prevent chemical burns, avoid contact with skin and eyes.
Avoid the exposure of reagent TMB/H2O2 to directed sunlight, metals or oxidants. Do not freeze thesolution.
This method allows the determination of Cortisol from 0.47 ng/ml to 200 ng/ml.

5. PRECAUTIONS-----

Please adhere strictly to the sequence of pipetting steps provided in this protocol.
The performance data represented here were obtained using specific reagents listed in this Instruction forUse.
All reagents should be stored refrigerated at 2 °C - 8 °C in their original container. Any exceptions areclearly indicated. The reagents are stable until the expiry date when stored and handled as indicated.
Allow all kit components and specimens to reach room temperature (22 °C - 28 °C) and mix well prior touse.
Do not interchange kit components from different lots. The expiry date printed on box and vials labels
must be observed. Do not use any kit component beyond their expiry date.
If you use automated equipment, the user has the responsibility to make sure that the kit has been
appropriately tested.
The incomplete or inaccurate liquid removal from the wells could influence the assay precision and/orincrease the background.
To improve the performance of the kit on automatic systems it is recommended to increase the numberof washes.
It is important that the time of reaction in each well is held constant for reproducible results. Pipetting ofsamples should not extend beyond ten minutes to avoid assay drift. If more than 10 minutes are needed,follow the same order of dispensation. If more than one plate is used, it is recommended to repeat thedose response curve in each plate
Addition of the TMB Substrate solution initiates a kinetic reaction, which is terminated by the addition ofthe Stop Solution. Therefore, the TMB Substrate and the Stop Solution should be added in the same
sequence to eliminate any time deviation during the reaction.
Observe the guidelines for performing quality control in medical laboratories by assaying controls
samples.
Maximum precision is required for reconstitution and dispensation of reagents.
Plate readers measure vertically. Do not touch the bottom of the wells.

6. STORAGE AND STABILITYStore the at 2 - 8 °C; the kit is stable until the expiry date claimed on the kit label and the QC-Report.
Do not use the kit or its components after the expiry date.
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7. PROCEDURE
7.1 Preparation of the Standard and Controls
Before use, leave 5 minutes on a rotating mixer.
The standards are ready to use and have the following concentration of Cortisol:ng/mlStandard A
0Standard B
1Standard C
5Standard D
30Standard E
200

The Controls are ready to use.
Once opened the standards and controls are stable 6 months at 2 °C - 8 °C.
7.2 Preparation of Conjugate
The Conjugate is ready to use.
Once opened, it stable 6 months at 2 °C - 8 °C.
7.3 Preparation of the Sample
The determination of Cortisol with this kit should be performed in urine samples.
Important note: The kit has been designed to be used on untreated urine samples; acidification
treatments of the urine that lead the pH to values below 5.0 could interfere with the assay and produce
aberrant results.
It is not necessary to dilute the sample.
The total volume of urine excreted during 24 hours should be collected and mixed in a single container.
Urine samples which are not to be assayed immediately should be stored at 2 °C - 8 °C or at -20 °C for
longer periods (maximum 6 months).
Samples with concentration greater than 200 ng/ml have not to be diluted; such samples have to bereported as "> 200 ng/ml".
7.4 Preparation of Wash Solution
Dilute the content of each vial of the “Wash Solution" 10X concentrate with distilled water to a final volumeof 500 ml prior to use.
For smaller volumes respect the 1:10 dilution ratio.
The diluted wash solution is stable for 30 days at 2 °C - 8 °C.
In concentrated wash solution is possible to observe the presence of crystals; in this case mix at room
temperature until the complete dissolution of crystals; for greater accuracy, dilute the whole bottle ofconcentrated wash solution to 500 ml, taking care to transfer completely the crystals, then mix until crystals
are completely dissolved.

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7.5 Procedure
Allow all reagents to reach room temperature (22 °C - 28 °C) for at least 30 minutes.
Unused coated microwell strips should be released securely in the foil pouch containing desiccant and stored
at 2 °C - 8 °C.
To avoid potential microbial and/or chemical contamination, unused reagents should never be transferred
into the original vials.
As it is necessary to perform the determination in duplicate in order to improve accuracy of the test results,
prepare two wells for each point of the standard curve (A - E), two for each Control, two for each sample,
one for Blank.
Reagent
Standard A - E

Standards

Samples/Controls

10 µl

Samples/Controls
ConjugateBlank

10 µl
300 µl300 µl

Incubate at 37 °C for 1 hour.
Remove the contents from each well; wash the wells 3 times with 350 µl of diluted wash solution.
Important note: during each washing step, gently shake the plate for 5 seconds and remove excess
solution by tapping the inverted plate on an absorbent paper towel.
Automatic washer: in case you use an automatic washer, it is advised to do 6 washing steps.
Substrate Solution

100 µl100 µl100 µlIncubate at room temperature (22 °C – 28 °C) for 15 minutes in the dark
Stop Solution

