Instructions for use

Corticosterone ELISAMS E-5400R

Corticosterone EILSA
1 INTRODUCTION
1.1 Intended Use
The Corticosterone ELISA is an enzyme immunoassay for the quantitative measurement of Corticosterone inserum or plasma (lithium heparin or citrate plasma).
2 PRINCIPLE OF THE TESTThe Corticosterone ELISA is a solid phase enzyme-linked immunosorbent assay (ELISA), based on theprinciple of competitive binding.
The microtiter wells are coated with a polyclonal antibody directed towards a unique antigenic site of thecorticosterone molecule. During the first incubation, the corticosterone in the added sample competes for thebinding to the coated antibody with the added conjugate molecule, which is a Corticosterone molecule
conjugated to a horse-radish peroxidase. After incubation the unbound conjugate and sample are washed off.
Following the addition of the substrate solution, the intensity of colour developed is inversely proportional to theconcentration of corticosterone in the sample. After incubation of substrate solution, the colorimetric reaction isabruptly stopped by adding stop solution.
WARNINGS AND PRECAUTIONS
1.
2.

This kit is for research use only.
All reagents of this test kit which contain human serum or plasma have been tested and confirmed
negative for HIV I/II, HBsAg and HCV by FDA approved procedures. All reagents, however, should betreated as potential biohazards in use and for disposal.
3. Before starting the assay, read the instructions completely and carefully. Use the valid version ofinstructions for use provided with the kit. Be sure that everything is understood.
4. The microplate contains snap-off strips. Unused wells must be stored at 2 °C to 8 °C in the sealed foil
pouch and used in the frame provided.
5. Pipetting of samples and reagents must be done as quickly as possible and in the same sequence for eachstep.
6. Use reservoirs only for single reagents. This especially applies to the substrate reservoirs. Using areservoir for dispensing a substrate solution that had previously been used for the conjugate solution may
turn solution colored. Do not pour reagents back into vials as reagent contamination may occur.
7. Mix the contents of the microplate wells thoroughly to ensure good test results. Do not reuse microwells.
8. Do not let wells dry during assay; add reagents immediately after completing the rinsing steps.
9. Allow the reagents to reach room temperature (21 °C to 26 °C) before starting the test. Temperature willaffect the absorbance readings of the assay. However, values for the samples will not be affected.
10. Never pipet by mouth and avoid contact of reagents and specimens with skin and mucous membranes.
11. Do not smoke, eat, drink or apply cosmetics in areas where specimens or kit reagents are handled.
12. Wear disposable latex gloves when handling specimens and reagents. Microbial contamination of reagentsor specimens may give false results.
13. Handling should be done in accordance with the procedures defined by an appropriate national biohazard
safety guideline or regulation.
14. Do not use reagents beyond expiry date as shown on the kit labels.
15. All indicated volumes have to be performed according to the protocol. Optimal test results are only
obtained when using calibrated pipettes and microtiter plate readers.
16. Do not mix or use components from kits with different lot numbers. It is advised not to exchange wells ofdifferent plates even of the same lot. The kits may have been shipped or stored under different conditions
and the binding characteristics of the plates may result slightly different.
17. Avoid contact with Stop Solution containing 0.5 M H2SO4. It may cause skin irritation and burns.
18. Some reagents contain Proclin 300, BND and/or MIT as preservatives. In case of contact with eyes or skin,flush immediately with water.
19. TMB substrate has an irritant effect on skin and mucosa. In case of possible contact, wash eyes with anabundant volume of water and skin with soap and abundant water. Wash contaminated objects before
reusing them. If inhaled, take the person to open air.
20. Chemicals and prepared or used reagents have to be treated as hazardous waste according to the national
biohazard safety guideline or regulation.
21. For information on hazardous substances included in the kit please refer to Safety Data Sheets.
Safety Data Sheets for this product are available upon request.

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4 REAGENTS
4.1 Reagents provided
MS E-5431
Content:

Microtiterwells
12x8 (break apart) strips, 96 wells; Wells coated with an anti-Corticosterone antibody(polyclonal).

Standards and Controls - ready to use
Cat. no.

ComponentStandard

Concentration nmol/lVolume / vial

MS E-5402
MS E-5403
MS E-5404
MS E-5405
MS E-5406
MS E-5407

Standard A
Standard B
Standard C
Standard D
Standard E
Standard F
Standard G

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

MS E-5151

Control 1

MS E-5152

Control 205
153060120240
For control values andranges please refer to viallabel or QC-Datasheet.

MS E-5401

Conversion:

1 nmol/l

1 ml
1 ml= 34.646 ng/dl= 0.34646 ng/ml
The standards are calibrated against the following reference material: Cerilliant, code C-117
Contain non-mercury preservative.
Enzyme Conjugate - 250X concentrate

MS E-5440
Content:

Corticosterone conjugated to horseradish peroxidase; see „Reagent Preparation“.

Volume:1 x 150 µl
Conjugate Diluent - ready to use

MS E-5461
Content:

Contains non-mercury preservative.

Volume:1 x 25 ml
Substrate Solution - ready to use

SA E-0055
Content:

Tetramethylbenzidine (TMB)

Volume:1 x 25 ml
Stop Solution - ready to use

FR E-0080
Content:

contains 0.5 M H2SO4.
Avoid contact with the stop solution. It may cause skin irritations and burns.

Volume:1 x 14 ml

Hazards
identification:
H290 May be corrosive to metals.
H314 Causes severe skin burns and eye damage.
FR E-0030
Volume:Wash Solution - 40X concentrated
1 x 30 ml
see „ Reagent Preparation “Note: Additional Standard A for sample dilution is available upon request.
4.2 Materials required but not provided

A calibrated microtiter plate reader (450 nm, with reference wavelength at 620 nm – 630 nm)
Calibrated variable precision micropipettes
Absorbent paper
Distilled waterTimer
Graph paper or software for data reduction

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4.3 Storage Conditions
When stored at 2°C to 8°C unopened reagents will retain reactivity until expiration date. Do not use reagents
beyond this date.
Opened reagents must be stored at 2°C to 8°C. Microtiter wells must be stored at 2°C to 8°C. Once the foil
bag has been opened, care should be taken to close it tightly again.
Opened kits retain activity for 8 weeks if stored as described above.
4.4 Reagent Preparation
Bring all reagents and required number of strips to room temperature prior to use.
Wash Solution
Add deionized water to the 40X concentrated Wash Solution.
Dilute 30 ml of concentrated Wash Solution with 1170 ml distilled water to a final volume of 1200 ml.
The diluted Wash Solution is stable for 2 weeks at room temperature.
Enzyme Conjugate
Dilute Enzyme Conjugate concentrate 1 + 250 in Conjugate Diluent.
This solution should be prepared freshly.
If the whole plate is used, dilute 100 µl Enzyme Conjugate with 25 ml Conjugate Diluent.
If the whole plate is not used at once prepare only the required quantity of Enzyme Conjugate.
4.5 Disposal of the Kit
The disposal of the kit and all used materials/reagents must be made according to the national regulations.
Special information for this product is given in the Safety Data Sheet, section 13.
4.6 Damaged Test Kits
In case of any damage to the test kit or components, the manufacturer must be informed in writing, at thelatest one week after receiving the kit. Damaged single components should not be used for a test run. They
have to be stored until a final solution has been found. After this, they should be disposed according to theofficial regulations.
5 SPECIMEN COLLECTION AND PREPARATION
Serum or plasma (lithium heparin or citrate plasma) can be used in this assay.
Note: Samples containing sodium azide should not be used in the assay.
In general it should be avoided to use haemolytic, icteric or lipaemic specimens. For further information refer tochapter “Interfering Substances”.
5.1 Specimen Collection
Serum: Collect blood by venipuncture (e.g. Sarstedt Monovette for serum), allow to clot, and separate serumby centrifugation at room temperature. Do not centrifuge before complete clotting has occurred. Donors
receiving anticoagulant therapy may require increased clotting time.
Plasma: Whole blood should be collected into centrifuge tubes containing anti-coagulant (e.g. Sarstedt
Monovette with the appropiate plasma preparation) and centrifuged immediately after collection.
5.2 Specimen Storage and Preparation
Specimens should be capped and may be stored for up to 2 days at 2 °C to 8 °C prior to assaying.
Specimens held for a longer time (up to 15 months) should be frozen only once at -20 °C prior to assay.
Thawed samples should be inverted several times prior to testing.
5.3 Specimen Dilution
If in an initial assay, a specimen is found to contain more than the highest standard, the specimen can bediluted with Standard A and re-assayed as described in “Assay Procedure”.
For the calculation of the concentrations this dilution factor has to be taken into account.
Example:a) Dilution 1:10: 10 µl Serum + 90 µl Standard A (mix thoroughly)b) Dilution 1:100: 10 µl dilution a) 1:10 + 90 µl Standard A (mix thoroughly).

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6 ASSAY PROCEDURE
6.1 General Remarks

All reagents and specimens must be allowed to come to room temperature before use. All reagents must bemixed without foaming.
Once the test has been started, all steps should be completed without interruption.
Use new disposal plastic pipette tips for each standard, control or sample in order to avoid crosscontamination.
Absorbance is a function of the incubation time and temperature. Before starting the assay, it isrecommended that all reagents are ready, caps removed, all needed wells secured in holder, etc. This will
ensure equal elapsed time for each pipetting step without interruption.
As a general rule the enzymatic reaction is linearly proportional to time and temperature.

