Bio-Synthesis provides a wide array of methods to conduct antibody purification. The correct choice of purification methods depends upon the class/subclass of the antibody, the species which raised it, and the intended use of the antibodies. We will purify antibodies from bioreactors, supernatants, ascites, and serum using a variety of methods such as:
- Immunoaffinity purification
- Protein A/G purification
- Ion exchange chromatography
- Procedures and methodology used for the purification and a Quality BSI provides control reports (including analysis of yield, purity, and immunoreactivity) with each purified product
General description on the type of antibody purification:
Antisera Purification: Various purification techniques are available if a high background is observed in assays using the antisera. First, it is essential to check that the background is non-specific and not due to the response against the peptide. Peptide blocking studies check that the response against the target protein is not a background artifact. Performing a competitive peptide blocking study allows determining the background noise.
Ammonium Sulfate Precipitation: Ammonium sulfate precipitation is a commonly used method for removing proteins from a solution. The technique is a somewhat crude, non-specific purification method that eliminates 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 the solution. However, ammonium sulfate precipitation will not result in highly purified antibodies. The contaminants can consist of other high-molecular-weight proteins and trapped proteins in the large flocculent precipitates. We recommended using ammonium sulfate precipitation 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 originates from Staphylococcus aureus. It can 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 similarly to Protein A. Protein A, and G have differing binding efficiencies for IgG from different species. It is essential 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. Ensure when eluting the antibody from the column to avoid denaturation of the antibody.
Immunoaffinity Purification: Immunoaffinity purification is the most commonly used method to purify antigen-specific antibodies from crude sera. Unlike Protein A or G, the non-specific Ig fraction is not retained. In this procedure, the peptide antigen is bound covalently to solid support—antibodies within the polyclonal sample specific to 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 results in 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 essential to compare the response generated by the purified sample against the response generated by the crude sera.