GLP-2 ELISA Kit

Catalog No. ABIN956056

Rat GLP-2 ELISA Kit, Colorimetric assay for quantification of Rat GLP-2.

Quick Overview for GLP-2 ELISA Kit (ABIN956056)

Target: GLP-2 (Glucagon-like peptide 2 (GLP-2))

Reactivity: Rat

Detection Method: Colorimetric

Method Type: Competition ELISA

Application: ELISA

Product Details GLP-2 ELISA Kit

Purpose: The Rat GLP-2 (Glucagon-Like Peptide-2) EIA is for the quantitative determination of GLP-2 in rat serum or plasma.

Analytical Method: Quantitative

Specificity: Does not cross-react with rat Glucagon and rat GLP-1 within the range of 300 pmol/mL.

Characteristics: The proglucagon gene is expressed in both pancreatic A cells and intestinal L cells. Tissue-specific post-translational processing of proglucagon by the prohormone convertase produces the different proglucagon derived peptides (PGDPs ) in both pancreas and intestine. The most notable pancreatic PGDP is glucagon, whereas the L cells produce several structurally related peptides, including glucagon-like peptide (GLP)-1 and GLP-2, as well as glicentin and oxyntomodulin, which contain glucagon sequence in their molecules. Among PGDPs, GLP-2 has recently been found to show intestinal epithelial proliferation. Advantages of this assay include sensitive quantification, high specificity and no interference from other body fluid factors or physiologically active substances. The Rat GLP-2 Calibrator is highly purified synthetic product.This EIA kit is based on a competitive enzyme immunoassay using a highly specific antibody to rat GLP-2 and a biotin/avidin-affinity system. The 96-well plate is coated with goat anti-rabbit IgG antibody. Rat GLP-2 Calibrator or samples, biotinylated rat GLP-2 and anti-rat GLP-2 polyclonal antibody antibody are added to the wells for a competitive immuno-reaction. After plate rinsing, HRP-labeled streptoavidins are added to bind to the antigen-antibody complex so that HRP-labeled streptoavidin-biotinylated rat GLP-2-antibody complexes are formed on the surface of the wells. Finally, excess HRP-labeled streptoavidins are rinsed, HRP-enzyme activity is determined, and the concentration of rat GLP-2 is calculated.

Components: 1. Antibody-Coated Plate MTP: 1 plate (96-well) Goat Anti-rabbit IgG
2. GLP-2 Calibrator Lyophilized: 1 vial (50 ng/vial) Synthetic rat GLP-2
3. Labeled Antigen Lyophilized: 1 vial Biotinylated rat GLP-2
4. GLP-2 Antibody Liquid: 1 bottle (6 mL) Rabbit anti-rat GLP-2
5. SA-HRP Concentrate Liquid: 1 bottle (0.2 mL) HRP-labeled streptoavidin
6. Diluent for SA-HRP 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 hydrochloride
9. Stop Solution Liquid: 1 bottle (12 mL) 2 N H2SO4
10. Buffer Solution Liquid: 1 bottle (35 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 492 nm
Rotator for microtiter plate
Washing device for microtiter plate and dispenser for approximately 0.3 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

Application Details

Plate: Pre-coated

Reagent Preparation:

1. Preparation of Calibrator Solutions: Reconstitute the GLP-2 Calibrator (lyophilized rat GLP-2, 50 ng/vial) with 0.5 mL of Buffer Solution, giving a 100 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 33.33 ng/mL Calibrator Solution. Repeat the serial dilution to make Calibrator Solutions at 11.11, 3.704, 1.235, 0.412 and 0.137 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 Labeled Antigen with 9 mL of Buffer Solution. Note: Labeled Antigen must be prepared immediately before assay. Use clean test tubes or vessels.
   
   3. Preparation of SA-HRP diluted solution: Add 0.12 mL of SA-HRP Concentrate to the bottle of SA-HRP Diluent (12 mL) and mix thoroughly. Note: 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) before beginning the test.
   2. Add 300 µL of Wash Solution into wells. Aspirate the Wash Solution in the wells. Repeat this washing procedure twice.
   3. Add 75 µL of Labeled Antigen solution into the wells. Then add 25 µL of the prepared Calibrator Solutions (0, 0.137, 0.412, 1.235, 3.704, 11.11, 33.33, 100 ng/mL) or samples. Next, add 50 µL of rat GLP-2 Antibody to the wells.
   4. Cover the plate with the Plate Seal and incubate at 4°C overnight (16 -18 hours).
   5. Remove the Plate Seal and aspirate the solution in the wells. Wash the wells three times with approximately 0.3 mL/well of 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. During the incubation, the plate should be rotated on a plate rotator.
   8. Remove the Plate Seal, aspirate and wash the wells five times with approximately 0.3 mL/well of Wash Solution.
   9. Add 100 µL of Substrate Solution 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. During continuous rotation of test plate, the plate rotator may be heated. It is recommended to place polystyrene foam or plywood between the plate and the rotator.

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-2 concentrations in samples from the corresponding absorbance values. When a sample value exceeds 100 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

Target Details for GLP-2

Target: GLP-2 (Glucagon-like peptide 2 (GLP-2))

Alternative Name: GLP-2