Human GLP-2 ELISA Assay

Additional Information

Assay Background

Proglucagon gene is expressed in both pancreatic A cell and intestinal L cell. Tissue-specific posttranslational processing of proglucagon by prohormone convertase produces different proglucagon derived peptides (PGDPs) in both pancreas and intestine. The most notable pancreatic PGDP is glucagon, whereas intestinal L cell produces several structurally related peptides, including glucagon-like peptide 1 (GLP-1) and 2 (GLP-2), as well as glicentin and oxyntomodulin which contain glucagon sequence in their molecules. Among PGDPs, GLP-2 has been found to show intestinal epithelial proliferation.

Assay Procedure

1. Bring all the reagents and samples to room temperature (20-30°C) at least 1 hour before starting assay.
2. Add 0.35mL/well of washing solution into the wells of the plate, and then aspirate the solution. Repeat this washing procedure further twice (total 3 times). 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. Fill 40uL of labeled antigen solution into the wells first, then introduce 25uL each of standard solutions (0, 0.412, 1.235, 3.704, 11.11, 33,33 and 100 ng/mL) or samples and finally add 50uL of GLP-2 antibody into the wells.
4. Cover the plate with adhesive foil and incubate it at 4ºC for 16 ~ 18 hours. (Not shaken)
5. After incubation, take off the adhesive foil, aspirate the solution in the wells and wash the wells 3 times with approximately 0.35 mL/well each of washing 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 100uL of SA-HRP solution into each of the wells.
7. Cover the plate with adhesive foil and incubate it at room temperature (20~30ºC) for 1 hour. During incubation, the plate should be shaken with a microtiter plate shaker.
8. Resolve one OPD tablet with 12 mL of substrate buffer. It should be prepared immediately before use.
9. Take off the adhesive foil, aspirate the solution in the wells and wash the wells 5 times with approximately 0.35 mL/well each of washing 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.
10. Add 100uL of substrate solution into each the wells, cover the plate with adhesive foil and incubate it for 30 minutes at room temperature.
11. Add 100uL of stopping solution into each of the wells to stop color reaction.
12. Read optical absorbance of the solution in the wells at 490 nm. Calculate mean absorbance values of standard solutions and plot a standard curve on semi logarithmic graph paper (abscissa: concentration of standard solution; ordinate: absorbance value). Use the average absorbance of each sample to determine the corresponding value by simple interpolation from this standard curve.
    

Typical Standard Curve

Precision and Reproducibility: