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Instructions for use

Corticosterone rat/mouse ELISAAR E-8100

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Corticosterone rat/mouse ELISA
1. INTRODUCTION
1.1 Intended Use
The Corticosterone rat/mouse ELISA is a competitive immunoassay for the measurement ofcorticosterone in rat and mouse serum or plasma. For research use only. Not for use in diagnostic
procedures.

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1.2 Summary and Explanation
Corticosterone is secreted by the adrenal cortex under control of the pituitary hormone ACTH via a negative
feedback mechanism. It is the most abundant circulating steroid in rats, since rodents are not able tosynthesize Cortisol, the major glucocorticoid in human, as a result of lacking the enzyme C17-Hydroxylase.
Corticosterone has a wide range of activities in rodents. It regulates carbohydrate, protein and fat
metabolism. It has also an influence on the hemopoietic system and reduces the total number oflymphocytes and eosinophils, but to a lesser extent than cortisol. In contrast to cortisol, corticosterone has
only minimal anti-inflammatory activity.
Corticosterone level in nocturnal animals like rats exhibit a distinct circadian variation with peak values in
the latter portion of the day, followed by a nadir in the morning (1) and is believed to play an important rolein sleep-wake cyclus (2). This is in contrast to diurnal mammals, where peak concentrations of
glucocorticoids are found in the morning. Enhanced corticosterone release by female compared to male rats
under basal and stress conditions has been observed (6).
Determination of corticosterone in rats is of interest to facilities conducting neurophysiological research, toacademic institutions and to pharmaceutical companies with drug research departments. Drugs that
influence the endocrine system can increase or reduce corticosteroid production in the adrenal cortex. Rat
serum corticosterone is therefore an ideal indicator of the side effects of a potential therapeutic agent. The
same constellations of effects seen in rats are generally seen in human. Plasma corticosterone in rats isoften used in connection with ACTH measurement as a stress indicator (3, 4). The effects of chronic stresson the function of the hypothalamic-pituitary-adrenocortical system are age-dependent. Recent studies
suggest that aging increases basal but not stress induced levels of corticosterone in the brain (5).

Inst

2. PRINCIPLE

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The Corticosterone rat/mouse ELISA Kit is a solid phase enzyme-linked immunosorbent assay (ELISA),
based on the principle of competitive binding. An unknown amount of corticosterone present in the sample
and a defined amount of corticosterone conjugated to horseradish peroxidase compete for the binding sitesof corticosterone antiserum coated to the wells of a microplate. After incubation on a shaker the microplateis washed four times. After addition of the substrate solution the concentration of corticosterone is inversely
proportional to the optical density measured.

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3. WARNINGS AND PRECAUTIONSThis kit is for research use only. Not for use in diagnostic procedures.

2.

Before starting the assay, read the instructions completely and carefully. Use the valid version of the
package insert provided with the kit. Be sure that everything is understood.

3.

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.

4.

Pipetting of samples and reagents must be done as quickly as possible and in the same sequence for each
step.

5.

Use reservoirs only for single reagents. This especially applies to the substrate reservoirs. Using a reservoir
for dispensing a substrate solution that had previously been used for the conjugate solution may turnsolution colored. Do not pour reagents back into vials as reagent contamination may occur.
Mix the contents of the microplate wells thoroughly to ensure good test results. Do not reuse microwells.

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1.

7.

Do not let wells dry during assay; add reagents immediately after completing the rinsing steps.

8.

Allow the reagents to reach room temperature (21 – 26 °C) before starting the test. Temperature will affectthe absorbance readings of the assay. However, values for the samples will not be affected.

9.

Never pipet by mouth and avoid contact of reagents and specimens with skin and mucous membranes.

10. Do not smoke, eat, drink or apply cosmetics in areas where specimens or kit reagents are handled.
11. Wear disposable latex gloves when handling specimens and reagents. Microbial contamination of reagentsor specimens may give false results.
12. Handling should be done in accordance with the procedures defined by an appropriate national biohazard
safety guideline or regulation.
13. Do not use reagents beyond expiry date as shown on the kit labels.
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14. All indicated volumes have to be performed according to the protocol. Optimal test results are only obtained
when using calibrated pipettes and microtiterplate readers.
15. 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.
16. Avoid contact with Stop Solution. It may cause skin irritation and burns.
17. Chemicals and prepared or used reagents have to be treated as hazardous waste according to the national
biohazard safety guideline or regulation.
18. For information please refer to Material Safety Data Sheets. Safety Data Sheets for this product areavailable upon request directly from the manufacturer.

