Leptin ELISA Kit

Produktnummer ABIN956116

Ratte Leptin ELISA Kit, Colorimetric Assay für die Quantifizierung von Ratte Leptin.

Kurzübersicht für Leptin ELISA Kit (ABIN956116)

Target: Leptin (LEP)

Reaktivität: Ratte

Nachweismethode: Colorimetric

Methodentyp: Sandwich ELISA

Applikation: ELISA

Produktdetails Leptin ELISA Kit

Verwendungszweck: The Rat Leptin ELISA is for the quantitative determination of leptin in rat plasma, serum and culture supernatant.

Analytische Methode: Quantitative

Produktmerkmale: These findings have required the urgency to develop a highly sensitive immunoassay system specific to rat leptin. has developed an ELISA kit that is a stable and convenient assay system for rat leptin in plasma, serum and culture supernatant. Advantages of this kit include good quantification, no influence with other body fluid factors or physiological active substances and needlessness of sample pretreatment.This kit for the determination of leptin in rat plasma, serum and culture supernatant samples is based on a sandwich enzyme immunoassay. The 96-well plate is coated with anti-rat leptin monoclonal antibody. Rat leptin calibrator or samples and HRP-labeled anti-rat leptin polyclonal antibody are added to the wells for a one step sandwich immunoreaction. During this immunoreaction, monoclonal antibody - antigen - HRP-labeled antibody complex is formed. After rinsing out excess HRP-labeled antibody, HRP enzyme activity is determined and the concentration of rat leptin is calculated.

Bestandteile: 1. Antibody-Coated Plate MTP: 1 plate (96-well) Anti-Rat Leptin mAb
2. Leptin Calibrator Lyophilized: 1 vial (20 ng) Rat Leptin
3. HRP-Labeled Ab Liquid: 1 bottle (6 mL) HRP-labeled antibody from rabbit
4. Substrate Buffer Liquid: 1 bottle (24 mL) 0.015% Hydrogen peroxide
5. OPD Tablet: 2 tablets o-Phenylenediamine hydrochloride
6. Stop Solution Liquid: 1 bottle (12 mL) 2 N H2SO4
7. Buffer Solution A Liquid: 1 bottle (20 mL) Phosphate buffer including animal serum
8. Buffer Solution B Liquid: 1 bottle (15 mL) Phosphate buffer including surfactant
9. Wash Solution Liquid: 1 bottle (50 mL) Concentrated saline Concentrate
10. Plate Seal: 2 sheets

Benötigtes Material: Photometer for microtiter plate (plate reader), which can read absorbance up to 2.5 at 490 nm
Rotator for microtiter plate
Washing device for microtiter plate and dispenser for approximately 0.35 mL with aspiration system
Micropipettes, multi-channel pipettes for 8 wells or 12 wells and their tips
Test tubes for preparation of Calibrator Solution
Graduated cylinder (1,000 mL)
Distilled water or de-ionized water

Anwendungsinformationen

Plattentyp: Pre-coated

Aufbereitung der Reagenzien:

1. Preparation of Calibrator: (A) Sample volume 20 µL (plasma and serum): Reconstitute the Rat Leptin Calibrator (lyophilized, 20 ng/vial) with 1 mL of buffer solution A, which affords a 20,000 pg/mL calibrator solution. Then, 0.5 mL of the calibrator solution is diluted with 0.5 mL of buffer solution A, that yields a 10,000 pg/mL calibrator solution. Repeat the dilution to make calibrators of 5,000, 2,500, 1,250, 625 and 312.5 pg/mL. Buffer solution A is used as the 0 pg/mL calibrator. (B) Sample volume 50 µL (non-plasma and non-serum): Reconstitute the Rat Leptin Calibrator (lyophilized, 20 ng/vial) with 1 mL of buffer solution B and 0.5 mL of the reconstituted calibrator solution is diluted with 1.5 mL of buffer solution B which affords a 5,000 pg/mL calibrator solution. Then, 0.5 mL of the calibrator solution is diluted with 0.5 mL of buffer solution B, that yields a 2,500 pg/mL calibrator solution. Repeat the dilution to make calibrators of 1,250, 625, 312.5, 156.2 and 78.1 pg/mL. Buffer solution B is used as the 0 pg/mL calibrator. Note: Calibrator Solution must be prepared immediately before assay. Use clean test tubes or vessels.
   2. Preparation of Substrate Solution: Dissolve one OPD Tablet in 11 mL of Substrate Buffer. Note: Substrate Solution must be prepared immediately before assay. Use clean test tubes or vessels.
   3. 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.
   4. Other reagents are ready for use.

