GLP-2 ELISA Kit

Catalog No. ABIN956066

Product Details GLP-2 ELISA Kit

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

Reactivity: Human

Detection Method: Colorimetric

Method Type: Competition ELISA

Application: ELISA

Purpose: The Human GLP-2 EIA is for the quantitative determination of GLP-2 in human plasma and serum.

Analytical Method: Quantitative

Specificity: No cross-reactivity with neither glucagon (rat, mouse, or human) nor GLP-1, even at a concentration 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 sample constituents. The Human GLP-2 Calibrator is a highly purified synthetic product (purity: > 98%).This EIA kit is based on a competitive enzyme immunoassay using a highly specific antibody to human GLP-2 combined with a biotin-avidin affinity system. The 96-well plate is coated with goat anti-rabbit IgG and GLP-2 Calibrators or samples, biotinylated human GLP-2 and rabbit anti-GLP-2 antibody are added to the wells for a competitive immuno-reaction. After incubation and washing, HRP-labeled streptoavidin (SA-HRP) is added to form HRP-labeled SA-biotinylated GLP-2- antibody complex on the surface of the wells. Finally, HRP enzyme activity is determined by o-Phenylenediamine dihydrochloride (OPD) and the concentration of human GLP-2 is calculated.

Components: 1. Antibody-Coated Plate Microtiter Plate: 1 plate (96-well) Goat anti-rabbit IgG antibody
2. GLP-2 Calibrator Lyophilized: 1 vial (50 ng/vial) Synthetic human GLP-2
3. Labeled Antigen Lyophilized: 1 vial Biotinylated human GLP-2
4. GLP-2 Antibody Liquid: 1 bottle (6 mL) Rabbit anti-human GLP-2 antibody
5. SA-HRP Solution Liquid: 1 bottle (12 mL) HRP-labeled streptoavidin (SA)
6. Substrate Buffer Liquid: 1 bottle (26 mL) Citrate buffer with 0.015% hydrogen peroxide
7. OPD Tablet: 2 tablets o-Phenylenediamine dihydrochloride
8. Stop Solution Liquid: 1 bottle (12 mL) 1 M H2SO4
9. Buffer Solution Liquid: 1 bottle (25 mL) Phosphate buffer
10. Wash Solution Liquid: 1 bottle (50 mL) Concentrated saline Concentrate
11. 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 with an aspiration system
Micropipettes, multi-channel pipettes for 8 wells or 12 wells and their tips
Glass 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 human GLP-2 Calibrator (lyophilized 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, and 0.412 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 Buffer Solution. Note: Labeled Antigen must be prepared immediately before assay. Use clean test tubes or vessels.
   
   3. 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.
   
   4. 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.
   
   5. 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 0.35 mL/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 onto an absorbent surface, such as paper toweling, to ensure removal of most of the residual Wash Solution.
   3. Add 40 µL of Labeled Antigen Solution into wells. Then add 25 µL of the prepared Calibrator Solutions (0, 0.412, 1.235, 3.704, 11.11, 33.33, and 100 ng/mL) or samples. Next, add 50 µL of GLP-2 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 3 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 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 onto an absorbent surface, such as paper toweling, to ensure removal of most of the residual Wash 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 492 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-2 concentrations in samples from the corresponding absorbance values. When a sample value is expected to exceed 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 (GLP-2 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