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4. REAGENTSw
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Microtiterplate - Ready to use

AR E-8131

12 x 8 (break apart) strips with 96 wells; wells coated with anti-corticosterone
antibody

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4.1 Reagents Provided

Standards

Concentration

AR E-8101

Standard A

0 ng/ml

AR E-8102

Standard B

15 ng/ml

0.3 mlAR E-8103

Standard C

50 ng/ml

0.3 mlAR E-8104

Standard D

185 ng/ml

0.3 mlAR E-8105

Standard E

AR E-8106

Standard F

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Volume/Vial
0.3 ml0.3 ml2250 ng/ml

0.3 mlstru
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640 ng/ml

Incubation Buffer - Ready to use

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AR E-8113

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1 x 11 mloft

Volume:ov

Standards - ready to use.
Cat. no.
SymbolEnzyme Conjugate - Ready to use

AR E-8140

Corticosterone conjugated to horseradish peroxidise.

Volume:1 x 7 ml

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Content:

Substrate Solution - Ready to use

AR E-0055

contains tetramethylbenzidine (TMB) and hydrogen peroxide in a buffered matrix.

Volume:1 x 22 ml

Stop Solution - Ready to use

Content:

contains 2 N Hydrochloric Acid solution.

only

AR E-0080

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Content:

Volume:1 x 7 ml

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Hazards
identification:

H290 May be corrosive to metals.
H314 Cause severe skin burns and eye damage.
H335 May cause respiratory irritation.
Wash Solution - 10 x concentrated

AR E-0030
Volume:1 x 50 ml
See “Preparation of Reagents”

Note: Additional Standard A for sample dilution is available upon request.

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4.2 Materials Required but not Provided

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A microtiter plate reader capable for endpoint measurement at 450 nm
Microplate mixer operating more than 600 rpm
Calibrated variable precision micropipettes (10 µl, 50 µl, 100 µl, 200 µl).
Absorbent paper
Distilled or deionized water
Timer
Semi logarithmic graph paper or software for data reduction

4.3 Reagent Preparation
All reagents should be at room temperature before use.

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Wash Solution:
Dilute 50 ml of 10X concentrated Wash Solution with 450 ml deionized water to a final volume of 500 ml.
The diluted Wash Solution is stable for at least 3 months at room temperature.

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4.4 Storage Conditions

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When stored at 2°C to 8°C unopened reagents will be stable until expiration date. Do not use reagents
beyond this date. Opened reagents must be stored at 2°-8°C. After first opening the reagents are stable for
30 days if used and stored properly.
Microtiter wells must be stored at 2°C to 8°C. Take care that the foil bag is sealed tightly.

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4.5 Disposal of the Kits

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The disposal of the kit must be made according to the national regulations. Special information for thisproduct is given in the Material Safety Data Sheet.

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4.6 Damaged Test Kits

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In case of any severe damage of the test kit or components, the manufactuer has to be informed written,
latest one week after receiving the kit. Severely 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 the official regulations.

he5. SAMPLE

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For determination of Corticosterone rat/mouse serum and plasma can be used. The procedure calls for
10 µl matrix per well. The samples should assay immediately or aliquot and stored at -20°C. Avoid repeatedfreeze-thaw cycles. Samples expected to contain rat/mouse Corticosterone concentrations higher than thehighest standard (2250 ng/ml) should be diluted with the Standard A before assay. The additional dilution
step has to be taken into account for the calculation of the results.

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All reagents and samples must be allowed to come to room temperature before use. All reagents must be
mixed without foaming.
Once the test has been started, all steps should be completed without interruption.
Use new disposal plastic pipette tips for each standard and sample in order to avoid cross contamination.
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.
Respect the incubation times as stated in this instructions for use.
For internal quality control we suggest to use Rat Control Set (AR K-8000) For more information
please contact the manufacturer.

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6.1 General Remarkse

6. ASSAY PROCEDURE

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Please note: The use of plasma as specimen can result in a diminished precision of this assay.

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

Prepare a sufficient number of microplate wells to accommodate calibrators and samples in duplicates.

2.

Dispense 10 µl of each Standard, Sample and Control with new disposable tips into appropriate
wells

3.

Dispense 100 µl of Incubation Buffer into each well.

4.

Add 50 µl Enzyme Conjugate into each well5.

Incubate for 2 hours at room temperature on a microplate mixer (> 600 rpm)
Important Note:
Optimal reaction in this assay is markedly dependent on shaking of the microplate!
Discard the content of the wells and rinse the wells 4 times with diluted Wash Solution (300 µl perwell). Remove as much Wash Solution as possible by beating the microplate on absorbent paper.

7.

Add 200 µl of Substrate Solution to each well.

8.

Incubate without shaking for 30 minutes in the dark at room temperature.

9.