6.2 Test Procedure
Each run must include a standard curve.
1.

Secure the desired number of Microtiter wells in the frame holder.

2.

Dispense 20 µl of each Standard, Control and samples with new disposable tips into appropriatewells.

3.

Dispense 200 µl Enzyme Conjugate into each well.
Thoroughly mix for 10 seconds. It is important to have a complete mixing in this step.

4.

Incubate for 60 minutes at room temperature.

5.

Briskly shake out the contents of the wells.
Rinse the wells 3 times with 400 µl diluted Wash Solution per well, if a plate washer is used – or –rinse the wells 3 times with 300 µl diluted Wash Solution per well for manual washing.
Strike the wells sharply on absorbent paper to remove residual droplets.
Important note:
The sensitivity and precision of this assay is markedly influenced by the correct performance of thewashing procedure!

6.

Add 100 µl of Substrate Solution to each well.

7.

Incubate for 15 minutes at room temperature.

8.

Stop the enzymatic reaction by adding 50 µl of Stop Solution to each well.

9.

Determine the absorbance (OD) of the solution in each well at 450 nm (reading) and at 620 - 630 nm(background subtraction, recommended) with a microtiter plate reader.
It is recommended that the wells be read within 10 minutes after adding the Stop Solution.

6.3 Calculation of Results
1. Calculate the average absorbance values for each set of standards, controls and samples.
2. Using semi logarithmic graph paper, construct a standard curve by plotting the mean absorbance obtained
from each standard against its concentration with absorbance value on the vertical (Y) axis andconcentration on the horizontal (X) axis.
3. Using the mean absorbance value for each sample determine the corresponding concentration from thestandard curve.
4. Automated method: The results in the Instructions for Use have been calculated automatically using a 4Parameter curve fit (4 Parameter Rodboard or 4 Parameter Marquardt are the preferred methods.) Other
data reduction functions may give slightly different results.
5. The concentration of the samples can be read directly from this standard curve. Samples with concentrations
higher than that of the highest standard have to be further diluted or reported as > 240 nmol/l. For thecalculation of the concentrations this dilution factor has to be taken into account.
6.3.1 Example of Typical Standard Curve
The following data is for demonstration only and cannot be used in place of data generations at the time ofassay.
Standard

Optical Units (450 nm)

Standard A (0 nmol/l)Standard B (5 nmol/l)Standard C (15 nmol/l)Standard D (30 nmol/l)Standard E (60 nmol/l)Standard F (120 nmol/l)Standard G (240 nmol/l)

2.31
1.69
1.35
1.10
0.87
0.63
0.48

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7 EXPECTED NORMAL VALUES
It is strongly recommended that each laboratory should determine its own normal and abnormal values.
In a study conducted with apparently normal healthy adults, using the Corticosterone ELISA the following data
are observed:
PopulationnMean(nmol/l)Median(nmol/l)

5th - 95th Percentile(nmol/l)

Males

62

14.4

11.7

5.0 - 32.7

3.6 - 36.9

2.78 - 52.0

Females

64

11.7

9.4

3.0 - 29.6

2.1 - 41.7

1.6 - 50.0

Total

126

13.0

10.3

3.4 - 32.5

2.8 - 40.8

1.6 - 52.0

2.5th - 97.5th Percentile Range (min. - max.)(nmol/l)(nmol/l)The values in ng/dl were calculated by multiplying the measured values (in nmol/l) by 34.646 as described inchapter 4.1.
PopulationnMean(ng/dl)

Median(ng/dl)

5th - 95th Percentile(ng/dl)Males

62494.9

402.0

173.7 - 1128.3

123.2 - 1272.6

95.5 - 1791.4

Females

64

401.5

322.2

104.9 - 1019.6

71.6 - 1436.9

55.1 - 1724.2

Total

126

447.4

355.0

117.1 - 1118.3

96.5 - 1405.7

55.1 - 1791.4

2.5th - 97.5th Percentile Range (min. - max.)(ng/dl)(ng/dl)

8 QUALITY CONTROLGood laboratory practice requires that controls be run with each standard curve. A statistically significant
number of controls should be assayed to establish mean values and acceptable ranges to assure properperformance.
It is recommended to use control samples according to state and federal regulations. The use of control
samples is advised to assure the day to day validity of results. Use controls at both normal and pathologicallevels.
The controls and the corresponding results of the QC-Laboratory are stated in the QC certificate added to thekit. The values and ranges stated on the QC sheet always refer to the current kit lot and should be used for
direct comparison of the results.
It is also recommended to make use of national or international Quality Assessment programs in order toensure the accuracy of the results.
Employ appropriate statistical methods for analysing control values and trends. If the results of the assay do
not fit to the established acceptable ranges of control materials sample results should be considered invalid.
In this case, please check the following technical areas: Pipetting and timing devices; photometer, expiration
dates of reagents, storage and incubation conditions, aspiration and washing methods.
After checking the above mentioned items without finding any error contact your distributor or themanufacturer directly.

