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For quantitative determination of urocortin 3 in mouse/rat plasma or serum samples

Mouse/rat Urocortin 3 EIA Kit

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.1) 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. 2) 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.2) 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.3) Furthermore, immunoreactive Ucn3 was present in human plasma 51.8 pmol/L and urine 266 pmol/L obtained from healthy subjects.3) It was also detected in human rectum 15.4 pmol/g wet weight and sigmoid colon 6.5 pmol/g wet weight.4) 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 Ucn1.4)

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.5) 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.5)

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.6) Pancreatic Ucn3 acting through CRFR2 was suggested to be involved in the local regulation of glucagon and insulin secretion.6)

Treatment with Ucn3 (SCP) or Ucn2 (SRP) suppressed food intake, delayed gastric emptying and decreased heat-induced edema.7) 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.7) 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.8)

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. 9) Ucn3 (SCP) is expressed in rat cardiomyocytes and the levels of Ucn3 (SCP) and Ucn2 (SRP) were increased by hypoxic stress.8) All these three peptide were shown to have potent cardioprotective effects in cells exposed to hypoxia/reoxygenation. 9)

We have already developed mouse urocortin 2 EIA kit(YK190), and this time urocortin 3 EIA kit (YK200) is been developing in our laboratory which highly specific for mouse/rat urocortin 3 with almost no cross reaction 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 or serum with high sensitivity. It will be a specifically useful tool for Ucn3 researches.

<Reference>
ReferencePDF

Feature and Advantages

This EIA kit is used for quantitative determination of urocortin 3 in mouse/rat plasma & serum samples. 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 assay kit can measure mouse/rat urocortin 3 in the range of 0.41-100 ng/mL
The assay completes within 16-18 hr.+3 hr.
With one assay kit, 41 samples can be measured in duplicate.
Test sample: Mouse/rat plasma & serum Sample volume: 25 μL
The 96-well plate in kit is consisted by 8-wells strips. The kit can be used separately.
Precision and reproducibility (Mouse/rat plasma & serum) Intra-assay CV(%) 5.10〜13.58 Inter-assay CV(%) 5.69〜10.24
Stability and Storage Store all of the components at 2-8°C. 6 months from the date of manufacturing. The expiry date is described on the label of kit.

<Specificity>
The EIA kit has high specificity to mouse/rat urocortin 3 and shows cross reactivity neither Ucn1 (mouse, rat), Ucn1 (human), Ucn2 (mouse), Ucn2 (rat), ACTH (mouse, rat), ACTH (human) nor CRF (mouse, rat, human).

<Test Principle>
This EIA 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. The 96 wells plate is coated with rabbit anti mouse/rat urocortin 3 antibody. Mouse/rat urocortin 3 standard or samples, labeled antigen are added to the wells for competitive immunoreaction. After incubation and plate washing, HRP labeled streptavidin (SA-HRP) are added to form HRP labeled streptavidin-biotinylated mouse/rat urocortin 3-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.

Composition

    Form Quantity Main Ingredient
1. Antibody coated plate MTP*1 1 plate (96 wells) Rabbit anti mouse/rat urocortin 3 antibody
2. Standard lyophilized 1 vial (100ng) Synthetic mouse/rat urocortin 3
3. Labeled antigen lyophilized 1 vial Biotinylated mouse/rat urocortin 3
4. SA-HRP solution Liquid 1 bottle (12 mL) HRP labeled streptavidin
5. Substrate buffer Liquid 1 bottle (24 mL) 0.015% Hydrogen peroxide
6. OPD tablet tablet 2 tablets o-Phenylenediamine dihydrochloride
7. Stopping solution Liquid 1 bottle (12 mL) 1 M H2SO4
8. Buffer solution Liquid 1 bottle (15 mL) Phosphate buffer
9. Washing solution (Concentrated) Liquid 1 bottle (50 mL) Concentrated saline
10. Adhesive foil   3 sheets  

MTP*1 ….. Microtiter plate

References

1. Lewis K, Li C et al: (2001) Identification of urocortin III, an additional member of the corticotropin-releasing factor (CRF) family with high affinity for the CRF2 receptor. Proc Natl Acad Sci USA. 98, 7570-7575
2. Fukuta T, Takahashi K et al: (2005) Urocortin 1, urocortin3/stresscopin, and corticotropin- releasing factor receptors in human adrenal and its disorders. J Clin Endocrinol Metab. 90, 4671-4678
3. Takahashi K, Totsune K et al: (2004) Expression of urocortin III/stresscopin in human heart and kidney. J Clin Endocrinol Metab. 89, 1897-1903
4. Saruta M, Takahashi K et al: (2005) Urocortin3/stresscopin in human colon: possible modulators of gastrointestinal function during stressful conditions. Peptide. 26, 1196-1206
5. Li C, Vaughan J et al: (2002) Urocortin III-immunoreactive projections in rat brain: partial overlap with sites of type 2 corticotrophin-releasing factor receptor expression.
J Neurosci. 22, 991-1001
6. Li C, Chen P et al: (2003) Urocortin III is expressed in pancreatic beta-cells and stimulates insulin and glucagon secretin. Endocrinology. 144, 3216-3224
7. Hsu SY and Hsueh AJ (2001) Human stresscopin and stresscopin-related peptide are selective ligands for the type 2 corticotropin-releasing hormone receptor. Nat Med. 7, 605-611
8. Brar BK, Jonassen AK et al: (2004) Urocortin-II and urocortin-III are cardioprotective against ischemia reperfusion injury: an essential endogenous cardioprotective role for corticotropin releasing factor receptor type 2 in the murine heart. Endocrinology. 145, 24-35
9. Chanalaris A , Lawrence KM et al: (2003) Protective effects of the urocortin homologues stresscopin (SCP) and stresscopin-related peptide (SRP) against hypoxia/reoxygenation injury in rat neonatal cardiomyocytes. J Mol Cell Cardiol. 35, 1295-1305

Product List

Product Name Cat# Quantity Price

Urocortin 3, EIA Kit

YII-YK200-EX 1KIT

¥ 76,000
$ 1014
€ 760

1. Lewis K, Li C et al: (2001) Identification of urocortin III, an additional member of the corticotropin-releasing factor (CRF) family with high affinity for the CRF2 receptor. Proc Natl Acad Sci USA. 98, 7570-7575 2. Fukuta T, Takahashi K et al: (2005)