ANTIBODY-BASED TECHNIQUES - A Guide to Methods in the Biomedical Sciences - page 75

media, respectively. Although there are a number of methods that can
be used, the most common is to take advantage of one of two bacterial
proteins which bind antibodies (particularly IgG antibody classes) with
high specificity. The antibodies can then be eluted (using low pH buffers)
in pure form. These are protein A and protein G.
Protein A
Protein A is a cell wall constituent produced by the bacterium Staphy-
lococcus aureus (the Cowan I strain is most commonly used). It is a pro-
tein of 42 kDa that binds to the Fc region of certain antibody molecules,
specifically some of the IgG subclasses. The use of Protein A to purify
IgG was introduced in 1975 (30). Protein A is used to detect or purify
IgG or to isolate IgG-antigen complexes. It is often covalently bound
to sepharose beads and can be reused a number of times due to its
stability and resistance to denaturants.
Protein G
Protein G is a cell wall protein isolated from group G streptococci.
It binds to IgG subclasses not recognized by protein A, and binds IgM
antibodies as well. In its native form, protein G also binds albumin, Fab
regions of antibodies, and constituents of cell membranes. To improve
specificity, recombinant forms of protein G, in which only the Fc bind-
ing sites have been retained, have been produced and are now com-
mercially available. Recombinant protein G is used in ways similar to
protein A.
Antibodies as probes
The amazing versatility of antibodies is not only due to their remark-
able variability and their high degree of specificity, but also due to the
fact that the can be modified by covalent attachment of reagents that
allow for their detection in a variety of assays. A few of the most common
forms of modifications, and some of their uses, is listed below.
lodinated antibodies
One of the first labeling techniques for antibodies was to covalently la-
bel them with a radioactive isotope of iodine, such as can also be
used). Antibodies can be labeled to a high specific activity with with-
out compromising antigen binding or biological activity. They can be used
for both in vivo and in vitro studies. The use of radioactively-labeled
antibodies was crucial to the development of the radioimmunoassay