Custom Services- Affinity Purification
The purified egg yolk IgY fraction, as well as containing IgY directed against the immunizing antigen, contains all the other antibody specificities that were present in the animal’s circulation at the time the eggs were formed. The IgY fraction, depending on the purification method, is approximately 85-90% pure (as determined by SDS polyacrylamide gel electrophoresis).
The specific activity (percentage of immunoglobulins that is directed against your antigen) of this IgY fraction depends on the immunogenicity of your antigen and the genetic make-up of the immunized animal. It is reasonable to expect between 2 and 10% specific activity of IgY from an animal immunized with a protein greater than 50 kilodaltons. Expect less than 1% specific activity to a peptide from an animal immunized with peptide KLH (there will be a much higher specific activity directed to the carrier KLH portion of the immunogen). For many applications, this non-affinity purified antibody fraction will work just fine and it is unnecessary to further purify your IgY. In fact, further purification may result in the loss of your highest avidity antibodies.
However, sometimes, it is necessary to remove the antibodies that are not antigen specific and this is done by making an antigen column to which the whole IgY fraction is applied. The IgY you receive is the antigen-specific fraction that is eluted from the column. To make the affinity column, we will need 5 mg protein antigen (in addition to that supplied for immunizations) at a concentration greater than 3 mg/ml.
Our custom affinity purification service includes:
- making an affinity column with your antigen or peptide
- antibody affinity purification of antigen- or peptide-specific IgY from a 0.5 gram purified chicken IgY antibody fraction.