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

10.

Determine the absorbance of each well at 450 nm. It is recommended to read the wells within
15 minutes.

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6.

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6.3 Calculation of Results

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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 absorbanceobtained 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 IFU have been calculated automatically using a 4 PL (4 Parameter
Logistics) curve fit. 4 Parameter Logistics is the preferred calculation method. Other data reduction
functions may give slightly different results.
5. The concentration of the samples can be determined directly from this standard curve. Samples withconcentrations higher than that of the highest standard have to be further diluted. For the calculation of
the concentrations, this dilution factor has to be taken into account.

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Conversion to SI units:
Corticosterone (ng/ml) x 2.886 = nmol/l

6.3.1 Example of Typical Standard Curve

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Following data are intended for illustration only and should not be used to calculate results from another
run.

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Standard
Standard A(0 ng/ml)Standard B(15 ng/ml)
Standard C(50 ng/ml)
Standard D(185 ng/ml)
Standard E(640 ng/ml)
Standard F(2250 ng/ml)

Absorbance Units
3.004
2.817
2.505
1.620
0.723
0.297

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7. EXPECTED NORMAL VALUES
In order to determine the normal range of serum corticosterone in rat, samples of male and female rats
were collected in the morning (7.00 – 9.00 a.m.) as well as in the late afternoon (5.00 – 6.00 p.m.) andanalyzed using the Corticosterone rat/mouse ELISA kit. The following ranges are calculated with the resultsof this study.

Male rats ♂
Female rats ♀
n.d. non detectable

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Range (ng/ml)
Morning
n.d. – 11.4
53.9 – 332.1

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Range (ng/ml)
Late afternoon
172.6 – 245.4
292.5 – 819.0

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In further studies serum samples of 23 mice were collected between 11.00 a.m. and 2.00 p.m. und analyzed
in a similar manner.
Range (ng/ml)
Male mice ♂
47 – 159
It is recommended that each laboratory establish its own normal range since corticosterone levels can vary
due to handling and sampling techniques.
8. PERFORMANCE CHARACTERISTICS
8.1 Analytical Sensitivitykit

The lowest analytical detectable level of corticosterone that can be distinguished from the Standard A is 6.1
ng/ml at the 2SD confidence limit.

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8.2 SpecifictiyDehydroepiandrosterone
Estrone

<0.1

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12.5
<0.1

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11-Deoxycorticosterone

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2.3

Cortisold% Cross reaction
0.3

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Steroid
Aldosteronew
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The following materials have been evaluated for cross reactivity. The percentage indicates cross reactivity at
50% displacement compared to corticosterone.

<0.1

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Estradiol<0.1s17-Hydroxyprogesterone

ionProgesteroneruct

Testosterone
5α-Dihydrotestosterone

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<0.1

1.1

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Pregnenolone

<0.1

<0.1

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Androsterone

<0.1

<0.1

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Androstenedionest5α-Androstane

6.2

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8.3 REPRODUCIBILITY
8.3.1 Intra-Assay

Mean (ng/ml)

62.8

126.0

271.4

SD

5.6

9.2

16.0

CV (%)

8.9

7.3

5.9

n=

20

20

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The intra-assay variation was determined by 20 replicate measurements of three serum samples within one
run. The within-assay variability is shown below:

se8.3.2 Inter-AssayPlea

The inter-assay (between-run) variation was determined by duplicate measurements of three serum
samples.

Version: 7.0-c

Mean (ng/ml)

59.3

113.2

257.4

SD

4.3

9.3

19.4

CV (%)

7.2

8.2

7.5

n=

10

10

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8.4 Recovery
Using a steroid-free serum a spiking solution was prepared (5000 ng/mL). Aliquots of 20, 40, 60 and 80 µL,
respectively, were spiked into 480, 460, 440 µL and 420 µL of three rat serum pools leaving the serum
matrix of the spiked samples relatively intact. All samples were then measured by the Corticosterone
rat/mouse ELISA Procedure.

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./.
89%
103%
100%
./.
82%
95%
93%
./.
107%
120%
114%

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Recovery(%)

d2Expected(ng/mL)
./.
229.1
429.1
629.1
./.
322.5
522.5
722.5
./.
537.3
737.3
937.3

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Observed(ng/mL)
29.1
204.1
444.0
629.5
122.5
265.4
497.6
672.0
137.3
572.8
883.1
1068.5ov

Spiking(ng/mL)
200
400
600
200
400
600
400
600
800

Serum

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8.5 Linearity

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Expecteds(ng/mL)
./.
325,0
162,5
81,2
./.
202,9
101,4
50,7
./.
239,0
119,5
59,7
./.
207,8
103,9
51,9

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Linearity
(%)
./.
94%
97%
83%
./.
104%
107%
110%
./.
98%
90%
81%
./.
90%
77%
73%

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(ng/mL)
650,0
304,5
157,6
67,2
405,7
210,2
108,5
55,7
477,9
235,4
107,1
48,1
415,5
186,3
79,7
37,7

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native1 in 2
1 in 41 in 8native1 in 2
1 in 41 in 8native1 in 2
1 in 41 in 8native1 in 2
1 in 41 in 8ObservedionDilution

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Four native serum samples were assayed undiluted and diluted with the standard matrix.

