Chicken IgY Antigen Capture ELISA
Because of the high concentration of IgY found in egg yolks, eggs are a convenient and safe source of antibodies for study and to demonstrate the power off the ELISA technique. Virusys has developed an easy to use ELISA that permits the sensitive detection and quantitation of chicken IgY in egg yolks. The assay has been developed for educational use to be included in a biotechnology oriented curriculum. It may also find use in quantitation of chicken antibody based products.
The assay is based on the "antigen capture" technique:
- Purified rabbit anti-chicken IgY ("capture" antibody) is attached to the surface of a plastic well (supplied as 96-well ELISA plates).
- A sample that may or may not contain chicken IgY is then added and allowed to incubate for 15 min. If present in the sample, the chicken IgY binds to or is "captured" by the rabbit anti-chicken IgY, while other macromolecules in the sample are not (lock and key concept).
- The sample is then removed from the well and the well is washed to remove residual sample components.
- Purified goat anti-chicken IgY is then added to the well (detection antibody) and incubated for 15 min. This material not only will bind to chicken IgY (if it was captured from the sample), but it contains an additional modification; chemically attached to the goat anti-chicken IgY protein is an enzyme (Horseradish Peroxidase; HRP). If IgY was captured from the sample, then the detection antibody (goat anti-chicken IgY-HRP) will bind to the captured IgY in an amount that is proportional to the captured IgY.
- The unbound detection antibody is then removed and the well is washed again. This is a very important wash step, because it is necessary to remove unbound detection antibody (and therefore HRP), from the well.
- Chromagen is then added to the well. The enzyme reaction will convert the chromagen (enzyme substrate) to a colored (blue) reaction product. The amount of reaction product that is produced is directly proportional to the amount of goat anti-chicken IgY-HRP in the well, which in turn, is proportional to the amount of chicken IgY that was captured from the sample.
- Since the enzyme reaction will continue as long as chromagen is available, a stop reagent is added to the well after 15 min. that inhibits the enzyme, changes the blue color to yellow, and stabilizes the yellow color.
- The yellow reaction product can then be quantified visually or using an instrument.