Rodent Urocortin 3 ELISA Assay


This Eagle Biosciences Mouse/Rat Urocortin 3 ELISA Assay kit is used for quantitative determination of urocortin 3 in mouse/rat plasma, serum and their brain tissue extracts. The kit is characterized for sensitive quantification, high specificity and no influence with other components in samples. Mouse/rat urocortin 3 standard is highly purified synthetic product. The Eagle Biosciences Human Mouse/Rat Urocortin 3 ELISA Assay is for Research Use Only and should not be used for diagnostic procedures.

Rodent Urocortin 3 ELISA Assay

The Rodent Urocortin 3 ELISA Assay is For Research Use Only

Size: 1×96 wells
Dynamic Range: 0.41.9 – 100 ng/mL
Incubation Time: 21 hours
Sample Type: Mouse or Rat Serum, Plasma
Sample Size: 25μL
Alternative Names: Mouse/Rat Urocortin 3, Mouse Rat UCN 3, Rodent Stresscopin, SCP

Assay Background

Urocortin 3 (Ucn3) or stresscopin (SCP) is a new member of the corticotropin-releasing factor (CRF) peptide family identified in the mouse and human. The CRF family of neuropeptides includes mammalian peptides CRF, urocortin 1(Ucn1) and urocortin 2 (Ucn2) or stress-related peptide (SRP), as well as piscine urotensin 1 and frog sauvagine. Mouse and human Ucn3 share 90% identity in the 38-aa putative mature peptide.

In the human, Ucn1 immunoreactivity was marked in the medulla, whereas Ucn3 was immunostained mostly in the cortex. The receptors for Ucn1, Ucn2, Ucn3 and CRF are all expressed in human adrenal cortex and medulla2), therefore these peptides are expected to play important roles in physiological adrenal functions. Ucn3 was also detected by RIA in human heart 0.74-1.15 pmol/g wet weight, kidney 1.21 pmol /g wet weight, pituitary 2.72 pmol /g wet weight and brain tissues 1-2 pmol /g wet weight. Furthermore, immunoreactive Ucn3 was present in human plasma 51.8 pmol/L and urine 266 pmol/L obtained from healthy subjects. It was also detected in human rectum 15.4 pmol/g wet weight and sigmoid colon 6.5 pmol/g wet weight. These data suggest that Ucn3 regulates the cardiac and renal functions as a local factor and a circulating hormone and plays some physiological or pathological roles in the modulation of gastrointestinal functions during stressful conditions in different manners from those of Ucn1.

Pharmacological studies showed that Ucn3 is a high-affinity ligand for the type 2 CRF receptor (CRFR2). In the rat, Ucn3-positive neurons were found predominantly within the hypothalamus and medial amygdala.5) Ucn3 fibers were distributed mainly in the hypothalamus and limbic structures. These data support that Ucn3 is an endogenous ligand for CRFR2 in these areas. The results also suggest that Ucn3 is positioned to play a role in mediating physiological functions, including food intake and neuroendocrine regulation.

In the mouse, Ucn3 was expressed in pancreatic beta-cells and in a mouse beta cell line, MIN6. High potassium, forskolin or high glucose could stimulate Ucn3 secretion from these cells.6) Ucn3 injections to the rat resulted in significant increase of plasma insulin level.6) Ucn3 also stimulated glucagon and insulin release from isolated rat islets. Pancreatic Ucn3 acting through CRFR2 was suggested to be involved in the local regulation of glucagon and insulin secretion.

Treatment with Ucn3 (SCP) or Ucn2 (SRP) suppressed food intake, delayed gastric emptying and decreased heat-induced edema. Thus Ucn3 (SCP) and Ucn2 (SRP) might represent endogenous ligands for maintaining homeostasis after stress, and could allow the design of drugs to ameliorate stress-related diseases. The use of CRFR2 selective agonists, Ucn2 and Ucn3, to treat ischemic heart disease was proposed because of their potent cardioprotective effects in murine heart and their minimal impact on the hypothalamic stress axis.

Ucn1 is able to bind to two types of G-protein coupled receptors: CRFR1 and CRFR2, whereas Ucn3 (SCP) and Ucn2 (SRP) bind exclusively and with high affinity to CRFR2.  Ucn3 (SCP) is expressed in rat cardiomyocytes and the levels of Ucn3 (SCP) and Ucn2 (SRP) were increased by hypoxic stress. All these three peptide were shown to have potent cardioprotective effects in cells exposed to hypoxia/reoxygenation.

