Antisera Purification
If a high background is observed in assays using the antisera, various purification techniques are available. It is important to first check that the background is non-specific and not due to the response against the peptide. This can be determined by performing a competitive peptide blocking study. Peptide blocking studies check that the response against the target protein is not a background artifact.
Ammonium sulfate precipitation: This is a commonly used method for removing protein from solution. The method is a fairly crude, non-specific purification that removes the majority of plasma proteins and leaves the immunoglobulin fraction. When in solution, proteins form hydrogen bonds with water through their exposed polar and ionic groups. Adding small ions such as ammonium or sulfate removes water molecules from the protein, resulting in precipitation of the protein out of solution. It should be stated that ammonium sulfate precipitation will not result in highly purified antibodies. The contaminants will consist of other high-molecular-weight proteins and proteins that are trapped in the large flocculent precipitates. It is recommended that ammonium sulfate precipitation be used as part of a purification scheme involving further purification steps.
Protein A/G: Protein A or Protein G purification removes the IgG fraction based on the specificity of these proteins for the Fc portion of the IgG. Protein A is produced from Staphylococcus aureus. It has the capacity to bind at least two molecules of IgG. The binding is specific to the Fc portion and does not affect the antigen binding sites. Protein G is isolated from Group G streptococci and binds the Fc region of the IgG in a similar manner to Protein A. Protein A and G have differing binding efficiencies for IgG from different species. It is important to check this before deciding which method to use. For example, Protein G works well with sheep Ig, but Protein A does not. Neither Protein A nor Protein G will bind to chicken Ig. Care should be taken when eluting the antibody from the column to avoid denaturation of the antibody.
Immunoaffinity Purification:
Also called monospecific purification. The most commonly used method to purify antigen-specific antibodies from crude sera is immunoaffinity purification. Unlike Protein A or G, the non-specific Ig fraction is not retained. In this procedure,
peptide antigen
is bound covalently to a solid support. The antibodies within the polyclonal sample that are specific for the peptide antigen bind to the support column. The unbound antibodies are removed from the column by washing and the specific antibodies are eluted from the column. The product of immunoaffinity purification is highly specific antibodies. Immunoaffinity purification can occasionally cause denaturation of the antibody due to the conditions used to elute the bound antibody from the column. It is important to compare the response generated by the purified sample against the response generated by the crude sera.