Corticosterone ELISA Kit

Produktnummer ABIN956191

Produktdetails Corticosterone ELISA Kit

Target: Corticosterone (CORT)

Reaktivität: Ratte, Maus

Nachweismethode: Colorimetric

Methodentyp: Competition ELISA

Applikation: ELISA

Verwendungszweck: The Mouse and Rat Corticosterone ELISA is for the quantitative determination of corticosterone in mouse and rat biological fluids.

Proben: Plasma, Serum

Analytische Methode: Quantitative

Produktmerkmale: This ELISA kit is used for the quantitative determination of corticosterone in biological fluids such as plasma, serum or urine samples of mice, rats and other species, and also cell or tissue culture supernatant. It has various advantages, such as no extraction procedure of samples, short assay time, and practically no influences of other body fluids or physiological active substances coexisting in samples assayed. This ELISA kit for the determination of corticosterone is based on a competitive enzyme immunoassay using a combination of specific antibodies to corticosterone and corticosterone-HRP conjugate (HRP-labeled corticosterone) system. The 96 well plate is coated with goat anti rabbit IgG, to which corticosterone calibrator or samples, HRP-labeled corticosterone and specific antibody are added for competitive immunoreaction. After incubation and plate washing, HRP enzyme activity is determined by 3,3',5,5'-tetramethylbenzidine (TMB) and the concentration of corticosterone is calculated.

Bestandteile: 1. Antibody-Coated Plate Microtiter plate 1 plate (96-well) Goat anti-rabbit IgG
2. Corticosterone Calibrator Lyophilized 1 vial (50 ng) Synthetic corticosterone
3. HRP-Labeled Corticosterone Liquid 1 vial (0.3 mL) HRP conjugated corticosterone
4. Specific Antibody Liquid: 1 bottle (7 mL) Rabbit anti-corticosterone antibody
5. TMB Substrate Liquid: 1 bottle (12 mL) TMB (3,3',5,5'-tetramethylbenzidine)
6. Stop Solution Liquid: 1 bottle (12 mL) 1M H2SO4
7. Buffer Solution Liquid: 1 bottle (10 mL) PBS buffer containing protein stabilizer
8. Sample Diluent Liquid: 1 bottle ( 50 mL) A specially formulated displacer of CBG
9. Wash Solution Concentrate Liquid: 1 bottle (25 mL) Concentrated saline
10. Adhesive Foil 2 sheets.

Benötigtes Material: Photometer for microtiter plate (plate reader), which can read extinction 2.5 at 450 nm
Washing device for microtiter plate and dispenser with aspiration system (optional)
Micropipettes for volumes between 10 µL - 1,000 µL
Multi-channel pipettes for 8 wells or 12 wells and their tips
Glass test tubes for preparation of calibrator and sample solutions
Graduated cylinder (500 mL or 1,000 mL)
Distilled or de-ionized water
A microplate shaker (210-240 rpm)

Anwendungsinformationen

Plattentyp: Pre-coated

Aufbereitung der Reagenzien:

1. Preparation of Calibrator Solution: Reconstitute the lyophilized Corticosterone Calibrator (50 ng/vial) with 1 mL of Sample Diluent, which affords a 50 ng/mL Calibrator Solution. Dilute 0.2 mL of the 50 ng/mL Calibrator Solution with 0.4 mL of Sample Diluent, which yields a 16.67 ng/mL Calibrator Solution. Repeat the same dilution procedure to make 5.56, 1.85, 0.62, and 0.21 ng/mL Calibrator Solutions. Sample Diluent is used as the 0 ng/mL Calibrator.
   2. Preparation of HRP-labeled corticosterone solution: Take 0.25 mL of HRP-labeled Corticosterone from the labeled vial and dilute with 7 mL of Buffer Solution.
   3. Dilution of Wash Solution Concentrate: Dilute one bottle of Wash Solution Concentrate (25 mL) to 500 mL with distilled or de-ionized water.
   4. The other reagents are ready for use.

Aufbereitung der Proben:

1. For mouse/rat plasma and serum: Dilute 10 µL of plasma or serum sample with 400 µL of Sample Diluent in a test tube. Mix the diluted solution and allow it to stand for 10 minutes at room temperature.
   2. For mouse/rat urine: Dilute 10 µL of urine sample (40 to 100 fold) with Sample Diluent in a test tube. Mix the diluted solution and allow it to stand for 10 minutes at room temperature.
   3. For culture supernatant (RPMI1640 with or without FCS): Dilute 50 µL of supernatant with 250 µL of Sample Diluent in a test tube. Mix the diluted solution and allow it to stand for 10 minutes at room temperature.
   4. For other species or matrix samples: Because corticosterone concentrations are different significantly in various species of animals, it is recommended that a series of diluted samples are prepared and tested to find the optimal dilution ratio before assay.

