GLP-1 ELISA Kit

Catalog No. ABIN956065

Product Details GLP-1 ELISA Kit

Target: GLP-1 (Glucagon-like peptide 1 (GLP-1))

Reactivity: Human, Mouse, Rat

Detection Method: Colorimetric

Method Type: Competition ELISA

Application: ELISA

Purpose: The Rat, Mouse and Human GLP-1 EIA is for the quantitative determination of GLP-1 in rat, mouse and human plasma.

Analytical Method: Quantitative

Specificity: No cross-reactivity with rat, human or mouse glucagons, human glicentin and rat, mouse, human GLP-2.

Characteristics: GLP-1 is a peptide hormone from the intestinal mucosa, which is produced from its precursor, proglucagon, by post- translational processing. The mammalian proglucagon is synthesized in the neuroendocrine L-cell of the intestine and the alpha-cells of the pancreas. It contains within its structure the sequences of glucagon and two glucagon-like peptides (GLP-1 and GLP-2) in tandem flanked at their amino and carboxyl termini by dibasic residues. GLP-1 is a 37 amino acid peptide and is produced in the human small intestine and pancreas, in either C-terminal-amidated or glycine-extended form. GLP-1 (7-36) amide and its receptor are present in several brain regions and may play a role in the physiological control of feeding. Several reports have been presented as follows as to the biological activities of GLP-1. GLP-1 (7-37) and (7- 36) amide is known as one of the most potent insulin secretagogues. GLP-1 (7-36) amide was supposed to improve glycemic control in patients with type 2 diabetes by increasing insulin secretion, by inhibiting glucagon secretion and by delaying gastric draining rather than by altering extrapancreatic glucose metabolism. Intravenous GLP-1 (7-37) and (7-36) amide could normalize fasting hyperglycemia in type 2 diabetic patients. Hyperglycemia during parenteral nutrition could be controlled by exogenous GLP-1, whereas the chronic therapy of type 2 diabetes required GLP-1 derivatives with longer duration of action. Recombinant GLP-1 (7-36) amide was recently shown to cause significant weight loss in type 2 diabetics when administered for 6 weeks as a continuous subcutaneous infusion, 5-day treatment of hereby obese human subjects with GLP-1 at high doses by prandial subcutaneous infusion promptly slowed gastric emptying as a probable mechanism of action of increased satiety, decreased hunger and reduced food intake with an ensuing weight loss. A G-protein-coupled receptor, GPR120, which is abundantly expressed in the intestine, functions as a receptor for unsaturated long-chain FFAs (free fatty acids). The stimulation of GPR120 by FFAs promotes the secretion of GLP-1 in vitro (measured by KT-384) and in vivo, and increases circulation insulin, indicate that GPR120-mediated GLP-1 secretion induced by dietary FFAs is important in the treatment of diabetes. All these approaches have shown remarkable efficacy in both experimental and clinical studies. The GLP-1-based therapy of type 2 diabetes, therefore, represents a new and attractive alternative. Advantages of this assay include high sensitivity, high specificity and no interference from other components in plasma samples. The GLP-1 calibrator of this kit is a highly purified synthetic product.This EIA kit is based on a competitive enzyme immunoassay using a highly specific antibody combined with a biotin- avidin affinity system. The 96 well plate is coated with goat anti-rabbit IgG and GLP-1 Calibrators or samples, biotinylated human GLP-1 and GLP-1 antibody are added to the wells for a competitive immuno-reaction. After incubation and rinsing excess GLP-1, HRP-labeled streptoavidins (SA-HRP) are added to bind to the antigen-antibody complex so that HRP- labeled streptoavidin-biotinylated GLP-1-antibody complexes are formed on the surface of the wells. Finally, HRP enzyme activity is determined by o-Phenylenediamine dihydrochloride (OPD) and the concentration of GLP-1 is calculated.

