1 Purification of Antibodies

Antibodies have become useful, versatile reagents and essential tools in the scientific laboratory. The different sources of antibodies include polyclonal antisera from immunized animals and monoclonal antibodies from cells in culture or from ascites in animals.

Compared to monoclonal antibodies polyclonal antisera takes less time, effort and technical skill to produce. The serum from immunized animals contains antibodies with specificity to many different antigens or even to different antigenic groups (epitopes) in the antigen molecule. Even after hyperimmunization only a small fraction (usually a few percent) of the total IgG binds to the immunizing antigen. The variability in the response of the immunized animal accounts for differences in the binding characteristics of different batches of polyclonal antibodies. Polyclonal antibodies can be produced in nearly any species.

In contrast, monoclonal antibodies require a fusion cell partner and they can only be made with cells of a few species. They also require tissue culture facilities and skills, but the outcome is an essentially limitless supply of uniform antibodies that recognize a single epitope. They can also be produced in large quantities with uniform characteristics. Among the advantages of the use of monoclonal antibodies, is the specificity of their binding: even impure antigens or whole cells can be used for immunization and the resulting monoclonal antibodies recognize one epitope and therefore one molecule. This is a very powerful tool for identifying unique populations of cells or molecules. Other rewards are that monoclonal antibodies are homogeneous in nature and can be produced in unlimited quantities.

Both polyclonal and monoclonal antibodies can be used in many of the same application such as immuno-histochemistry, immunoblotting, immunoprecipitation, and enzyme-linked immunosorbent assays (ELISAs).

Some assays or applications do not require a purified antibody. In this case the polyclonal antibodies can be used as antiserum and the monoclonals as either ascitic fluid or supernatants. Other assays require that the antibodies present in the serum, ascites or supernatants be in purified form; some examples include the following: (1) when the antibodies are used after chemical modifications such as labeling with fluorescent probes or isotopes; (2) for the preparation of IgG fragments such as F(ab′)2 or Fab; (3) when accurate concentrations of the antibody are required.

IgG can be purified, as described in the Chapters 3–5, by a variety of methods: Ammonium sulfate precipitation followed by size–exclusion (SE) chromatography are the least expensive option available for the purification of antibodies. Protein A- and protein G-affinity chromatography are the fastest methods for purifying antibodies, but they are not effective for all subclasses of antibodies. Ion-exchange (IEX) chromatography is indicated for purifying intact monoclonal and polyclonal antibodies and antibody fragments. A protocol for affinity chromatography purification of polyclonal antibodies with defined specificity or immunoglobulin class is also provided.

IgG purification from whole serum (such as by ammonium sulfate precipitation) effectively removes other contaminating proteins. However such IgG preparations will still contain both the specific (to the desired antigen) and non-specific IgG molecules. If necessary, this step can be followed by size-exclusion or ion exchange chromatography. Alternatively, Protein A, protein G or one of the newer commercial proprietary affinity chromatography columns can be used.

When antibodies are being purified, an assay for antibody activity and a method for determining the purity of the antibody are required. Contamination with other serum proteins can easily be checked by SDS–PAGE and staining the gels with Coomassie Blue. The activity of the antibodies can be determined by immunoassays such as any one of the methods that are described in this book.

There are commercially available kits primarily designed for the purification of IgG and IgM classes of antibodies derived from all common animal species. These kits are in general very reliable and work well, although they are more expensive than using the classical methods of purification described in these chapters. There are also kits for the fragmentation of antibodies (for example see Pierce Biotechnology, www.piercenet.com). Table 1 gives a list of some of the immunoglobulin purification kits that are available; it is by no means complete, but shows the diversity of products commercially available.

Table 1 Commonly used kits for antibody purification
Full size table