Protein Protocols Protein Protocols

1122 Schwarz
Table 1
Affinity of Protein A for IgG for Different Species and Subclasses
IgG species/subclass Affinity Binding pH Elution pH
Human IgG1 High 7.5 3
Human IgG2 High 7.5 3
Human IgG3 Moderate 8 4–5
Human IgG4 High 7.5 3
Mouse IgG1 Low 8.5 5–6
Mouse IgG2a Moderate 8 4–5
Mouse IgG2b High 7.5 3
Mouse IgG3 Moderate 8 4–5
Rat IgG1 Low 8.5 5–6
Rat IgG2a None–low
Rat IgG2b Low 8.5 5–6
The affinity between protein A and the antibody is due mainly to hydrophobic interactions
at the binding sites (1). The interaction can be strengthened by the inclusion of
higher concentrations of chaotropic salts such as sodium citrate or sodium sulfate.
Although the addition of chaotropic salts is unnecessary in the case of high-affinity
antibodies, it allows weakly binding antibodies such as mouse IgG1 to strongly interact
with protein A. This purification scheme is outlined in Subheading 3.2. The different
affinities resulting from minor differences in the Fc region can also be utilized for the
separation of subclasses or the separation of different species in hybridoma supernatant
using a pH gradient. However, this is possible only if the difference in affinity is large
enough like in the case of the separation of mouse IgG2b from bovine IgG. If fetal calf
serum supplemented growth media is used, bovine IgG will contaminate the monoclonal
antibody if the method described in Subheading 3.1. or 3.2. is used. An
improved protocol utilizing the different affinities is described in Subheading 3.3.
1.2. Thiophilic Adsorption Chromatography
The term thiophilic adsorption chromatography was coined for gels, which contain
low molecular weight sulfur-containing ligands such as divinyl sulfone structures or
mercapto-heterocyclic structures (2,3). The precise binding mechanism of proteins to
these gels is not well understood. However, thiophilic chromatography can be regarded
as a variation of hydrophobic interaction chromatography inasmuch as chaotropic salts
need to be added to facilitate binding of antibodies to the gel. Therefore, the interaction
between the ligands and the antibody is likely to be mediated through accessible aromatic
groups on the surface of the antibody.
The major advantage of these thiophilic gels over protein A gels is that they bind all
antibodies with sufficiently high capacity and very little discrimination between sub-