Rodent Urocortin 1 ELISA Assay

Additional Information

Assay Principle

Urocortin (Urocortin1: Ucn1) is first identified in rat, and later in human and mouse3. It is the second mammalian member of the CRF family. Rat and mouse Ucn1 have the same amino acid sequence and display 95% structure homology to human Ucn1, 45% to CRF and 63% to urotensin.

In the rat, Ucn1 immunoreactivity (IR) was shown to distribute widely in central nervous system, endocrine organs, and digestive system and its concentration was highest in pituitary (11 pmol/g, w.w.).  Koziez, Yanaihara et al. used a polyclonal antibody against rat Ucn1 to define the distribution of Ucn1-IR in rat central nervous system and found a large number of neurons with Ucn1-IR in rat brain. 

Synthetic human Ucn1 binds with high affinity to CRF receptor type 1(CRFR1), 2 alpha(CRFR2α) and 2 beta (CRFR2β). CRFR1 and CRFR2 have been shown to link to the development of stress-related disorders, such as mood and eating disorders. CRFR1 is expressed predominantly in the brain and pituitary, whereas CRFR2 expression is limited to particular brain areas and to some peripheral organs. Data were also presented supporting the hypothesis that this peptide is the endogenous ligand for the CRFR2.

Assay Procedure

1. Before starting the assay, bring all the reagents and samples to room temperature (20~30ºC).
2. Fill 0.3 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 40 uL of buffer solution to the wells first, then introduce 10 uL of each of standard solutions (0, 1.563, 3.125, 6.25, 12.5, 25, 50 and 100 ng/mL) or samples and finally add 50uL 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 0.3 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 100uL 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. Take off the adhesive foil, aspirate and wash the wells 4 times with 0.3 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.
9. Add 100uL of Enzyme substrate solution (TMB) to each of the wells, cover the plate with adhesive foil and keep it for 30 minutes at room temperature in a dark place for color reaction (keep still, plate shaker not need).
10. Add 100uL of stopping solution to each of the wells to stop color reaction.
    Read the optical absorbance of the solution in the wells at 450 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