Antibody Purification

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Introduction
The diversity of the antibody-antigen interaction and our ability to manipulate the characteristics
of the interaction has created many uses for antibodies and antibody fragments,
both for immunochemical techniques within general research and for therapeutic and
diagnostic applications.
The use of recombinant technology opens up the potential to create an infinite number of
combinations between immunoglobulins, immunoglobulin fragments, tags and selected
proteins, further manipulating these molecules to our advantage.



Polyclonal antibodies
Most frequently, a host will produce a large number of antibodies that recognizes independent
epitopes (the antibody binding site) on the antigen. Each specific antibody is produced by a
different clone of plasma cells. Serum is a very good source of polyclonal antibodies. These
antibodies are commonly used as reagents in immunochemical techniques, using crude serum
as the source. Further purification may be required, either to isolate the group of polyclonal
antibodies or to isolate a specific antibody from the group.

Monoclonal antibodies
Hybridoma cells are created by isolating plasma cell precursors which are then fused with
immortal cells. The hybridoma cells can be single cell cloned and expanded as individual
clones that secrete only one antibody type, a monoclonal antibody. The high specificity of
a monoclonal antibody is a significant advantage, particularly in therapeutic applications.
Monoclonal antibodies are frequently used in the form of tissue culture supernatants harvested
from the hybridoma culture, or as crude extracts produced from hybridoma cells grown as
tumours in syngenic mice. Production of monoclonal antibodies using hybridoma technology
has been successful for the production of mouse monoclonal antibodies, but this has meant
that therapeutic applications have always been associated with the risk of immunogenic
reactions (only human antibodies are non-immunogenic to humans).

Genetically engineered sources
Recombinant technology is used increasingly for the manipulation and production of
antibodies and their fragments.
For antibodies to be most effective when used as a therapeutic agent they should have a
long serum half-life, low immunogenicity, a high affinity for the antigen, and be able to
neutralize the antigen's activity. These are all features that can be enhanced by genetic
manipulation. To reduce immunogenicity, mouse-human chimeric antibodies have been
produced, containing some human constant region sequences along with the mouse V
regions.


Tagged fusion antibodies and fragments
Amplification of a protein containing a tag of known size and biological function greatly
simplifies subsequent isolation, purification and detection. For example, (His)6 or GST tags
are now in common use to enable simple affinity purification at any scale. In some cases
the protein yield can also be increased. Adding tags of this type is also extremely useful if
the target molecule has no Fc region (an Fc region enables purification with Protein A
Sepharose™ or Protein G Sepharose affinity media).