Human PYY ELISA Assay

Additional Information

Assay Principle

This Eagle Biosciences Human PYY ELISA Assay kit is for the determination of human PYY in samples is based on a competitive enzyme immunoassay using combination of highly specific antibody to human PYY and biotin-avidin affinity system. To the wells of plate coated with rabbit anti human PYY antibody, standard or samples, labeled antigen are added for competitive immunoreaction. After incubation and plate washing, horse radish peroxidase (HRP) labeled streptoavidin (SA) is added to form HRP labeled streptoavidin-biotinylated antigen-antibody complex on the surface of the wells. Finally, HRP enzyme activity is determined by 3,3’,5,5’-Tetramethyl- benzidine (TMB) and the concentration of human PYY is calculated.

Assay Procedure

1. Before start assay, bring all the reagents and samples to room temperature (20～30ºC).
2. Add 0.3 mL/well of washing solution into the wells and aspirate the washing solution in the wells. 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 25uL of buffer solution into the wells first, then introduce 50L of each of standard solutions (0, 0.082, 0.247, 0.741, 2.222, 6.667, 20 ng/mL) or samples and finally add 25uL of labeled antigen into the wells. The total pipetting time of standard solutions and samples for a whole plate should not exceed 30 min.
4. Cover the plate with adhesive foil and incubate it at 4ºC overnight for 16 ~ 18 hours. (Still, plate shaker not need.)
5. After incubation, move the plate back to room temperature keeping for about 40 minutes and take off the adhesive foil, aspirate and wash the wells 4 times with approximately 0.3 mL/well of washing solution.
6. Finally, invert the plate and tap it onto an absorbent surface, such as paper toweling, to ensure blotting free of most residual washing solution.
7. Pipette 100uL of SA-HRP solution into each of the wells.
8. Cover the plate with adhesive foil and incubate it at room temperature (20 ~ 30ºC) for 2 hour. During the incubation, the plate should be shake with a plate shaker.
9. Take off the adhesive foil, aspirate and wash the wells 4 times with approximately 0.3 mL/well 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 enzyme substrate solution (TMB) to each of the well, cover the plate with adhesive foil and keep it for 30 minutes at room temperature in a dark place for color reaction. (Still, plate shaker not need.)
11. Add 100uL of stopping solution into each of the wells to stop color reaction.
12. Read the optical absorbance of the solution in the wells at 450 nm. The dose-response curve of this assay fits best to a 4 (or 5)-parameter logistic equation. The results of unknown samples can be calculated with any computer program having a 4 (or 5)-parameter logistic function.
    Otherwise calculate mean absorbance values of wells containing standards and plot a standard curve on semilogarithmic graph paper (abscissa: concentration of standard; ordinate: absorbance values). Use the average absorbance of each sample to determine the corresponding value by simple interpolation from this standard curve.

Typical Standard Curve

Precision & Reproducibility