Human Obestatin ELISA Assay
The Human Obestatin ELISA Assay is For Research Use Only
Size: 1×96 wells
Dynamic Range: 0.231 – 25 ng/mL
Incubation Time: 24 hours
Sample Type: Human plasma, serum
Sample Size: 20 μL
Specificity: The Eagle Biosciences Human Obestatin ELISA Assay kit shows cross-reactivity of 100% to human obestatin, 37.3% to mouse/rat obestatin, 25.2% to human obestatin (11-23)-NH2, less than 0.02% to human/mouse/rat obestatin (1-10), and no cross-reactivity to mouse/rat obestatin (11-23)-NH2. It shows no crossreactivity to human ghrelin and human des-octanoyl ghrelin in the range of standard concentrations.
This Human Obestatin ELISA for determination of obestatin in human plasma or serum samples is based on a competitive enzyme immunoassay using the combination of highly specific antibody to human obestatin and biotin–avidin affinity system. The 96 wells plate is coated with goat anti rabbit IgG, to which biotinylated human obestatin, human obestatin standard or samples and rabbit anti human obestatin antibody are added for competitive immunoreaction. After incubation and plate washing, horse radish peroxidase (HRP) labeled streptavidin (SA) is added, so that HRP labeled SA-biotinylated human obestatin-antibody complex is formed on the surface of the wells. Finally, HRP enzyme activity is determined by 3,3’,5,5’-Tetramethylbenzidine (TMB) and the concentration of human obestatin is calculated.
Rodent Obestatin ELISA Assay
Human PYY ELISA Assay
Human GIP (Active) ELISA Assay Kit
Obestatin is a 23 amino acid residues peptide isolated from the rat stomach. The peptide shares the precursor with a food intake stimulating peptide, ghrelin, but possesses reducing effects on food intake, gut motility and body weight (1). With the use of an antiserum directed against the mouse/rat obestatin, obestatin immunoreactivity (irOBS) was detected in cells of the gastric mucosa and myenteric plexus and in Leydig cells of the testis in Sprague–Dawley rats. Double labeling of myenteric plexus with antisera against obestatin and choline acetyltransferase (ChAT) revealed that nearly all irOBS neurons were ChAT positive and vice versa (2). Obestatin (100nM) added to dissociated and cultured rat cerebral cortical neurons elevated cytosolic calcium concentrations [Ca+2]i in a population of cortical neurons (2).
Intracerebroventricular admini- stration of obestatin inhibited water drinking in ad libitum fed and watered rats, and in food and water deprived animals. In addition, obestatin inhibited angiotensin II-induced water drinking in animals provided free access to water and food (3). Obestatin peptides had no effect on insulin sensitivity as revealed by hypoglycaemic response when co-administered with insulin, supporting a role of obestatin in regulating metabolism through changes of appetite, but indicating no direct actions on glucose homeostasis or insulin secretion (4). It is supposed that in rats the effects of obestatin on food intake may be secondary to an action of the peptide to inhibit water drinking (3). Recently, it is reported affording cardioprotection to ischemic- reperfused isolated rat heart, inhibiting apoptosis in culture of similarly stressed cardiomyocytes(5) and inhibiting dopamine release in rat hypothalamus(6).
The obestatin concerning study for energy homeostasis and body weight regulation could be expected to have a large development in the future. The Eagle Biosciences Human Obestatin ELISA Assay kit developed by our laboratory can be used for direct determination of blood obestatin level’s variations and will be a useful tool for further development of obestatin research.
- Add 300µL of washing solution to each well and keep it for at least 30 seconds, then aspirate or decant the washing solution in the wells.
- Invert the plate and tap it firmly on a lint free paper towel to ensure blotting free of most residual washing solution.
- Fill 50µL of labeled antigen solution into each well first, then introduce 20µL of each of standard solutions (0, 0.231, 0.463, 0.926, 2.778, 8.333, 25ng/mL) or samples and finally add 50µL of human obestatin antibody solution into each well.
- Cover the plate with Adhesive Foil and incubate it at 4°C for 20 – 22 hours (still).
- After incubation, take off the Adhesive Foil, aspirate the contents, then add 300µL of washing solution to each well and aspirate. Repeat the wash step for total of five times with approximately 300µL/well of washing solution each time and finally invert the plate and tap it firmly on a lint free paper towel to ensure blotting free of most residual washing solution.
- Pipette 100µL of SA-HRP Solution into each well.
Cover the plate with Adhesive Foil and incubate it at room temperature for 1 hour with shaking.
- Take off the Adhesive Foil, aspirate and wash the wells five times as Procedure 4.
- Add 100µL of TMB Substrate into each well; cover the plate with Adhesive Foil and keep it for 30 minutes with shaking at room temperature under a light proof condition (please refer to Section 7 Notes, 7. for more information).
- Add 100µL of Reaction Stopping Solution into each well to stop coloring reaction.
- Read the optical absorbance of the wells at 450nm. Calculate mean optical density values of wells containing standard solutions or their bound percentage (B/Bo%) to Bo wells (0 ng/mL standard as Bo) and plot a standard curve on a semi-logarithmic graph paper (abscissa: concentrations of standard; ordinate: optical density or B/Bo%). Use the average optical density or B/Bo% of each sample to determine the corresponding value by simple interpolation from the standard curve.
Typical Standard Curve
Precision & Reproducibility
Intra-assay CV（％）: 3.5 ~ 9.9
Inter-assay CV（％）: 5.6 ~ 9.0
Analytical RecoveryHuman serum: 101.5~113.2% (n=7)
Human plasma: 106.1~118.9% (n=7)