Components: 1. Antibody-Coated Plate MTP: 1 plate (96-well) Goat Anti-Rabbit IgG
2. GLP-1 Calibrator Lyophilized: 1 vial (25 ng/vial) Synthetic GLP-1 (7-36) amide
3. Labeled Antigen Lyophilized: 1 vial Biotinylated GLP-1 (7-36) amide
4. GLP-1 Antibody Liquid: 1 vial (6 mL) Rabbit anti-GLP-1 (7-36) amide
5. SA-HRP Liquid 1 tube (0.2 mL) HRP-Labeled Streptoavidin
6. SA-HRP Diluent Liquid: 1 bottle (12 mL) Phosphate buffer
7. Substrate Buffer Liquid: 1 bottle (26 mL) 0.015% Hydrogen peroxide
8. OPD Tablet: 2 tablets o-Phenylenediamine dihydrochloride
9. Stop Solution Liquid: 1 bottle (12 mL) 1M H2SO4
10. Buffer Solution Liquid: 1 bottle (10 mL) Phosphate buffer
11. Wash Solution Liquid: 1 bottle (50 mL) Concentrated saline Concentrate 12. Plate Seal: 3 sheets

Material not included: 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 with an 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

Application Details

Plate: Pre-coated

Reagent Preparation:

1. Preparation of Calibrator Solutions: Reconstitute the GLP-1 Calibrator (lyophilized GLP-1, 25 ng/vial) with 0.5 mL of Buffer Solution, giving a 50 ng/mL Calibrator Solution after reconstitution. 0.1 mL of the reconstituted Calibrator Solution is diluted with 0.2 mL of Buffer Solution to yield a 16.67 ng/mL Calibrator Solution. Repeat the serial dilution to make Calibrator Solutions at 5.556, 1.852, 0.617, 0.206 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.
   
   2. Preparation of Labeled Antigen: Reconstitute the Labeled Antigen with 6 mL of distilled water. Note: Labeled Antigen must be prepared immediately before assay. Use clean test tubes or vessels.
   
   3. Preparation of SA-HRP Solution: Add 120 µL of SA-HRP into the bottle of SA-HRP Diluent and mix well. Note: Diluted SA-HRP Solution must be prepared immediately before assay. Use clean test tubes or vessels.
   
   4. Preparation of Substrate Solution: Dissolve one OPD Tablet in 12 mL of Substrate Buffer. Note: Substrate Solution must be prepared immediately before assay. Use clean test tubes or vessels.
   
   5. 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.
   6. Other reagents are ready for use.

Assay Procedure:

1. Warm the reagents and samples to room temperature (20-30°C) at least 1 hour before beginning the test.
   2. Add 350 µL/well of diluted Wash Solution into the wells and aspirate the Washing Solution. Repeat this washing procedure twice, for a total of 3 washing steps. 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 µL of Labeled Antigen Solution into wells. Then add 30 µL of the prepared Calibrator Solutions (0, 0.206, 0.617, 1.852, 5.556, 16.67, 50 ng/mL) or samples. Next, add 40 µL of GLP-1 Antibody into the wells.
   4. Cover the plate with a Plate Seal and incubate at 4°C overnight (16-18 hours). Plate rotator is not needed for this incubation.
   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 it onto an absorbent surface, such as paper toweling, to ensure blotting free of most residual washing solution.
   6. Pipette 100 µL of diluted SA-HRP Solution into each of the wells.
   7. Cover the plate with a Plate Seal and incubate at room temperature for 1 hour. During the incubation, the plate should be rotated on a plate rotator.
   8. Remove the Plate Seal, aspirate and wash the wells 5 times with approximately 0.35 mL/well of diluted Wash 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 100 µL of Substrate Solution (dissolved OPD tablet) into the wells, cover the plate with a Plate Seal and incubate for 30 minutes at room temperature.
   10. Add 100 µL of Stop Solution into the wells to stop the reaction.
   11. Read the optical absorbance of the wells at 490 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.

Calculation of Results:

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 GLP-1 concentrations in samples from the corresponding absorbance values. When a sample value exceeds 50 ng/mL, it must be diluted with Buffer Solution and re-assayed until the sample value is within the assay range.

Restrictions: For Research Use only

Handling

Storage: 4 °C

Storage Comment: Store kit at 4°C. This kit can be used dividedly in strips of the plate. In such case, the rest of reconstituted reagents (calibrator and labeled antigen solutions) should be stored at 4°C and used within 2 weeks, or stored at or below -30°C and used within 1 month.

Target Details for GLP-1

Target: GLP-1 (Glucagon-like peptide 1 (GLP-1))

Alternative Name: GLP-1 (GLP-1 Products)

Synonyms: GLP1 ELISA Kit, GLP2 ELISA Kit, GRPP ELISA Kit, GLP-1 ELISA Kit, Glu ELISA Kit, PPG ELISA Kit, glucagon ELISA Kit, GCG ELISA Kit, Gcg ELISA Kit