Affinity Chromatography Handbook

26
Characteristic IgG
Antibody classes
IgM
IgA
IgE IgD
Heavy chain
g
m
a
e
d
Light chain k or l k or l k or l k or l k or l
Y structure
Fig. 10. Antibody classes.
IgG, IgG fragments and subclasses
The basis for purification of IgG, IgG fragments and subclasses is the high affinity of protein A
and protein G for the Fc region of polyclonal and monoclonal IgG-type antibodies, see
Figure 9.
Protein A and protein G are bacterial proteins (from Staphylococcus aureus and Streptococcus,
respectively) which, when coupled to Sepharose, create extremely useful, easy to use media
for many routine applications. Examples include the purification of monoclonal IgG-type
antibodies, purification of polyclonal IgG subclasses, and the adsorption and purification
of immune complexes involving IgG. IgG subclasses can be isolated from ascites fluid, cell
culture supernatants and serum.
Table 2 shows a comparison of the relative binding strengths of protein A and protein G to
different immunoglobulins compiled from various publications.
A useful reference on this subject is also: Structure of the IgG-binding regions of streptococcal
Protein G, EMBO J., 5, 1567–1575 (1986).
Binding strengths are tested with free protein A or protein G and can be used as a guide to
predict the binding behaviour to a protein A or protein G purification medium. However,
when coupled to an affinity matrix, the interaction may be altered. For example, rat IgG 1
does not bind to protein A, but does bind to Protein A Sepharose.