100 µl100 µl100 µlShake the microplate gently. Read the absorbance (E) at 450 nm against a reference wavelength of620 - 630 nm or against Blank within 5 minutes.
8. QUALITY CONTROL
Each laboratory should assay controls at normal, high and low levels range of Urinary Cortisol for monitoring
assay performance. These controls should be treated as unknowns and values determined in every test
procedure performed. Quality control charts should be maintained to follow the performance of the supplied
reagents. Pertinent statistical methods should be employed to ascertain trends. The individual laboratory
should set acceptable assay performance limits. Other parameters that should be monitored include the 80,
50 and 20% intercepts of the standard curve for run-to-run reproducibility. In addition, maximum
absorbance should be consistent with past experience. Significant deviation from established performancecan indicate unnoticed change in experimental conditions or degradation of kit reagents. Fresh reagentsshould be used to determine the reason for the variations.
9. RESULTS
9.1 Mean AbsorbanceCalculate the mean of the absorbance (Em) for each point of the standard curve and of each sample.
9.2 Standard Curve
Plot the values of absorbance (Em) of the standards (A - E) against concentration. Draw the best-fit curvethrough the plotted points (e.g.: Four Parameter Logistic).
9.3 Calculation of Results
Interpolate the values of the samples on the standard curve to obtain the corresponding values of theconcentrations expressed in ng/ml.
To calculate the cortisol concentration in urine, calculate as above and correct for total volume of volume ofurine collected in 24 hours:
ng/ml x Vol (ml) urine 24 h / 1000 = µg Cortisol/24

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10.

REFERENCE VALUES

To determine the normal range for urine samples, 128 apparently healthy male and female adults weretested.
Result:Normal range urine (24 h)
1.5 µg/24h - 63 µg/24h
11. PERFORMANCE AND CHARACTERISTICS
11.1 Analytical Sensitivity
Analytical Sensitivity was investigated through the LOB (white limit) the LOD (detection limit), the LOQ(quantification limit) and the anal sensitivity (A.S.).
The following table shows the criteria of the study and the results obtained.
LOBLOD
LOQ
A.S.

Criteria
60 replicates of Standard A, used as “Blank” have been investigated in
5 different sessions over 3 days
6 urine samples with low cortisol concentration have been investigate
over 10 assays in duplicate, performed in 5 days.
6 urine samples with low cortisol concentration have been investigate
over 10 assays in duplicate, performed in 5 days
20 replicates of Standard A and replicates Standard B have beenassayed.
A.S has been calculated by linear regression.

Results (ng/ml)
0.280.47
0.56
0.22

11.2 Precision and reproducibility (complex precision)
Precision and reproducibility have been assessed through 6 different urine samples with different
concentration of Cortisol.
The table below shows the Within Run and Total CV%.
SamplePS 2
PS 4
CT High
PS 5
PS 6
CT Low

n20
2020
20
2020

Mean (ng/ml)
112.14164.56350.57725.8789.269
3.438

Within Run CV%
6.6 %
8.1 %
7.3 %
7.6 %
7.6 %
7.0 %

Total CV%
12 %
12 %
11 %
10 %
11 %
9%

11.3 Analytical specificity
Interference for Albumin, Acetylsalicylic Acid, Ibuprofen and Ascorbic Acid were studied by adding theinterfering substance to the urine sample with a low and high Cortisol concentration, and by comparing its
concentration to the unspiked sample.
The interference has been evaluated as "significant" if it causes a concentration bias > 10% between spikedand unspiked sample.
The following table shows the results obtained:
Substance
AlbuminAcetylsalicylic acidIbuprofen
Ascorbic Acid

Concentration
5 mg/dl
3.62 mmol/l
2.42 mmol/l
5 mg/lInterferenceNoNoNoNoConclusion: no interference has been found for Albumin, Acetylsalicylic Acid, Ibuprofen and Ascorbic Acid.
11.4 Correlation
137 urine samples were tested with the Cortisol Urine ELISA kit and with a LC-MS method (reference)
The linear regression curve is:y = 1,008x - 0.5019
r2 = 0.83
12. WASTE MANAGEMENT
Reagents must be disposed off in accordance with local regulations.
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13. BIBLIOGRAPHY
1. Foster, L. B. and Dunn, R. T. Clin. Chem. 20/3 365(1974)
2. De Lacerda, et al J. Clin. Endocr. and Metab. 36,227 (1973)
3. Rolleri, E., et al Clin. Chim. Acta 66 319 (1976)
4. Kobayashi, Y., et al Steroids, 32 no 1(1978)
5. Akarawa, et al Anal. Biochem. 97 248 (1979)
14. TROUBLESHOOTINGERRORS / POSSIBLE CAUSES / SUGGESTIONS
No colorimetric reaction- no conjugate pipetted- contamination of conjugates and/or of substrate- errors in performing the assay procedure (e.g. accidental pipetting of reagents in a wrong sequence orfrom the wrong vial, etc.)
Too low reaction (too low ODs)
- incorrect conjugate (e.g. not from original kit)
- incubation time too short, incubation temperature too low
Too high reaction (too high ODs)
- incorrect conjugate (e.g. not from original kit)
- incubation time too long, incubation temperature too high- water quality for wash buffer insufficient (low grade of deionization)- insufficient washing (conjugates not properly removed)
Unexplainable outliers- contamination of pipettes, tips or containers- insufficient washing (conjugates not properly removed)
too high within-run CV%- reagents and/or strips not pre-warmed to room temperature prior to use- plate washer is not washing correctly (suggestion: clean washer head)
too high between-run CV %- incubation conditions not constant (time, temperature)- controls and samples not dispensed at the same time (with the same intervals) (check pipetting order)- person-related variation

Symbols:

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Storagetemperature

Manufacturer

Expiry date

Batch code

Consult instructions
for useContentCaution

Catalogue
numberEffective: 2019-05-16

Contains sufficient for<n> tests

For research use
only!
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