Leptin Kit ELISA

N° du produit ABIN956062

Détail du produit Leptin Kit ELISA

Antigène: Leptin (LEP)

Reactivité: Souris

Méthode de détection: Colorimetric

Type de méthode: Sandwich ELISA

Application: ELISA

Fonction: The Mouse Leptin ELISA is for the quantitative determination of Leptin in mouse serum.

Analytical Method: Quantitative

Specificité: Mouse Leptin: 100% Human Leptin: 0% Rat Leptin: 33.8%

Attributs du produit: These findings support the need of a highly sensitive immuno-assay system specific to mouse Leptin. Advantages of this assay include sensitive quantification, high specificity, no interference from other sample components, and no sample pretreatment. The Calibrator is a recombinant mouse Leptin.This kit is based on a sandwich enzyme immuno-assay. The 96-well plate is coated with highly purified anti-mouse Leptin antibody and Leptin Calibrator or sample is added for the first step of the immuno-reaction. After incubation and plate washing, biotinylated rabbit anti-mouse Leptin antibody is added to form Leptin antibody-antigen-biotinylated Leptin antibody complexes on the surface of the wells. After incubation and washing, HRP-Labeled streptoavidin (SA) is added to bind to the biotinylated Leptin antibody. Finally, HRP enzyme activity is determined by 3,3',5,5'-tetramethylbenzidine (TMB) and the concentration of mouse Leptin is calculated.

Ingrédients: 1. Antibody-Coated Plate Microtiter Plate: 1 plate (96-well) Rabbit anti-mouse Leptin antibody
2. Leptin Calibrator Lyophilized: 1 vial (20 ng) Recombinant mouse Leptin
3. Labeled Antibody Solution Liquid: 1 bottle (12 mL) Biotinylated rabbit anti-mouse Leptin pAb
4. SA-HRP Solution Liquid: 1 bottle (12 mL) HRP-labeled SA
5. TMB Substrate Solution Liquid: 1 bottle (12 mL) 3,3',5,5'-tetramethylbenzidine (TMB)
6. Stop Solution Liquid: 1 bottle (12 mL) 1 M H2SO4
7. Buffer Solution Liquid: 1 bottle (15 mL) Phosphate buffer
8. Wash Solution Liquid: 1 bottle (50 mL) Concentrated saline Concentrate
9. Plate Seal: 3 sheets.

Matériel non inclus: Photometer for microtiter plate (plate reader), which can read absorbance up to 2.5 at 450 nm
Rotator for microtiter plate
Washing device for microtiter plate with aspiration system and dispenser
Micropipettes, multi-channel pipettes for 8 wells or 12 wells and their tips
Polypropylene or glass test tubes for preparation of Calibrator Solution
Graduated cylinder (1,000 mL)
Distilled water or de-ionized water

Information d'application

Plaque: Pre-coated

Préparation des réactifs:

1. Preparation of Calibrator Solutions: Reconstitute the Leptin Calibrator (lyophilized Mouse Leptin, 20 ng/vial) with 1 mL of Buffer Solution, giving a 20 ng/mL Calibrator Solution after reconstitution. 0.2 mL of the reconstituted Calibrator Solution is diluted with 0.2 mL of Buffer Solution to yield a 10 ng/mL Calibrator Solution. Repeat the serial dilution to make Calibrator Solutions at 5, 2.5, 1.25, 0.625, and 0.313 ng/mL. Buffer Solution is used as the zero calibrator (0 ng/mL). Note: Calibrator Solution must be prepared immediately before assay. Use clean test tubes or vessels. In the case of multiple assays, the prepared Calibrator Solutions should be store at < -30°C. When a sample is predicted to be below 0.313 ng/mL, another calibrator may be prepared to decrease the lower limit of detection. Continue the serial dilution by diluting the 0.313 ng/mL Calibrator Solution to make a 0.156 ng/mL Calibrator Solution. However, the assay precision below 0.313 ng/mL may not be as good as the assay precision between 0.313 and 20 ng/mL.
   
   2. Preparation of Wash Solution: Dilute 50 mL of Wash Solution Concentrate to 1,000 mL with distilled or de-ionized water. Diluted Wash Solution is stable for 6 months at 4°C. Note: During storage of the Wash Solution Concentrate at 4°C, precipitates may be observed, however, they will dissolve when diluted.
   