9 PERFORMANCE CHARACTERISTICS
9.1 Assay Dynamic Range
The range of the assay is between 1.68 – 240.0 nmol/l.
9.2 Specificity of Antibodies (Cross-Reactivity)
The following substances were tested for cross-reactivity of the assay:
Component
Corticosterone
Progesterone
Deoxycorticosterone
11-Dehydrocorticosterone
Cortisol
Pregnenolone
Other steroids

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Cross reactivity
100 %
7.4 %
3.4 %
1.6 %
0.3 %
0.3 %
<0.1 %

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9.3 Sensitivity
The analytical sensitivity of the Corticosterone ELISA was calculated by subtracting 2 standard deviations fromthe mean of 20 replicate analyses of Standard A and was found to be 0.589 nmol/l.
The Limit of Blank (LoB) is 0.527 nmol/l.
The Limit of Detection (LoD) is 1.680 nmol/l.
The Limit of Quantification (LoQ) is 4.462 nmol/l.
9.4 Reproducibility
9.4.1 Intra Assay
The within assay variability was determined by measuring each sample 10 times per run (n = 10):Sample
nMean (nmol/l)
CV (%)
1106.8
7.7
210
62.3
3.8
310
122.5
3.3
410
160.0
5.89.4.2 Inter Assay
The between assay variability was determine by measuring each sample 10 times per run for 3 days (n = 30):
SamplenMean (nmol/l)
CV (%)
130
7.0
10.8
230
63.0
6.1
330
115.6
6.4
430
153.9
5.0
9.4.3 Inter-Lot
The inter-assay (between-lots) variation was determined by measuring each sample 6 times with 3 different kit
lots (n = 18):
SamplenMean (nmol/l)
CV (%)
118

7.2

3.4

218

55.6

13.7

318

61.7

1.3

418

159.4

4.3

9.5 Recovery
Samples have been spiked by adding corticosterone solutions with known concentrations.
The recovery (%) was calculated by multiplying the ratio of measured and expected values with 100.
Sample 1

Sample 2

Sample 3

Sample 4

Concentration (nmol/l)

48.2

66.8

115.0

118.5

Average Recovery (%)

112.1

108.6

109.2

107.9

from

110.1

107.3

103.6

99.0

to113.8

110.5

114.2

114.9

Range of Recovery (%)
9.6 Linearity

Samples were measured undiluted and in serial dilutions with Standard A. The recovery (%) was calculated bymultiplying the ratio of expected and measured values with 100.
Sample 1

Sample 2

Sample 3

Sample 4

Concentration (nmol/l)

49.4

117.3

147.4

238.9

Average Recovery (%)

101.1

104.6

97.4

106.0

from96.2

100.6

91.8

95.0

to108.9

111.3

100.6

114.9

Range of Recovery (%)

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10 LIMITATIONS OF USE
Reliable and reproducible results will be obtained when the assay procedure is performed with a complete
understanding of the package insert instruction and with adherence to good laboratory practice.
Any improper handling of samples or modification of this test might influence the results.
10.1 Interfering Substances
Haemoglobin (up to 1 mg/ml), Bilirubin (up to 0.5 mg/ml) and Triglyceride (up to 7.5 mg/ml) have no influenceon the assay results.
10.2 Drug Interferences
Until today no substances (drugs) are known to us, which have an influence to the measurement ofcorticosterone in a sample.
10.3 High-Dose-Hook Effect
A High-Dose-Hook Effect is not known for competitive assays.
11 REFERENCES/LITERATURE
1. Hupe, J.M., et al, Nature, 1998, 394, 784-787.
2. Kitaysky A.S., et al, J. Comp. Physiol, 2001, 171, 701-709.
3. Thellin O, Noel G, Khuana S, Ogle CK and Horseman N, Shock, 2001, 16(5), 393-397.
4. Krame, K.M., Sothern R.B., Chronobiol. Int., 2001, 18(6), 933-945.
5. Vazquez-Palacios G, et al, Pharmacol. Biochem Behavior, 2001, 70(2-3), 305-310.
6. Rød A, et al, Scientific Reports, 2017, 7 (6748)

Symbols:

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Manufacturer

Expiry date

Batch code

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for useContentCaution

Catalogue
numberEffective 2020-02-12

Contains sufficient for<n> tests

For research use
only!
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