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.
3. WARNINGS AND PRECAUTIONS
- This kit is for research use only. Not for use in diagnostic procedures.
- 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.
- 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.
- Pipetting of samples and reagents must be done as quickly as possible and in the same sequence for each step.
- 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.
- Mix the contents of the microplate wells thoroughly to ensure good test results. Do not reuse microwells.
- Do not let wells dry during assay; add reagents immediately after completing the rinsing steps.
- 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.
- Never pipet by mouth and avoid contact of reagents and specimens with skin and mucous membranes.
- Do not smoke, eat, drink or apply cosmetics in areas where specimens or kit reagents are handled.
- Wear disposable latex gloves when handling specimens and reagents. Microbial contamination of reagents or specimens may give false results.
- Handling should be done in accordance with the procedures defined by an appropriate national biohazard safety guideline or regulation.
- 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
- All indicated volumes have to be performed according to the protocol. Optimal test results are only obtained when using calibrated pipettes and microtiterplate readers.
- 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.
- Avoid contact with Stop Solution. It may cause skin irritation and burns.
- Chemicals and prepared or used reagents have to be treated as hazardous waste according to the national biohazard safety guideline or regulation.
- 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
A 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.
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. ASSAY PROCEDURE
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 a 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.
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.) 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 a 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. PERFORMANCE CHARACTERISTICS
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.
The following materials have been evaluated for cross-reactivity. The percentage indicates cross reactivity at 50% displacement compared to corticosterone
The intra-assay variation was determined by 20 replicate measurements of three serum samples within one run. The within-assay variability is shown below:
The inter-assay (between-run) variation was determined by duplicate measurements of three serum samples.
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
Four native serum samples were assayed undiluted and diluted with the standard matrix.
9. LIMITATIONS OF PROCEDURE
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. LEGAL ASPECTS
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.
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.
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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. 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 a 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 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 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. (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