   3. Other reagents are ready for use.

Procédure de l'essai:

1. Warm the reagents and samples to room temperature (20-30°C) at least 1 hour before beginning the test.
   2. Add 0.35 mL of diluted Wash Solution into wells and aspirate the solution. Repeat this washing procedure twice for a total of 3 wash steps. 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 45 µL of Buffer Solution into each of the wells. Then add 25 µL of the prepared Calibrator Solutions (0, 0.313, 0.625, 1.25, 2.5, 5, 10, and 20 ng/mL) or samples into the wells.
   4. Cover the plate with the Plate Seal and incubate at room temperature for 3 hours. During incubation, the plate should be rotated on a plate rotator.
   5. Remove the Plate Seal and aspirate the solution in the wells. Wash the wells 4 times with approximately 0.35 mL/well of diluted Wash Solution. Finally, invert the plate and tap onto an absorbent surface, such as paper toweling, to ensure removal of most of the residual Wash Solution.
   6. Pipette 100 µL of Labeled Antibody Solution into each of the wells.
   7. Cover the plate with a Plate Seal and incubate at room temperature for 2 hours. During the incubation, the plate should be rotated on a plate rotator.
   8. Remove the Plate Seal and aspirate the solution in the wells. Wash the wells 4 times with approximately 0.35 mL/well of diluted Wash Solution. Finally, invert the plate and tap onto an absorbent surface, such as paper toweling, to ensure removal of most of the residual Wash Solution.
   9. Add 100 µL of SA-HRP solution into each of the wells.
   10. Cover the plate with the Plate Seal and incubate at room temperature for 1 hour. During incubation, the plate should be rotated on a plate rotator.
   11. Transfer the required volume of the TMB Substrate Solution into a vessel, and bring to room temperature protected from light for 1 hour before use. Store the remaining TMB Substrate Solution at 4°C.
   12. Remove the Plate Seal and aspirate the solution in the wells. Wash the wells 4 times with approximately 0.35 mL/well of diluted Wash Solution. Finally, invert the plate and tap onto an absorbent surface, such as paper toweling, to ensure removal of most of the residual Wash Solution.
   13. Add 100 µL of TMB Substrate Solution into each well, cover the plate with a Plate Seal and incubate for 30 minutes at room temperature, protected from light.
   14. Add 100 µL of Stop Solution into the wells to stop the reaction.
   15. Read the optical absorbance of the wells at 450 nm. The optical absorbance of reaction solution in wells should be read as soon as possible after stopping the color reaction. Note: Perform all determinations in duplicate.

Calcul des résultats:

Calculate mean absorbance values of wells containing the Calibrators and plot a calibration curve on semilogarithmic graph paper (abscissa: concentration of Calibrators, ordinate: absorbance values of Calibrators). Use the calibration curve to read Leptin concentrations in samples from the corresponding absorbance values. When the concentration of Leptin is expected to exceed 20 ng/mL, the sample must be diluted with Buffer Solution until the result is within the assay range.

Restrictions: For Research Use only

Stockage

Stock: 4 °C

Détail du antigène

Antigène: Leptin (LEP)

Autre désignation: Leptin (LEP Produits)

Synonymes: ob Kit ELISA, obese Kit ELISA, LEPD Kit ELISA, OB Kit ELISA, OBS Kit ELISA, leptin Kit ELISA, Lep Kit ELISA, LEP Kit ELISA, lep Kit ELISA

Sujet: Leptin, which is a product of the ob gene, is a protein consisting of 146 amino acids and is secreted from white adipose tissue. It is known that Leptin acts on the hypothalamus to decrease food intake and to reduce body weight, body fat, blood sugar and blood insulin in healthy and ob/ob mice. Further, gene expression of neuropeptide Y (NPY) is suppressed by Leptin. Recently, radioimmunoassays for Leptin determination in human plasma have become available and Leptin levels in a human patient group with obesity was found to increase in comparison with that of a normal group. The Leptin plasma levels correlated with body fat. These observations clearly show that Leptin concentration in human plasma reflects the weight of tissue fat. Therefore, the measurement of plasma or serum Leptin may be a good index of obesity. Although mouse Leptin shows a high homology (96%) with rat Leptin, it is observed that substitution of several amino acid residues occurs between human and mouse Leptin.

Pathways: Signalistation JAK/STAT, AMPK Signaling, Hormone Transport, Peptide Hormone Metabolism, Hormone Activity, Negative Regulation of Hormone Secretion, Regulation of Carbohydrate Metabolic Process, Feeding Behaviour, Monocarboxylic Acid Catabolic Process