Class-specific affinity purification

The first role of antibodies is to protect their host organism from the action of foreign molecules and organisms. B-cells have a great capacity to adapt themselves as they encounter and bind those contaminants, and especially to produce and secrete large quantities of antibodies in the plasma upon differentiation into plasmocytes. However, some pathogenic microorganisms have evolved over time following the same principle, and now produce molecules which are able to counter antibodies actions by sequestrating them and thus protect themselves from higher organisms immune system.

To take advantage of these characteristics, several antibody-binding proteins have been extensively characterised and engineered. Smaller-sized recombinant forms as well as hybrids are nowadays widely used as immunological tools, especially in antibody purification, for their ease of use and high flexibility to purify antibodies from almost all species and regardless of antigen specificity.

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Protein A

Protein A is a cell wall component of Staphylococcus aureus which binds to the Fc region of immunoglobulins, especially IgG. The interaction with IgG vary between species, and even within a species (see Table 1). For example, human IgG1, IgG2 and IgG4 bind strongly to protein A, while IgG3 does not. Thanks to its low price, this protein is widely used for large-scale purification of cell culture supernatants and ascitic fluids, and usually yields high purity and recovery rate.

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Protein G

Protein G is a bacterial cell wall protein isolated from group B streptococci. Like protein A, protein G binds to most mammalian immunoglobulins through their Fc regions. More specifically, protein G is able to bind strongly to all human and mouse subclasses. The unique immunoglobulin-binding characteristics of protein G can be used for the purification of mammalian monoclonal and polyclonal antibodies that do not bind well to protein A.

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Protein L

Protein L is a surface component of Peptostreptococcus magnus which contrary to protein A and G does not bind immunoglobulins through the Fc region but through the light chains, without hindering their antigen-binding capacity. This protein is thus able to bind not only a wider range of antibody classes including IgA, IgD, IgE, IgG and IgM but also antigen-binding antibody fragments such as Fab, F(ab’)2 or scFV.

However, binding is restricted to certain subtypes of kappa light chains, and should therefore be restricted to the purification of samples of monoclonal antibodies whose light chain isotype is known.

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Antibody-binding protein binding strength

Table 1: Binding capacity of usual antibody-binding proteins

The indicated values for protein L refer only to antibody species and subtypes having appropriate kappa light chains. Lambda light chains and some kappa light chains do not bind.

Purification of IgG using protein A, G or L

According to the host species, class and subclass, the antibody-binding protein exhibiting the stronger binding capacity will be used to purify IgG from biological samples. Those samples can come from either a polyclonal antibody development project (final bleed) or monoclonal antibody production (ascitic fluid or cell culture supernatant) we previously performed, or from your own laboratory. Depending on the nature of the sample, the expected antibody concentrations are as follows:

  • serum: from 1 to 3 mg antibody / mL serum
  • ascites: from 1 to 5 mg antibody / mL ascitic fluid
  • cell culture (flask): from 5 to 20 µg antibody / mL supernatant
  • cell culture (high density device): up to 1 mg antibody / mL supernatant

Protein A or G purified IgG are delivered in a glycine buffer (glycine 0.1 M, Tris 0.1 M, NaCl 3 M, pH 7.8) containing 0.02% sodium azide. If you do not wish azide to be added, please inform us while placing your order.

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