We have already developed mouse/rat urocortin 1 EIA kit (YK210), mouse urocortin 2 EIA kit (YK190) and rat urocortin 2 EIA kit (YK191). This time, as a part of tools for urocortin research, our laboratory developed mouse/rat urocortin 3 EIA kit (YK200), which is highly specific for mouse/rat urocortin 3 with almost no crossreaction to Ucn1 (mouse, rat), Ucn1 (human), Ucn2 (mouse), Ucn2 (rat), ACTH (mouse, rat), ACTH (human) and CRF (mouse, rat, human). The kit can be used for measurement of Ucn3 in mouse/rat plasma, serum and their brain tissue extracts with high sensitivity (The brain tissue extracts need to be treated with solid-phase extraction cartridges). It will be a specifically useful tool for Ucn3 researches.

Related Products

Mouse Rat Urocortin 1 ELISA Assay Kit
Rat Urocortin 2 ELISA Assay
Mouse Urocortin 2 ELISA Assay

Additional Information

Assay Principle

This Mouse/Rat Urocortin 3 ELISA Assay kit for determination of mouse/rat urocortin 3 in samples is based on a competitive enzyme immunoassay using combination of highly specific antibody to mouse/rat urocortin 3 with biotin-avidin affinity system. To the wells of plate coated with rabbit anti mouse/rat urocortin 3 antibody, standards or samples and labeled antigen (biotinylated antigen) are added for competitive immunoreaction. After incubation and plate washing, horseradish peroxidase (HRP) labeled streptoavidin (SA) is added to form HRP labeled SA-labeled antigen-antibody complex on the surface of the wells. Finally, HRP enzyme activity is determined by o-Phenylenediamine dihydrochloride (OPD) and the concentration of mouse/rat urocortin 3 is calculated.

Assay Procedure

  1. Before starting the assay, bring all the reagents and samples to room temperature (20 ~ 30ºC).
  2. Fill 0.35 mL/well of washing solution into the wells and aspirate the washing solution in the wells. Repeat this washing procedure further twice (total 3 times). Finally, invert the plate and tap it onto an absorbent surface, such as paper toweling, to ensure blotting free of most residual washing solution.
  3. Add 25 uL of buffer solution to the wells first, then introduce 25 L of each of standard solutions (0, 0.41, 1.23, 3.70, 11.1, 33.3 and 100 ng/mL) or samples and finally add 50 uL of labeled antigen to the wells. The total pipetting time of standard solutions and samples for a whole plate should not exceed 30 minutes.
  4. Cover the plate with adhesive foil and incubate it at 4ºC for 16~18 hours (keep still, plate shaker not need).
  5. After incubation, move the plate back to room temperature keeping for approximately 40 minutes (keep still, plate shaker not need) and take off the adhesive foil, aspirate and wash the wells 4 times with 0.35 mL/well of washing solution. Finally, invert the plate and tap it onto an absorbent surface, such as paper toweling, to ensure blotting free of most residual washing solution.
  6. Add 100 uL of SA-HRP solution to each of the wells.
  7. Cover the plate with adhesive foil and incubate it at room temperature for 2 hours. During the incubation, the plate should be shaken with a plate shaker (approximately 100 rpm).
  8. Dissolve one OPD tablet with 11 mL of substrate buffer. It should be prepared immediately before use.
  9. Take off the adhesive foil, aspirate and wash the wells 4 times with 0.35 mL/well of washing solution. Finally, invert the plate and tap it onto an absorbent surface, such as paper toweling, to ensure blotting free of most residual washing solution.
  10. Add 100 uL of substrate solution to each of the wells, cover the plate with adhesive foil and keep it for 20 minutes at room temperature (keep still, plate shaker not need).
  11. Add 100 uL of stopping solution to each of the wells to stop color reaction.
  12. Read the optical absorbance of the wells at 490 nm (or 492 nm). The dose-response curve of this assay fits best to a 4 (or 5)-parameter logistic equation. The results of unknown samples can be calculated with any computer program having a 4 (or 5)-parameter logistic function. Otherwise calculate mean absorbance values of wells containing standards and plot a standard curve on semi logarithmic graph paper (abscissa: concentration of standard; ordinate: absorbance values). Use the average absorbance of each sample to determine the corresponding value by simple interpolation from this standard curve.

Typical Standard Curve

Rodent Urocortin 3 ELISA Assay


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