Once an adequate polyclonal response has been reached, the crude serum can be purified by two distinct methods:
- Protein A (or G) affinity chromatography is used for purifying all the IgGs present in the sample. For better results and to enrich for specific IgG antisera, we recommend the elimination of antibodies with low affinity and specificity but higher binding capacity, such as IgM by purification of the antiserum. This type of purification is useful for relatively non sensitive systems like Western Blotting.
- Immunoaffinity purification is recommended for purifying antigen specific antibodies from a pool of polyclonal antibodies, principally when they are to be used in immunocytochemistry or in immunofluorescence, where a high background would interfere with analysis. In this procedure, the pure antigen (free peptide) is covalently bound to a solid support: CNBr-Sepharose, then the specific antibodies within the pool bind themselves to the antigen-sepharose, while the unbound antibodies are removed by washing. Finally the specific antibodies are eluted from the column and collected in TRIS buffer; their positivity and titer are reconfirmed by ELISA testing. We normally purify half of the total serum and the purified antibodies are then shipped in solution.