Elisa Kit for Corticosterone Testing in Rats and Mice by Rocky Mountain Diagnostics

Overview and Instructions for use




1.1 Intended Use

The Corticosterone rat and mouse ELISA is a competitive immunoassay for the measurement of corticosterone in rat and mouse serum or plasma. For research use only. Not for use in diagnostic procedures.


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 to synthesize 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 of lymphocytes 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 role in 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, to academic 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 is often used in connection with ACTH measurement as a stress indicator (3, 4). The effects of chronic stress on 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).

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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 sites of corticosterone antiserum coated to the wells of a microplate. After incubation on a shaker the microplate is washed four times. After addition of the substrate solution the concentration of corticosterone is inversely proportional to the optical density measured.



  1. This 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 turn solution colored. Do not pour reagents back into vials as reagent contamination may occur.
  6. Mix the contents of the microplate wells thoroughly to ensure good test results. Do not reuse microwells.
  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 affect the 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 reagents or 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. Please use only the valid version of the Instructions for Use provided with the kit Version: 7.0-b Effective: 2018-06-06 3/8
  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 of different 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 are available upon request directly from the manufacturer.



4.1 Reagents Provided



4.2 Materials Required but not Provided

microtiter plate reader capable for endpoint measurement at 450 nm

Microplate mixer operating more than 600 rpm

Calibrated variable precision micropipettes (10µl,50µl,100µl,200µl).Absorbent paper

Distilled or deionized water


Semi logarithmic graph paper or software for data reduction


4.3 Reagent Preparation

All reagents should be at room temperature before use.

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.


4.4 Storage Conditions

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.


4.5 Disposal of the Kits

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


4.6 Damaged Test Kits

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.



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 repeated freeze-thaw  cycles.  Samples  expected  to  contain  rat/mouse  Corticosterone  concentrations  higher  than  the highest  standard(2250  ng/ml)  should  be  diluted  with  the  Standard  A  before  assay.  The  additional  dilution step has to betaken into account for the calculation of the results.

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




6.1 General Remarks

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 is recommended 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.


6.2 Assay Procedure

Each run must include standard curve.


6.3Calculation 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  and

concentration on the horizontal (X)axis.

3.  Using  the  mean  absorbance  value  for  each  sample,  determine  the  corresponding  concentration  from  the standard 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 gives lightly different results.

5.  The  concentration  of  the  samples  can  be  determined  directly  from  this  standard  curve.  Samples  with

concentrations  higher  than  that  of  the  highest  standard  have  to  be  further  diluted.  For  the  calculation  of the concentrations, this dilution factor has to betaken into account.

Conversion to SI units:

Corticosterone (ng/ml) x2.886=nmol/l


6.3.1 Example of Typical Standard Curve

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




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.)  and analyzed  using  the  Corticosterone  rat/mouse  ELISA  kit.  The  following  ranges  are  calculated  with  the  results of this study.


In further studies serum samples of 23 mice were collected between 11.00a.m. and 2.00p.m. undanalyzed in similar manner


It  is  recommended  that  each  laboratory  establish  its  own  normal  range  since  corticosterone  levels  can  vary due to handling and sampling techniques.




8.1 Analytical Sensitivity

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


8.2 Specifictiy

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




8.3.1 Intra-Assay

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


8.3.2 Inter-Assay

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



 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



 8.5 Linearity

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




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.   Anyimproperhandlingofsamplesormodificationofthistestmightinfluencetheresults.

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.



10.1 Reliability of Results

The  test  must  be  performed  exactly  as  per  the  manufacturer’s  instructions  for  use.  Moreover  the  user  must strictly  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  the test procedure,asufficientnumberofcontrolsforvalidatingtheaccuracyandprecisionofthetest.

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

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.   Such modification and/or exchanges invalid ate 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.Anydamagecausedtothetestkitduringtransportationisnotsubjecttotheliabilityofthemanufacturer.

 >>> CLICK HERE to Order the Corticosterone Rat & Mouse Elisa <<<




1.  D´Agostino  J,  Vaeth  GF  &  Henning  SJ  (1982):  Diurnal  rhythm  of  total  and  free  concentration  of  serum corticosterone in the rat. ActaEndocrinologica100,Vol.1,85-90.

2.  Vázquez-Palacios  G  et  al.  (2001):  Further  definition  of  the  effect  of  corticosterone  on  the  sleep-wake pattern in the male rat. Pharmacol.Biochem.Behav.70:305-310

3.  De  SouzaEB&  van  Loon GR(1982):Stress induced in hibition of the  plasma corticosterone  response  to subsequent  stress  in  the  rat:  A  non-adrenocorticotropin-mediated  mechanism.  Endocrinology  110,  1:  23-33

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  stress-induced levels of corticosterone in the brain of the awakerat. Neurobiol Aging2010 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. Behav28 (4): 464-76

7.  Bhattacharya   et   al.   (2017):   Genetically   Induced   Retrograde   Amnesia   of   Associative   Memories   After Neuroplast in 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,Volume9, Issue11

9.  Xie  L.,  Korkmaz  KS,  Braun  K.  &  Bock  J.  (2013):  Early  life  stress-induced  histone  acetylations  correlate with  activation  of  the  synaptic  plasticity  genes  Arc  and  Egr1  in  the  mouse  hippocampus.  Journal  of Neurochemistry. J. Neurochem. (2013125, 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