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9. LIMITATIONS OF PROCEDURE

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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.

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9.1 Drug Interferences
Until now no substances (drugs) are known influencing the measurement of rat or mouse corticosterone inserum. Lipemic and haemolysed samples can cause false results.

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10. LEGAL ASPECTS
10.1 Reliability of Results
The test must be performed exactly as per the manufacturer’s instructions for use. Moreover the user muststrictly adhere to the rules of GLP (Good Laboratory Practice) or other applicable national standards and/or
laws. This is especially relevant for the use of control reagents. It is important to always include, within thetest procedure, a sufficient number of controls for validating the accuracy and precision of the test.
The test results are valid only if all controls are within the specified ranges and if all other test parameters
are also within the given assay specifications. In case of any doubt or concern please contact the
manufacturer.
10.2 Liability

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Any modification of the test kit and/or exchange or mixture of any components of different lots from one
test kit to another could negatively affect the intended results and validity of the overall test. Suchmodification and/or exchanges invalidate any claim for replacement.
Regardless, in the event of any claim, the manufacturer’s liability is not to exceed the value of the test kit.
Any damage caused to the test kit during transportation is not subject to the liability of the manufacturer.

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11. REFERENCES

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1. D´Agostino J, Vaeth GF & Henning SJ (1982): Diurnal rhythm of total and free concentration of serum
corticosterone in the rat. Acta Endocrinologica 100, Vol. 1, 85-90.
2. Vázquez-Palacios G et al. (2001): Further definition of the effect of corticosterone on the sleep-wakepattern in the male rat. Pharmacol. Biochem. Behav. 70: 305-310
3. De Souza EB & van Loon GR (1982): Stress induced inhibition of the plasma corticosterone response to a
subsequent stress in the rat: A nonadrenocorticotropin-mediated mechanism. Endocrinology 110, 1: 2333
4. Kant GJ, Leu JR, Anderson SM & Mougey EH (1987): Effects of chronic stress on plasma corticosterone,
ACTH and prolactin. Physiology & Behaviour 40, 6: 775-779
5. Garrido P, de Blas M, Del Arco A, Segovia G & Mora F (2010): Aging increases basal but not stressinduced levels of corticosterone in the brain of the awake rat. Neurobiol Aging. 2010 Apr 21
6. Handa RJ, Burgess LH, Kerr JE, O'Keefe JA (1994): Gonadal Steroid Hormone Receptors and Sex
Differences in the Hypothalamo-Pituitary-Adrenal Axis. Hormon. Behav. 28 (4): 464-76
7. Bhattacharya et al. (2017): Genetically Induced Retrograde Amnesia of Associative Memories After
Neuroplastin Ablation. Biological Psychiatry January 15, 2017; 81:124-135
8. Petrella et al. (2014): Maternl Exposure to Low Levels of Corticosterone during Lactation Protects against
Experimental Inflammatory Colitis-Induced Damage in Adult Rat Offspring. PLOS ONE November 2014,
Volume 9, Issue 11
9. Xie L., Korkmaz KS, Braun K. & Bock J. (2013): Early life stress-induced histone acetylations correlatewith activation of the synaptic plasticity genes Arc and Egr1 in the mouse hippocampus. Journal of
Neurochemistry. J. Neurochem. (2013) 125, 457-464
10.
Van der Doelen et al. (2014): Early life adversity and serotonin transporter gene variation interact at
the level of the adrenal gland to affect the adult hypothalamo-pituitary-adrenal axis. Translational
Psychiatry (2014), 1-8
11.
Porcu et al. (2014): Failure of Acute Ethanol Adminstration to Alter Cerebrocortical and Hippocampal
Allopregnanolone Levels in C57BL/6J and DBA/2J Mice. Alcohol Clin Exp Res. Vol. 38, No. 4, 2014: pp
948-958

Plea

Symbols:

Version: 7.0-cStoragetemperature

Manufacturer

Expiry date

Batch code

Consult instructions
for useContent

CautionCatalogue
numberEffective: 2020-05-27

Contains sufficient for<n> tests

For research use
only!
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