Testdurchführung:

1. Warm the reagents and samples to room temperature (20-30°C) before beginning the test.
   2. Addition of calibrator solution and samples: (A) Sample volume 20 µL (plasma and serum): Add 50 µL of buffer solution A into wells first, then introduce 20 µL each of calibrator solutions ( 0, 312.5, 625, 1,250, 2,500, 5,000, 10,000 and 20,000 pg/mL) or samples, then add 50 µL of HRP-labeled antibody. Total 120 µL volume is introduced into the wells. (B) Sample volume 50 µL (non-plasma and non-serum): Add 50 µL each of calibrator solutions ( 0, 78.1, 156.2, 312.5, 625, 1,250, 2,500 and 5,000 pg/mL) or samples, then add 50 µL of HRP-labeled antibody. Total 100 µL volume is introduced into the wells.
   3. Cover the plate with the Plate Seal and incubate at room temperature for five hours. During incubation, the plate should be rotated on a plate rotator. During continuous rotation of test plate, the plate rotator may be heated up. It is recommended to place styrene form or plywood between the plate and the rotator. During incubation except for the color reaction, the test plate should be rotated gently by plate rotator to promote immunoreaction.
   4. Remove the Plate Seal and aspirate the solution in the wells. Wash the wells three times with approximately 0.35 mL/well of Wash Solution.
   5. Pipette 100 µL of substrate solution into the wells, cover the plate with the Plate Seal and incubate it for 10 minutes at room temperature.
   6. Add 100 µL of stop solution into the wells to stop reaction.
   7. Read the optical absorbance of the wells at 490 nm. Read optical absorbance of reaction solution in wells as soon as possible after stopping the color reaction. Note: Perform all determinations in duplicate.

Ergebnisberechnung:

Calculate mean absorbance values of wells containing the Calibrators and plot a calibration curve on logarithmic graph paper (abscissa: concentration of Calibrators, ordinate: absorbance values of Calibrators). Use the calibration curve to read rat leptin concentrations in samples from the corresponding absorbance values. When a sample value exceeds 20,000 pg/mL, it needs to be diluted with buffer solution A until the value is within the assay range.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Lagerung: 4 °C

Details zu Leptin

Target: Leptin (LEP)

Andere Bezeichnung: Leptin

Hintergrund: Leptin, which is a product of ob gene, is a protein consisting of 167 amino acids and it is secreted from white adipose tissue. It is known that leptin acts on hypothalamus to decrease food intake and to reduce body weight, body fat, blood sugar and blood insulin in a healthy and an ob/ob mouse. Further, gene expression of neuropeptide Y (NPY) is surpressed by leptin. Recently, a radioimmunoassay for leptin determination in human plasma has become available and the leptin level in a human group with obesity was found to increase in comparison with that of a normal group. The level correlates well with body fat and these observations show clearly that the leptin concentration in human plasma reflects the tissue fat weight. The measurement of plasma leptin may be an excellent index of obesity. Although rat leptin shows a high homology (96%) with mouse leptin, it is observed that substitution of several amino acid residues occurs at both end N- and C- terminal regions between human and rat leptin.

Pathways: JAK-STAT Signalweg, 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