Obestatin ELISA Kit

Catalog No. ABIN956170

Rat and Mouse Obestatin ELISA Kit, Colorimetric assay for quantification of Rat and Mouse Obestatin.

Quick Overview for Obestatin ELISA Kit (ABIN956170)

Target: Obestatin (OB)

Reactivity: Rat, Mouse

Detection Method: Colorimetric

Method Type: Competition ELISA

Application: ELISA

Product Details Obestatin ELISA Kit

Purpose: The Mouse and Rat Obestatin ELISA is for the quantitative determination of obestatin in mouse and rat serum.

Analytical Method: Quantitative

Characteristics: This ELISA kit for determination of obestatin in mouse/rat serum samples is based on a competitive enzyme immunoassay using the combination of highly specific antibody to mouse/rat obestatin and biotin-avidin affinity system. The 96 wells plate is coated with goat anti-rabbit IgG, to which biotinylated mouse/rat obestatin, mouse/rat obestatin calibrator or samples and rabbit anti-mouse/rat obestatin antibody are added for competitive immunoreaction. After incubation and plate washing, horseradish peroxidase (HRP) labeled streptavidin (SA) is added, so that HRP labeled SA- biotinylated mouse/rat 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 mouse/rat obestatin is calculated.

Components: 1. Antibody-Coated Plate Microtiter plate: 1 plate (96-well) Goat anti-rabbit IgG
2. Calibrator Lyophilized: 1 vial (20 ng) Synthetic mouse/rat obestatin
3. Labeled Antigen Lyophilized: 1 vial Biotinylated mouse/rat obestatin
4. Specific Antibody Liquid: 1 bottle (6 mL) Rabbit anti-mouse/rat obestatin antibody
5. SA-HRP Solution Liquid: 1 bottle (12 mL) HRP-labeled streptavidin
6. TMB Substrate Liquid: 1 bottle (12 mL) TMB (3,3',5,5'-tetramethylbenzidine)
7. Stop Solution Liquid: 1 bottle (12 mL) 1 M H2SO4
8. Buffer Solution Liquid: 1 bottle (25 mL) BSA containing saline buffer
9. Wash Solution Concentrate Liquid: 1 bottle (25 mL) Concentrated saline
10. Plate Seal: 3 sheets.

Material not included: Photometer for microtiter plate (plate reader), which can read absorbance up to 2.5 at 450 nm
Washing device for microtiter plate and dispenser with aspiration system
Micropipettes, multi-channel pipettes for 8 wells or 12 wells and their tips
Test tubes (glass or polypropylene) for preparation of calibrator solution
Graduated cylinder (500 mL or 1,000 mL)
Distilled water or de-ionized water

Application Details

Plate: Pre-coated

Reagent Preparation:

1. Preparation of Calibrator Solution: Reconstitute the Mouse/Rat Obestatin Calibrator with 1 mL of buffer solution, which makes a 20 ng/mL Calibrator Solution. Add 0.1 mL of the 20 ng/mL Calibrator Solution with 0.2 mL of Buffer Solution, which yields a 6.667 ng/mL Calibrator Solution. Repeat the same dilution procedure to make 2.222, 0.741, 0.247 and 0.082 ng/mL Calibrator Solutions. Buffer Solution is used as the 0 ng/mL Calibrator. Note: Calibrator Solution should be prepared immediately before use.
   2. Preparation of labeled antigen: Reconstitute labeled antigen with 6 mL of buffer solution. Note: Labeled Antigen Solution should be prepared immediately before use.
   3. Preparation of Wash Solution: Dilute 25 mL of Wash Solution Concentrate to 500 mL with distilled or de-ionized water. Note: During storage of Wash Solution Concentrate at 4°C, precipitates may be observed. However, they will be dissolved when diluted. Diluted Wash Solution is stable for 6 months at 4°C.
   4. The other reagents are ready for use.

Assay Procedure:

1. Bring all the reagents except samples to room temperature (20-30°C) before starting assay.
   2. Add 0.35 mL/well of diluted Wash Solution into the wells of the plate and keep it for about 30° Conds, then aspirate the solution. Repeat this washing procedure twice (total 3 times). Finally, invert the plate and tap onto an absorbent surface, such as paper toweling, to ensure removal of most of the residual Wash Solution.
   3. Add 50 µL of labeled antigen solution, and then add 25 µL of each prepared Calibrator Solution (0, 0.082, 0.247, 0.741, 2.222, 6.667 and 20 ng/mL) or samples and finally add 50 µL of mouse/rat obestatin antibody into the wells.
   4. Cover the plate with a Plate Seal and incubate at 4°C overnight for 18
   20 hours. (Still, plate shaker not needed.)
   5. After 4°C incubation, remove the Plate Seal, aspirate the solution in the wells and wash the wells 3 times as before with approximately 0.35 mL/well of diluted Wash Solution.
   6. Pipette 100 µL of SA-HRP Solution into each of the wells.
   7. Cover the plate with a Plate Seal and incubate at room temperature for 1 hour. (Still, plate shaker not needed.)
   8. Remove the Plate Seal and aspirate the solution in the wells and then wash the wells 5 times as before with approximately 0.35 mL/well of diluted Washing Solution.
   9. Add 100 µL of TMB solution into each of the wells, cover the plate with a Plate Seal and incubate for 30 minutes at room temperature, protected from light. (Still, plate shaker not needed.)
   10. Add 100 µL of Stop Solution into each of the wells.
   11. Read optical absorbance of the solution in the wells at 450 nm. Read plate optical absorbance of reaction solution in wells as soon as possible after stopping the color reaction. Notes: Perform all determinations in duplicate.

Calculation of Results:

Calculate mean absorbance values of calibrators and plot a calibration curve on semi-logarithmic graph paper (abscissa: concentration of calibrator, ordinate: absorbance values). Use the calibration curve to read mouse/rat obestatin concentrations in samples from the corresponding absorbance values.

Restrictions: For Research Use only

Handling

Storage: 4 °C

Target Details for Obestatin

Target: Obestatin (OB)

Alternative Name: Obestatin

Background: Obestatin is a 23 amino acid residue peptide isolated from 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. 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. Obestatin (100 nM) added to dissociated and cultured rat cerebral cortical neurons elevated cytosolic calcium concentrations [Ca+2]i in a population of cortical neurons. Intracerebroventricular administration 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. Obestatin peptides had no effect on insulin sensitivity as revealed by hypoglycemic 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. 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. The obestatin concerning study for energy homeostasis and body weight regulation could be expected to have a large development in the future. The Mouse and Rat Obestatin ELISA developed by our laboratory can be used for direct determination of serum obestatin level variations and will be a useful tool for further development of obestatin research.This ELISA kit is used for quantitative determination of obestatin in mouse/rat serum samples. It has various advantages, such as highly specific and sensitive quantification, no influences with other body fluids or physiological active substances and unnecessity of sample pretreatment. The mouse/rat obestatin calibrator of this kit is a highly purified synthetic product (purity: higher than 99%). The ELISA kit shows cross-reactivity of 100% to mouse/rat obestatin, 118.6% to mouse/rat obestatin (11-23)-NH2, 0.5% to mouse/rat obestatin (1-23)-OH and less than 0.39% to human/mouse/rat obestatin (1-10) and no cross-reactivity to human obestatin, human obestatin (11-23))-NH2, and mouse/rat ghrelin and mouse/rat des-octanoyl ghrelin.