Protein G and protein A bind to different IgG

Extracted from Affinity Chromatography Vol. 1: Antibodies, GE Healthcare, 2016
 

The high affinity of protein G and protein A for the Fc region of polyclonal and monoclonal IgG-type antibodies forms the basis for purification of IgG, IgG fragments containing the Fc region, and IgG subclasses.

Protein G and protein A are bacterial proteins from Group G Streptococci and Staphylococcus aureus, respectively. When coupled to Sepharose, protein G and protein A create extremely useful, easy-to-use chromatography media for routine purification of antibodies. Examples include the purification of monoclonal IgG-type antibodies, purification of polyclonal IgG and its subclasses, adsorption and purification of immune complexes involving IgG, and fusion proteins. IgG subclasses can be isolated from cell culture supernatants, serum, and ascites.

Table 3.1 shows a comparison of the relative binding strengths of protein G and protein A to different immunoglobulins. The information has been compiled from various publications. Binding strengths are tested with free protein G or protein A and can be used as guidelines to predict the binding behavior to a protein G or protein A purification medium. However, when coupled to an affinity matrix, the interaction can be altered. For example, rat IgG1 binds to Protein G Sepharose, but not to Protein A Sepharose.

Single-step purification of samples from native sources or calf serum-supplemented medium based on Fc region specificity will co-purify host IgG and can even bind trace amounts of serum proteins. To avoid trace amounts of contaminating IgG, consider alternative techniques such as immunospecific affinity using anti-host IgG antibodies coupled to, for example, NHS-activated Sepharose, ion exchange chromatography (IEX) with, for example, Capto™ adhere or hydrophobic interaction chromatography (HIC, see Chapter 6).


Table 3.1.
Relative binding strengths of antibodies from various species to protein G and protein A as measured in a competitive ELISA test. The amount of IgG required to give a 50% inhibition of binding of rabbit IgG conjugated with alkaline phosphatase was determined.

Species Subclass Protein G binding Protein A binding
Human IgA

IgD

IgE

IgG1

IgG2

IgG3

IgG4

IgM*






++++

++++

++++

++++

variable





++++

++++



++++

variable
Avian egg yolk IgY
Cow   ++++ ++
Dog   + ++
Goat   ++
Guinea pig IgG1 ++ ++++
Hamster   ++ +
Horse   ++++ ++
Koala   +
Llama   +
Monkey (rhesus)   ++++ ++++
Mouse IgG1

IgG2a

IgG2

IgG3

IgM*
++++

++++

+++

+++

+

++++

+++

++

variable
Pig   +++ +++
Rabbit   +++ ++++
Rat IgG1

IgG2a

IgG2

IgG3
+

++++

++

++






+
Sheep   ++ +/–

* Purified using HiTrap IgM Purification HP columns.
Purified using HiTrap IgY Purification HP columns.
++++ = strong binding.
++ = medium binding.
— = weak or no binding.

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