Testdurchführung:

1. Bring all the reagents, except test samples, to room temperature (22-25° C) before starting assay.
   2. Add 350 µL of diluted Wash Solution to each well and keep it for about 30° Conds, and then aspirate or decant the wash solution in the wells. Invert the plate and tap onto an absorbent surface, such as paper toweling, to ensure blotting free of most of the residual Wash Solution.
   3. Pipette 100 µL of corticosterone calibrator solutions (0, 0.21, 0.62, 1.85, 5.56, 16.67, and 50 ng/mL) or diluted samples, after being vortexed, into appropriate wells. Add 50 µL of HRP-labeled corticosterone solution into each well, and finally add 50 µL of Specific Antibody into each well.
   4. Cover the plate with adhesive foil and incubate it on a shaker at 210-220 rpm room temperature for two hours.
   5. After incubation, take off the adhesive foil, aspirate or decant the solutions in the wells. Add 350 µL of diluted wash solution to each well and keep it for about 30° Conds, and then aspirate or decant the wash solution in the wells. Repeat this wash process 4 times (total 5 times). Finally, invert the plate and tap onto an absorbent surface, such as paper toweling, to ensure blotting free of most of the residual Wash Solution.
   6. Add 100 µL of TMB Substrate into each well.
   7. Cover the plate with adhesive foil and incubate it on a shaker at 210-220 rpm at room temperature for 30 minutes.
   8. Add 100 µL of Stop Solution into each of the wells to stop color reaction.
   9. Read optical absorbance of the solution in the wells at 450 nm.

Ergebnisberechnung:

The assay fits best to a 4-parameter logistic equation, Y= (a-d)/(1+(x/c)^b)+d, here a,b,c,d represent constant parameter. Alternatively, calculate mean optical density values of wells containing calibrators or their percent bound to maximum binding wells (0 ng/mL) and plot a calibration curve on semilogarithmic graph paper (abscissa: concentration of calibrator, ordinate: optical density values or bound%). Use the average optical density or bound% of each sample to determine the corresponding value by simple interpolation from this calibration curve. The results should be multiplied by the dilution factor to obtain the actual concentrations for undiluted unknown samples. Perform all the determinations in duplicate.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Lagerung: 4 °C

Informationen zur Lagerung: Store kit at 4° C. The kit is stable until expiration date listed on box.

Haltbarkeit: The expiry date is stated on the label.

Details zu Corticosterone

Target: Corticosterone (CORT)

Andere Bezeichnung: Corticosterone (CORT Produkte)

Substanzklasse: Hormone

Hintergrund: Corticosterone (C21H30O4, also called 11beta,21-Dihydroxyprogesterone, Reichstein's Substance H, or Kendall's Compound B) is, like cortisol and cortisone, a glucocorticoid hormone secreted from the cortex of the adrenal gland. Corticosterone is derived from cholesterol through a series of enzymatically mediated steps and also serves as a precursor of aldosterone. It is a primary glucocorticoid in mice, rats and other animals (such as rabbits, birds, amphibians, and reptilians) in which the 17-hydroxylase is supposed to not exist in the adrenal gland. Corticosterone is produced under the control of ACTH and the production has a circadian rhythm with peak levels in the latter portion of the day in nocturnal animals like rats and is believed to play a decisive role in sleep-wake cycles. Corticosterone can be used as a non-invasive biomarker of stress study through the collection of urine and feces to avoid corticosterone increase in blood levels which is caused by normal invasive methods. Corticosterone is also being studied in different fields such as impairment of long-term memory retrieval, chronic corticosterone elevation due to dietary restrictions and response to burn injuries etc. Since most of corticosterone in blood is bound to a plasma protein called corticosterone-binding globulin (CBG), the determination of blood corticosterone with presently available commercial assay kits requires an initial extraction procedure. On the other hand, the present assay kit for corticosterone newly developed by our laboratory provides a tool for direct determination of corticosterone in blood by simple dilution of blood samples with the diluent included in the kit. Furthermore, assays using the kit can be completed within a short period. The corticosterone ELISA kit newly developed will be quite a useful tool for further development of corticosterone research.